Addressing COVID-19 Vaccine Hesitancy

COVID-19 vaccine hesitancy in the United States is a major roadblock in the fight against a raging pandemic. Education on mRNA vaccine technology, efficacy, and safety may help reduce public hesitancy. An educational handout was created and distributed at the Brookfield Family Practice to help alleviate COVID-19 vaccine hesitancy in the local community.https://scholarworks.uvm.edu/fmclerk/1707/thumbnail.jp

The Bioavailability of Soluble Cigarette Smoke Extract Is Reduced through Interactions with Cells and Affects the Cellular Response to CSE Exposure.

Cellular exposure to cigarette smoke leads to an array of complex responses including apoptosis, cellular senescence, telomere dysfunction, cellular aging, and neoplastic transformation. To study the cellular response to cigarette smoke, a common in vitro model exposes cultured cells to a nominal concentration (i.e. initial concentration) of soluble cigarette smoke extract (CSE). However, we report that use of the nominal concentration of CSE as the only measure of cellular exposure is inadequate. Instead, we demonstrate that cellular response to CSE exposure is dependent not only on the nominal concentration of CSE, but also on specific experimental variables, including the total cell number, and the volume of CSE solution used. As found in other similar xenobiotic assays, our work suggests that the effective dose of CSE is more accurately related to the amount of bioavailable chemicals per cell. In particular, interactions of CSE components both with cells and other physical factors limit CSE bioavailability, as demonstrated by a quantifiably reduced cellular response to CSE that is first modified by such interactions. This has broad implications for the nature of cellular response to CSE exposure, and for the design of in vitro assays using CSE

Modeling CSE bioavailability during cellular in vitro exposure.

<p>As illustrated, there are several different ways in which CSE bioavailability may be limited, including binding of CSE components to cell culture plastics, serum factors, or interactions with cells. Furthermore, CSE bioavailability may be reduced through either degradation or evaporation of CSE components.</p

Effects of varying cell type and cell count on cellular response to CSE exposure.

<p><b>(A)</b> NIH 3T3 mouse fibroblasts were plated at 10,000, 15,000, or 20,000 cells/well in a 48 well plate, allowed to grow for 24 hours, and exposed to CSE for 24 hours using 400 uL of 0%, 2%, 3%, 4%, or 5% CSE. An MTT viability assay was then used to assess relative cellular damage. (<b>B</b>) NMuMG mouse epithelial cells were plated at 10,000 or 20,000 cells/well in a 48 well plate, allowed to grow for 24 hours, and exposed to CSE for 24 hours using 400 uL of 0%, 2%, 4%, 6%, 8% and 10% CSE. An MTT viability assay was then used to assess relative cellular damage. Significant differences in cellular morphology between (<b>C</b>) NIH 3T3 mouse fibroblast cells and (<b>D</b>) NMuMG mouse epithelial cells are evident in photomicrographs, but do not alter the general trends observed above. In each graph, values are reported as % viability compared to cells not exposed to CSE, with error bars representing the standard error of the mean. * Compared to other cell counts exposed to the same percent CSE, p < 0.05.</p

Expression of select genes in cells exposed to different experimental conditions relative to cells not exposed to CSE.

<p>Expression of select genes in cells exposed to different experimental conditions relative to cells not exposed to CSE.</p

Morphological changes and altered expression of mRNA transcripts associated with cellular cytotoxicity are evident in the cellular response to CSE exposure for cells plated at 1 x 10⁶ cells/plate.

<p>Phase contrast images of (<b>A</b>) Undamaged cells (<b>B</b>) Cells exposed to 6% CSE at low volume (3 mL) (<b>C</b>) Cells exposed to 8% CSE at low volume (<b>D</b>) A scatterplot illustrating the general upward trend in expression of cellular cytotoxicity biomarkers at both CSE concentrations (6%, 8%) at either low volume (LV) or high volume (HV) (please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163182#pone.0163182.t002" target="_blank">Table 2</a> for more details) (<b>E</b>) Cells exposed to 6% CSE at high volume (6 mL) (<b>F</b>) Cells exposed to 8% CSE at high volume.</p

Comparison of cellular response to fresh CSE versus flash-frozen CSE.

<p>NIH 3T3 mouse fibroblasts were plated at 15,000 cells/well in a 48 well plate, allowed to grow for 24 hours, exposed to CSE for 24 hours using 400 uL of 0–7% CSE that was either used fresh (within 30 minutes of making) or flash-frozen, then subjected to an MTT viability assay to assess relative cellular damage. Values are reported as % viability compared to cells not exposed to CSE, with error bars representing the standard error of the mean.</p

Effects of varying CSE solution volume on cellular response to CSE exposure.

<p>(<b>A</b>) NIH 3T3 mouse fibroblasts were plated at 10,000, 15,000, or 20,000 cells/well in a 48 well plate, allowed to grow for 24 hours, and damaged for 24 hours using 5% CSE at varying volumes (0.2 mL, 0.4 mL, 0.6 mL and 1.0 mL). An MTT cell viability assay was then used to assess relative cellular damage. <b>(B)</b> Cells were plated at 10,000 cells/well (<b>C</b>) 15,000 cells/well or (<b>D</b>) 20,000 cells/well, allowed to grow for 24 hours, and damaged for 24 hours using 0%, 4%, 5% or 6% CSE at varying volumes (0.2 mL, 0.4 mL, 0.6 mL and 1.0 mL). An MTT viability assay was then used to assess relative cellular damage. In each graph, values are reported as % viability compared to cells not exposed to CSE, with error bars representing the standard error of the mean.</p

Relative bioavailability of CSE soluble components after exposure to different cell counts.

<p>(<b>A</b>) In this transfer assay, different cell numbers (0, 25,000; 50,000 cells/well) were exposed to one of two volumes of CSE (0.5 mL or 1 mL) for 60 minutes. After this short exposure, 400 uL of the resulting CSE solution was removed from the wells, and added to new undamaged cells (15,000 cells/well). These cells were exposed to the CSE solution for an additional 24 hours, before (<b>B</b>) analysis by MTT, and calculation of relative bioavailability compared to 100% bioavailability defined as equivalent to the cellular response to the amount of CSE available after transfer from wells with a high volume of CSE solution, and no cells present. Error bars represent the standard error of the mean. n.s. represents a lack of significance compared to other designated experimental conditions, p > 0.05.</p

Effects of cell count on cytotoxicity biomarkers after CSE exposure, using the LDH release assay.

<p>(<b>A</b>) NIH 3T3 mouse fibroblasts were plated at 10,000, 15,000, or 20,000 cells/well in a 48 well plate, allowed to grow for 24 hours, and and exposed to CSE for 24 hours using 400 uL of 0–7% CSE. An MTT viability assay was then used to assess relative cellular damage. Values are reported as % viability compared to cells not exposed to CSE <b>(B)</b> Cells were plated and exposed to CSE as stated above, then a lactate dehydrogenase assay was performed to assess the amount of lactate dehydrogenase released. Values are reported as % cytotoxicity compared to cells exposed to 20% CSE (which was considered maximum LDH release), with error bars representing the standard error of the mean. * Compared to other cell counts exposed to the same percent CSE, p < 0.05.</p