Transcriptome level changes in PHCM genes after exposure to <i>T</i>. <i>cruzi</i>.

<p>Intensity plot of significantly differentially expressed mRNA transcripts in PHCM at 60, 90 and 120 minutes post <i>T</i>. <i>cruzi</i> exposure shows a strong up-regulation profile of AP-1 transcription factor members (JUNB, FOSB, FOS), EGR family of transcription factors (EGR1, EGR3) and pro-fibrotic cytokines and chemokines (IL5, IL6, IL13, CCL11). Transcriptome profiles were generated from 3 biological replicates at each time point. Significant changes were defined as transcripts having greater than two-fold up or down regulation and a multiple testing corrected ANOVA p-value < 0.05. Red intensity bars show greater than two-fold up regulation while green intensity bars indicate a more than two-fold down-regulation.</p

Kinetics of fibrotic gene expression profile of PHCM exposed to <i>T</i>. <i>cruzi</i>.

<p>Serum starved PHCM monolayers exposed to invasive <i>T</i>. <i>cruzi</i> trypomastigotes at a ratio of 10 parasites/cell at different time points were used for evaluating protein levels of profibrotic TF by western blot assays. Cell lysates of PHCM exposed to <i>T</i>. <i>cruzi</i> at different time points were separated by SDS-PAGE, transferred to NC membrane, probed with (a) anti-SNAI1 antibody and developed by chemiluminescence. Blot showing the bands for SNAI1 and GAPDH is a representative of 3 independent experiments conducted with similar results. (b) The graph summarizes a densitometric analysis of 3 independent experiments showing SNAI1 protein expression kinetics normalized against GAPDH. (c) Cell lysate blots were probed with VDR antibody and developed by chemiluminescence. Blot showing the bands for VDR and GAPDH is a representative of 3 independent experiments conducted with similar results. (d) Column graph summarizes densitometric analysis of 3 independent experiments showing VDR protein expression kinetics normalized against GAPDH. (e) Cell lysates were also probed with EGR1 antibody and developed by chemiluminescence. Blot showing the bands for EGR1 and GAPDH is a representative of 3 independent experiments conducted with similar results. (f) Column graph summarizes densitometric analysis of 3 independent experiments showing EGR1 protein expression kinetics normalized against GAPDH. *P<0.05 vs control, **P<0.01 vs control. The data on each column graph (b, d and f) show a representative experiment of 3 independent experiments performed with similar results.</p

Biological interactions and connectivity network of PHCM transcripts regulated by <i>T</i>. <i>cruzi</i>.

<p>A biological interaction network was created using significantly changed transcriptome microarray and qPCR genes as primary seed nodes for querying multiple biological interaction and pathway centric databases out to one degree of interaction. Primary seed nodes are shown as black circles, interaction expansion nodes (gray circles) were added to the network based on confirmed interactions with at least two primary seed nodes. Red/pink connections (edges) between nodes denote interactions based on physical interactions database sources such as BIND, MINT and DIP. Blue edges between nodes denote pathway data base derived interactions from KEGG, REACTOME, BIOCARTA and the NCI Pathway Interaction Database. Orange edges denote nodes mapping to their associated KEGG pathways.</p

Pathway Enrichment analysis of significantly changed genes.

<p>Pathway Enrichment analysis of significantly changed genes.</p

Validation of selected fibrotic gene transcripts in PHCM exposed to <i>T</i>. <i>cruzi</i>.

<p>Validation of selected fibrotic gene transcripts in PHCM exposed to <i>T</i>. <i>cruzi</i>.</p

Invasive <i>T</i>. <i>cruzi</i> trypomastigotes upregulates nuclear translocation of JunB in PHCM.

<p>PHCM seeded onto Lab-Tek chamber slides were serum starved and exposed to transgenic <i>T</i>. <i>cruzi</i> expressing GFP at a ratio of 10 parasites/cell at different time points followed by washing with PBS. Cells were probed with mouse anti human-JunB Alexa Fluor-488 IgG (green), actin myofibrils were stained red with phalloidin, nuclei stained blue with DAPI and visualized by Confocal microscopy using 40X magnification. Panel (a), upper panel shows an increase in green fluorescent staining of JunB with time. Lower panel shows DAPI and phalloidin staining. (b) Column graph shows normalized quantitative measurement of fluorescence intensity using CAStream software. The fluorescence intensity was normalized with the control and plotted. This is a representative experiment of three independent experiments performed with similar results. *P<0.05 vs control, **P<0.01 vs control.</p

Pathway intersection graph of genes modulated by <i>T</i>. <i>cruzi</i> in PHCM.

<p>Transcriptome array and Real Time-PCR genes mapping to significantly enriched pathways and interactions groups show a highly connected and overlapping structure. Enriched KEGG pathways (TGF-β signaling, Jak-STAT signaling, Cytokine-cytokine receptor interaction and Chagas disease) share multiple genes between them as well as with the AP1 transcriptional factor network. Gene nodes are sectioned into three slices to denote significant expression changes at the 60, 90 and 120 minute time points. Pie slices in red denote a greater than two-fold up regulation, slices in green denote less than a negative two-fold down regulation.</p

Expression of cytokines/chemokines in PHCM conditioned media.

<p>Expression of cytokines/chemokines in PHCM conditioned media.</p

Virological properties of vectors used in this study.

<p>Virological properties of vectors used in this study.</p

Antigen capsid-incorporation vector elicits an <i>in vivo T. cruzi</i> humoral immune response.

<p>BALB/c and C57BL/6 mice (n = 7) were primed, boosted, and reboosted with 1×10¹⁰ VP of Ad vectors. A) Immunization timeline showing when immunizations were performed (solid arrows) and sera was collected (dashed arrows). B) Post-prime, post-boost, and post-reboost BALB/c serum or C) Post-prime, post-boost, and post-reboost C57BL/6 serum was collected for ELISA binding assays. Ten µM of purified gp83 (KIYWKQPVEGTKSWTLSK) antigenic peptide was bound to ELISA plates. The plates were then incubated with serial diluted concentrations of immunized mice serum and the binding antibodies were detected with HRP conjugated secondary antibody. The amount of anti-gp83 antibodies in the sera was calculated based on a standard curve of mouse IgG protein. The values are expressed as the mean ± standard deviation. (*) = <i>P</i>≤0.05, and (**) = <i>P</i>≤0.01.</p