Effects of Clostridium difficile Toxin A and B on Human T Lymphocyte Migration
"> Figure 1
<p>Effects of TcdA and TcdB on T cell viability. Human blood T cells were treated with TcdA (<b>A</b>) or TcdB (<b>B</b>) for 3 h and the cell viability was determined by trypan blue staining using a hemocytometer (at least 3 independent experiments for each condition). The circled toxin concentrations (<span class="html-italic">i.e.</span>, TcdA: 50 ng/mL; TcdB: 25 ng/mL) were selected for all subsequent experiments. (<span class="html-italic">i.e.</span>, the qualitative selection criteria for TcdA and TcdB concentration are (1) comparable viability level ~80%; (2) the concentration before the viability level drops relatively fast.).</p> "> Figure 2
<p>Effects of TcdA and TcdB on T cell motility and chemotaxis in transwell assays. (<b>A</b>) Migration of TcdA- or TcdB pre-treated T cells or untreated cells to either medium alone or medium containing CCL19 (100 nM), is presented as the percentage of input cells that migrated to the bottom well of the transwell assay; (<b>B</b>) Fold change of cell migration to 100 nM CCL19 comparing to medium alone; (<b>C</b>) Flowcytometric analysis of CCR7 expression on T cells with or without TcdA or TcdB treatment. T cells were incubated with 50 ng/mL TcdA or 25 ng/mL TcdB for 3 h before CCR7 antibody staining (anti-human CCR7-APC); (<b>D</b>) T cell migration to the medium control, 50 ng/mL TcdA or 25 ng/mL TcdB. Data are normalized to the percentage of T cells migrated to medium alone. All migration experiments (at least 3 independent experiments for each condition) were performed in RPMI containing 0.4% BSA for 90 min. The <span class="html-italic">p</span> values for each comparison from the 2-sample <span class="html-italic">t</span> test are shown: <b>**</b> <span class="html-italic">p <</span> 0.01.</p> "> Figure 3
<p>Effects of TcdA and TcdB on T cell chemotaxis to a CCL19 gradient in microfluidic devices. (<b>A</b>) Chemotactic Index (C.I.) and speed of T cells with or without toxin pre-treatment (TcdA: 50 ng/mL; TcdB: 25 ng/mL; 3 h treatment before the cell migration experiments) over a 35 min cell migration experiment in a 100 nM CCL19 gradient. Results are presented as average ± S.E.M. The percentage of cells migrating towards the CCL19 gradient is shown on the top of the C.I; (<b>B</b>–<b>D</b>) Images of T cells without toxin treatment (<b>B</b>), treated by TcdA (<b>C</b>) or TcdB (<b>D</b>) in microfluidic devices. The <span class="html-italic">p</span> values for each comparison from the 2-sample <span class="html-italic">t</span> test are shown. <b>*</b> <span class="html-italic">p</span> < 0.05; <b>**</b> <span class="html-italic">p</span> < 0.01. Three independent experiments were performed for each condition with similar results and one representative experiment for each condition is presented.</p> "> Figure 4
<p>Effects of TcdA or TcdB on T cell migration to HT-29 cell culture. (<b>A</b>) T cell migration to HT-29 cell culture treated with TcdA or TcdB for 24 h or to untreated HT-29 cell culture. Data are normalized to the percentage of T cells migrated to the untreated HT-29 cell culture; (<b>B</b>–<b>D</b>) Morphological changes of HT-29 cells without (<b>B</b>) or with TcdA (<b>C</b>) or TcdB (<b>D</b>) treatment after 24 h. All cell migration experiment were performed in RPMI with 10% FBS for 90 min. The <span class="html-italic">p</span> values for each comparison from the 2-sample <span class="html-italic">t</span> test are shown. <b>*</b> <span class="html-italic">p</span> < 0.05; <b>**</b> <span class="html-italic">p</span> < 0.01.</p> ">
Abstract
:1. Introduction
2. Results and Discussion
2.1. Effects of TcdA and TcdB on T Cell Viability
2.2. Effects of TcdA and TcdB on T Cell Motility Measured by Transwell Assays
2.3. Effects of TcdA and TcdB on T Cell Chemotaxis
2.4. Effects of TcdA and TcdB on T Cell Migration to HT-29 Cell Culture
3. Experimental Section
3.1. Reagents
3.2. Cells
3.3. Flow Cytometric Analysis of Surface CCR7 Expression
3.4. Transwell Cell Migration Assays
3.5. Microfluidic Device and Gradient Generation
3.6. Cell Migration Experiments Using Microfluidic Device
3.7. Analysis of Microfluidic-Based Cell Migration Data
4. Discussion and Conclusions
Acknowledgments
Conflict of Interest
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Wu, D.; Joyee, A.G.; Nandagopal, S.; Lopez, M.; Ma, X.; Berry, J.; Lin, F. Effects of Clostridium difficile Toxin A and B on Human T Lymphocyte Migration. Toxins 2013, 5, 926-938. https://doi.org/10.3390/toxins5050926
Wu D, Joyee AG, Nandagopal S, Lopez M, Ma X, Berry J, Lin F. Effects of Clostridium difficile Toxin A and B on Human T Lymphocyte Migration. Toxins. 2013; 5(5):926-938. https://doi.org/10.3390/toxins5050926
Chicago/Turabian StyleWu, Dan, Antony George Joyee, Saravanan Nandagopal, Marianela Lopez, Xiuli Ma, Jody Berry, and Francis Lin. 2013. "Effects of Clostridium difficile Toxin A and B on Human T Lymphocyte Migration" Toxins 5, no. 5: 926-938. https://doi.org/10.3390/toxins5050926
APA StyleWu, D., Joyee, A. G., Nandagopal, S., Lopez, M., Ma, X., Berry, J., & Lin, F. (2013). Effects of Clostridium difficile Toxin A and B on Human T Lymphocyte Migration. Toxins, 5(5), 926-938. https://doi.org/10.3390/toxins5050926