Susceptibility and Response of Human Blood Monocyte Subsets to Primary Dengue Virus Infection

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16− and CD16+ subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16− and CD16+ blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16+ monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16+ monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease

CD8 T cell mediated induction of interleukin-12p70 production by dendritic cells

Ph.DDOCTOR OF PHILOSOPH

Production of soluble factors associated with protection against dengue by monocyte subsets.

<p>Isolated monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Supernatants were harvested over the course of 6 days. (A) Levels of IFN-α were determined by a multi-subtype specific ELISA kit. (B and C) Levels of CXCL10 and TRAIL were determined using multiplex bead arrays. Results are mean ± SE for 6 different donors. There were no significant differences were found between infected CD16⁻ and CD16⁺ monocytes. (D) Supernatants from CD16⁻ and CD16⁺ monocytes exposed to dengue virus or medium without virus were harvested at day 6. These supernatants were passed through 100 kDa centrifuge filters to remove dengue virus. K562 cells were pretreated for 24 hours with either culture medium, supernatants of CD16⁻ or CD16⁺ monocytes with or without virus exposure. Pre-treated K562 cells were washed and infected with dengue virus at a MOI of 2. After 2 days, the extent of infection was determined by intracellular labeling of K562 cells with anti-NS1 antibody. The percentage of NS1⁺ K562 cells after 2 days is shown. Data are representative of 2 experiments using different donors. **, <i>p</i><0.005 between respective monocyte subset with and without virus.</p

IL-4 treatment enhances the susceptibility of the CD16⁺ monocyte subset to a greater extent.

<p>Isolated CD16⁻ or CD16⁺ monocyte subsets were pretreated with 25 ng/ml of IL-4 for two days. Cells were subsequently washed and harvested before exposure to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. Percentages of CD16⁻ and CD16⁺ monocytes that are (A) NS1⁺ or (B) E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 5 different donors. (C) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed IL-4 treated CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 4 different donors. <b>*</b><i>p</i><0.05, between IL-4 treated CD16⁺ and IL-4 treated CD16⁻ monocytes with virus.</p

Susceptibility of monocyte subsets to dengue virus infection.

<p>(A) Flow cytometric profile of CD16⁻ and CD16⁺ monocytes after isolation. (B) Isolated CD16⁻ or CD16⁺ monocyte subsets were either exposed to dengue virus (DENV2, NGC) at a MOI of 10 or medium without virus. After 2 days, monocytes were fixed, permeabilized and labeled with anti-E-protein and anti-NS1 specific antibodies. Quadrants for virus exposed monocytes (right panel) were set based on monocytes without virus exposure (left panel). Percentage positive cells in each quadrant are shown. Representative data for 6 different donors. (C and D) Percentages of CD16⁻ and CD16⁺ monocytes that are NS1⁺ or E-protein⁺ over the course of 6 days after virus exposure. Results are mean ± SE of 6 different donors. (E) Plaque assays with BHK-21 cells were performed with supernatants taken from virus exposed CD16⁻ or CD16⁺ monocytes over the course of 6 days. Results are mean ± SE from 5 different donors.</p

Production of inflammatory cytokines by monocyte subsets.

<p>Monocyte subsets were exposed to dengue virus or medium without virus. Supernatants were harvested over the course of 6 days. Levels of (A) IL-1β (B) TNF-α (C) IL-6 (D) CCL2 (E) CCL3 and (F) CCL4 were measured using multiplex bead arrays. Results are mean ± SE of 5 different donors. <b>*</b><i>p</i><0.05, **, <i>p</i><0.005 between CD16⁺ and CD16⁻ monocytes with virus.</p

Experimental assessment on feasibility of computed tomography-based thermometry for radiofrequency ablation on tissue equivalent polyacrylamide phantom

Purpose: This study aimed to evaluate the effects of various computed tomography (CT) acquisition parameters and metal artifacts on CT number measurement for CT thermometry during CT-guided thermal ablation. Methods: The effects of tube voltage (100–140 kVp), tube current (20–250 mAs), pitch (0.6–1.5) and gantry rotation time (0.5, 1.0 s) as well as metal artifacts from a radiofrequency ablation (RFA) needle on CT number were evaluated using liver tissue equivalent polyacrylamide (PAA) phantom. The correlation between CT number and temperature from 37 to 80 °C was studied on PAA phantom using optimum CT acquisition parameters. Results: No statistical significant difference (p > 0.05) was found on CT numbers under the variation of different acquisition parameters for the same temperature setting. On the other hand, the RFA needle has induced metal artifacts on the CT images of up to 8 mm. The CT numbers decreased linearly when the phantom temperature increased from 37 to 80 °C. A linear regression analysis on the CT numbers and temperature suggested that the CT thermal sensitivity was –0.521 ± 0.061 HU/°C (R2 = 0.998). Conclusion: CT thermometry is feasible for temperature assessment during RFA with the current CT technology, which produced a high CT number reproducibility and stable measurement at different CT acquisition parameters. Despite being affected by metal artifacts, the CT-based thermometry could be further developed as a tissue temperature monitoring tool during CT-guided thermal ablation. © 2019, © 2019 The Author(s). Published with license by Taylor & Francis Group, LLC

Paediatric COVID-19 mortality: a database analysis of the impact of health resource disparity

Background The impact of the COVID-19 pandemic on paediatric populations varied between high-income countries (HICs) versus low-income to middle-income countries (LMICs). We sought to investigate differences in paediatric clinical outcomes and identify factors contributing to disparity between countries.Methods The International Severe Acute Respiratory and Emerging Infections Consortium (ISARIC) COVID-19 database was queried to include children under 19 years of age admitted to hospital from January 2020 to April 2021 with suspected or confirmed COVID-19 diagnosis. Univariate and multivariable analysis of contributing factors for mortality were assessed by country group (HICs vs LMICs) as defined by the World Bank criteria.Results A total of 12 860 children (3819 from 21 HICs and 9041 from 15 LMICs) participated in this study. Of these, 8961 were laboratory-confirmed and 3899 suspected COVID-19 cases. About 52% of LMICs children were black, and more than 40% were infants and adolescent. Overall in-hospital mortality rate (95% CI) was 3.3% [=(3.0% to 3.6%), higher in LMICs than HICs (4.0% (3.6% to 4.4%) and 1.7% (1.3% to 2.1%), respectively). There were significant differences between country income groups in intervention profile, with higher use of antibiotics, antivirals, corticosteroids, prone positioning, high flow nasal cannula, non-invasive and invasive mechanical ventilation in HICs. Out of the 439 mechanically ventilated children, mortality occurred in 106 (24.1%) subjects, which was higher in LMICs than HICs (89 (43.6%) vs 17 (7.2%) respectively). Pre-existing infectious comorbidities (tuberculosis and HIV) and some complications (bacterial pneumonia, acute respiratory distress syndrome and myocarditis) were significantly higher in LMICs compared with HICs. On multivariable analysis, LMIC as country income group was associated with increased risk of mortality (adjusted HR 4.73 (3.16 to 7.10)).Conclusion Mortality and morbidities were higher in LMICs than HICs, and it may be attributable to differences in patient demographics, complications and access to supportive and treatment modalities