35 research outputs found
Capsule influences the deposition of critical complement C3 levels required for the killing of Burkholderia pseudomallei via NADPH-oxidase induction by human neutrophils.
Burkholderia pseudomallei is the causative agent of melioidosis and is a major mediator of sepsis in its endemic areas. Because of the low LD(50) via aerosols and resistance to multiple antibiotics, it is considered a Tier 1 select agent by the CDC and APHIS. B. pseudomallei is an encapsulated bacterium that can infect, multiply, and persist within a variety of host cell types. In vivo studies suggest that macrophages and neutrophils are important for controlling B. pseudomallei infections, however few details are known regarding how neutrophils respond to these bacteria. Our goal is to describe the capacity of human neutrophils to control highly virulent B. pseudomallei compared to the relatively avirulent, acapsular B. thailandensis using in vitro analyses. B. thailandensis was more readily phagocytosed than B. pseudomallei, but both displayed similar rates of persistence within neutrophils, indicating they possess similar inherent abilities to escape neutrophil clearance. Serum opsonization studies showed that both were resistant to direct killing by complement, although B. thailandensis acquired significantly more C3 on its surface than B. pseudomallei, whose polysaccharide capsule significantly decreased the levels of complement deposition on the bacterial surface. Both Burkholderia species showed significantly enhanced uptake and killing by neutrophils after critical levels of C3 were deposited. Serum-opsonized Burkholderia induced a significant respiratory burst by neutrophils compared to unopsonized bacteria, and neutrophil killing was prevented by inhibiting NADPH-oxidase. In summary, neutrophils can efficiently kill B. pseudomallei and B. thailandensis that possess a critical threshold of complement deposition, and the relative differences in their ability to resist surface opsonization may contribute to the distinct virulence phenotypes observed in vivo
Serum opsonization enhances internalization and clearance of <i>B. pseudomallei</i> and <i>B. thailandensis</i> by neutrophils.
<p>Isolated human neutrophils were treated with DPI or DMSO (vehicle control) for 20 min prior to bacterial infection. Neutrophils were incubated with <i>B. pseudomallei</i> and <i>B. thailandensis</i> for (A) 10 min to measure bacterial uptake or for (B) 2 h to measure bacterial survival. The bars represent the mean±SEM of three separate experiments using neutrophils from different donors and performed in duplicate. The single asterisk (*) indicates a significant difference (<i>P</i>≤0.05) compared to unopsonized bacteria. The double asterisks (**) indicate a significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i>. The triple asterisks (***) indicate a significant difference (<i>P</i>≤0.05) between 20% NHS opsonized <i>Burkholderia</i> species and all other conditions.</p
<i>B. pseudomallei</i> are resistant to C3 deposition by the alternative pathway of complement.
<p><i>B. pseudomallei</i> and <i>B. thailandensis</i> were incubated with the indicated serum concentrations in the presence or absence of MgEGTA or EDTA, washed, and stained with an anti-human C3 FITC-conjugated antibody. Samples were analyzed by flow cytometry and the data reported as mean fluorescence intensity (MFI). The bars represent the mean±SEM of three separate experiments performed with duplicate samples. The single asterisk (*) indicates a statistically significant increase (<i>P</i>≤0.05) over unopsonized bacteria. The double asterisks (**) indicate a statistically significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i> values.</p
Levels of endogenous <i>Burkholderia</i>-reactive antibodies present in normal human serum.
<p><i>B. pseudomallei,</i> B. <i>thailandensis</i>, and <i>E. coli</i> were incubated with the indicated serum concentrations, washed, and stained with both an APC-labeled anti-human IgM antibody and R-PE-labeled anti-human IgG antibody. Samples were analyzed by flow cytometry and the data reported as mean fluorescence intensity (MFI). The bars represent the mean±SEM of two separate experiments performed with duplicate samples. The single asterisk (*) indicates a statistically significant increase (<i>P</i>≤0.05) over unopsonized bacteria. The double asterisks (**) indicate a statistically significant difference (<i>P</i>≤0.05) over 20% NHS-opsonized <i>B. pseudomallei</i> and <i>B. thailandensis</i> values.</p
<i>B. pseudomallei</i> acquire less complement C3 on its surface than <i>B. thailandensis.</i>
<p>(A) <i>B. pseudomallei</i> 1026b and <i>B. thailandensis</i> E264, or (B) <i>B. pseudomallei</i> DD503, <i>B. pseudomallei</i> ΔCPS, and <i>B. pseudomallei</i> ΔLPS were opsonized for 30 min at the indicated serum concentrations, washed, and stained with an anti-human C3 FITC-conjugated antibody. Samples were analyzed by flow cytometry and the data reported as mean fluorescence intensity (MFI). The bars represent the mean±SEM of three separate experiments performed with duplicate samples. (A) * indicates a statistically significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i> values; ns  =  not significant. (B) * indicates a statistically significant difference (<i>P</i>≤0.05) compared to <i>B. pseudomallei</i> DD503 (wild-type).</p
<i>B. pseudomallei</i> and <i>B. thailandensis</i> are inherently resistant to killing by human neutrophils.
<p>(A) <i>B. pseudomallei</i> and <i>B. thailandensis</i> were either incubated with neutrophils for 10 min to measure bacterial uptake (% bacterial input) or (B) for 2 h to measure bacterial survival (fold change from respective uptake values) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052276#s2" target="_blank">Materials and Methods</a>. The bars represent the mean±SEM of three separate experiments using neutrophils from different donors, each performed in duplicate. * indicates a statistically significant difference (<i>P</i>≤0.05) between <i>B. pseudomallei</i> and <i>B. thailandensis</i> values.</p
<i>B. pseudomallei</i> and <i>B. thailandensis</i> are resistant to direct complement-mediated killing.
<p><i>B. pseudomallei</i> 1026b, <i>B. thailandensis, B. pseudomallei</i> DD503, <i>B. pseudomallei</i> ΔCPS, <i>B. pseudomallei</i> ΔLPS and <i>E. coli</i> were incubated at the indicated serum concentrations at 37°C. At the indicated times, an aliquot of each sample was serially diluted and plated on TSA to determine CFU/ml. Each condition was assessed as duplicate samples in all experiments. * indicates a significant difference (<i>P</i>≤0.05) between <i>E. coli</i> and both <i>Burkholderia</i> species.</p
Variation of stress levels, burnout, and resilience throughout the academic year in first-year medical students.
Medical student wellness is of great concern in the health care field. A growing number of studies point to increases in suicide, depression, anxiety, mood disorders, and burnout related to physician lifestyles. Mental health issues commencing in medical school have been suggested to have a significant impact on future physician lifestyle and burnout. Tracking the mental health of medical students at the University of Toledo College of Medicine and Life Sciences (UTCOMLS) with standardized indices will help elucidate triggers of poor mental health. Anonymous surveys were developed and distributed to preclinical medical students at five strategic time points throughout the 2018 2019 academic year. Surveys collected basic demographic information as well as inventories measuring perceived stress, burnout, resilience, and mindfulness. 172 M1s (83 males and 89 females) were included in the study and average response rate for the first 4 (out of 5) surveys averaged 74.8%. M1 males and females had on average increased personal burnout over time with females consistently scoring higher. Both males and females had an increase in stress from August to each subsequent month (p<0.05). Females reported a higher level of perceived stress than males in the beginning and middle of the academic year (p<0.05). Both males and females report a gradual decrease in resiliency throughout the academic year. These surveys demonstrated over half of males and females in medical school reported higher perceived stress scores than their gender-matched peers in the general United States population. Our study strengthens documented trends in resiliency, perceived stress, and burnout amongst medical students. More study in designing targeted approaches to ameliorate these findings in the medical student population is warranted