A working model of how chronic, latent <i>T. vaginalis</i> infection of prostate tissue up-regulates the signaling cascade leading to prostate carcinogenesis.

<p>Production of IL-6 leads to transcriptional activation of the STAT3-PIM1-HMGA1 cascade. In this case induced transcriptional activation of the <i>HMGA1</i> proto-oncogene contributes directly to prostate cancer progression via pathways involving COX2 and the prostate-specific membrane antigen. The inset shows a <i>T. vaginalis</i> organism adherent to a VEC, and the same mechanism of cytoadherence occurs for PECs.</p

Demonstration of elevated amounts of PIM1 and HMGA1 proteins in PECs after adherence by <i>T. vaginalis</i> (<i>Tv</i>).

<p>In this experiment, trichomonads were added to T25 confluent monolayers of PECs (lane labeled+) using a parasite to PEC ratio of 10∶1. PECs without added organisms are labeled with a minus sign (−). This ratio of 10∶1 was chosen because it has been shown to yield at least one parasite attached per epithelial cell in adherence assays and to optimally signal epithelial cells for up-regulation of expression of genes <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Kucknoor1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Garcia1" target="_blank">[27]</a>. After visible attachment to PECs, non-adherent organisms were decanted and fresh PEC medium added followed by incubation at 37°C for an additional 30 min. The flask was then placed directly in ice for detachment of organisms, after which PECs were washed and removed from the flask for preparation of total proteins for immunoblotting using established protocols, polyclonal rabbit antibodies produced in our laboratories, and equal loading of protein onto gels, as detailed previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Under conditions of exposure of PECs with or without <i>T. vaginalis</i>, no change in the amount of other cellular proteins was detected, as evidenced by no changes in the amounts of Akt and Bad, and this served to show equal amounts of total proteins loaded onto gels for SDS-PAGE and immunoblotting <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002801#ppat.1002801-Hu1" target="_blank">[28]</a>. Prebleed rabbit serum was used as the negative control and gave no reactivity.</p

Distamycin A attenuates inflammatory cytokine-induced neutrophil-endothelial interactions.

<p><b>A.</b> Wild type mice were subjected to intravital microscopy analysis at 3–4 hours following treatment with TNF-α/DistA or TNF-α/Vehicle (Veh) (n = 3 mice per treatment group with multiple vessels and vascular segments analyzed for each animal). Representative still photos of vascular segments are shown for the 4-hour time point. White round spots lining the vascular wall represent adherent leukocytes. <b>B.</b> Formal video analysis of the phases of neutrophil-endothelial interaction was performed, and values for all vascular segments within a particular treatment group were averaged for each time point. A significant reduction in rolling fraction and sticking efficiency was detected in the TNF-α/Dist A mice at 3–4 hours (p = 0.0001 and 0.0004, respectively), compared with the TNF-α/Vehicle mice.</p

Fecal Metabolome in <i>Hmga1</i> Transgenic Mice with Polyposis: Evidence for Potential Screen for Early Detection of Precursor Lesions in Colorectal Cancer

Because colorectal cancer (CRC) remains a leading cause of cancer mortality worldwide, more accessible screening tests are urgently needed to identify early stage lesions. We hypothesized that highly sensitive, metabolic profile analysis of stool samples will identify metabolites associated with early stage lesions and could serve as a noninvasive screening test. We therefore applied traveling wave ion mobility mass spectrometry (TWIMMS) coupled with ultraperformance liquid chromatography (UPLC) to investigate metabolic aberrations in stool samples in a transgenic model of premalignant polyposis aberrantly expressing the gene encoding the high mobility group A (Hmga1) chromatin remodeling protein. Here, we report for the first time that the fecal metabolome of Hmga1 mice is distinct from that of control mice and includes metabolites previously identified in human CRC. Significant alterations were observed in fatty acid metabolites and metabolites associated with bile acids (hypoxanthine xanthine, taurine) in Hmga1 mice compared to controls. Surprisingly, a marked increase in the levels of distinctive short, arginine-enriched, tetra-peptide fragments was observed in the transgenic mice. Together these findings suggest that specific metabolites are associated with Hmga1-induced polyposis and abnormal proliferation in intestinal epithelium. Although further studies are needed, these data provide a compelling rationale to develop fecal metabolomic analysis as a noninvasive screening tool to detect early precursor lesions to CRC in humans

Distamycin A decreases lung and liver P-selectin tissue staining.

<p><b>A.</b> Representative sections (200× magnification) of lung tissue harvested from C57BL/6 mice 24 h after treatment with Vehicle (Veh), LPS+Veh, or LPS+Dist A (Dist). Tissue was fixed, processed, and stained with a P-selectin antibody. Number of positively stained vessels was counted in sections from at least 5 mice in each treatment group. (Arrows demonstrate examples of positively stained vessels; *p<0.05 compared with Veh; **p<0.05 compared with LPS/Veh). <b>B.</b> Representative sections (200× magnification) of liver tissue harvested from mice 4 h after treatment with Vehicle (Veh), LPS+Vehicle (Veh) or LPS+Dist A (Dist). Tissue was fixed, processed, and stained with a P-selectin antibody. Using NIH Image Software, 5 fields (at 200× magnification) per liver section from mice from each treatment group were quantified, with brown pixels counted as positive staining. Results were expressed as % positively stained area per 200× field. (*p<0.05 compared with Veh; **p<0.05 compared with LPS/Veh).</p

Distamycin A attenuates endotoxin-induced lung and liver inflammation.

<p><b>A.</b> Lungs were harvested from C57BL/6 male mice 24 hours following treatment with Vehicle (Veh), LPS/Vehicle (LPS+Veh) or LPS/Dist A (LPS+Dist A) i.p. (n = 9 mice per group). Lungs were fixed, processed, sectioned, and stained for Gr-1 (a marker of neutrophils, positive staining indicated by brown cells). Representative sections for each condition are shown (200× magnification). Using NIH Image Software, 5 fields (at 200× magnification) per lung section from mice from each treatment group were quantified, with brown pixels counted as positive staining. Results were expressed as % positively stained area per 200× field. (*p<0.05 compared with Veh; **p<0.05 compared with LPS+Veh). <b>B.</b> Liver tissue was harvested from C57BL/6 mice 4 h after treatment with Vehicle (Veh), LPS/Vehicle (LPS+Veh) or LPS/Dist A (LPS+DistA) i.p., (n = at least 4 mice per group) then processed, stained for Gr-1 (neutrophil marker), and analyzed as described above for Fig. 1A. (*p<0.05 compared with Veh; **p<0.05 compared with LPS+Veh).</p

HMGA1 binds to the P-selectin promoter and is critical for full induction of P-selectin promoter activity.

<p><b>A.</b> BAEC cells were transiently transfected with a P-selectin promoter-reporter construct with the addition of a blank expression vector, an expression vector for HMGA1, and/or expression vectors for NF-κB family members (p50/p65). Transfected cells were harvested and assessed for luciferase activity (normalized for β-galactosidase content). Results are expressed as fold-change in luciferase activity relative to transfection with the P-selectin promoter and a blank expression vector. <b>B.</b> BAEC cells were transiently transfected with a P-selectin promoter-reporter construct and increasing concentrations of a vector expressing a dominant-negative form of HMGA1 (DN-HMGA1). Transfected cells were stimulated with TNF-α, then harvested and assessed for luciferase activity (normalized for β-galactosidase content). Results are expressed for each transfection condition as fold-change in luciferase activity as a result of TNF-α stimulation. (*p<0.05 for 0.5 µg of DN-HMGA1 as compared with empty vector control; **p<0.05 for 1.0 µg of DN-HMGA1 as compared with empty vector control). This experiment was repeated three separate times, with each condition performed in triplicate. <b>C.</b> An electrophoretic mobility shift assay (EMSA) was performed using the HMGA1 peptide and a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521) without (Lane 2) or with Dist A (10 µM, Lane 3). Lane 1 represents the radiolabeled probe in the absence of incubation with protein. (* represents the HMGA1-DNA complex in Lane 2 which is diminished in intensity following addition of Dist A in Lane 3). Binding studies were repeated at least two separate times. <b>D</b>. Chromatin Immunoprecipitation (ChIP) was performed on murine bEnd.3 endothelial cells without (Lane 1) or with 3 hours of exposure to Vehicle (Veh, Upper Panel and Lanes 2–3 Lower Panel), mTNF-α (TNF, Lane 4), Distamycin A (Dist A, Lane 5), mTNF-α/Vehicle (T+V, Lane 6), and mTNF-α/Dist A (T+D, Lane 7). Immunoprecipitation of crosslinked, sonicated cell lysates was carried out with an affinity-purified HMGA1 antibody (Upper panel and Lanes 3–7 Lower Panel) or IgG rabbit control antibody (Lanes 1–2 Lower Panel), with PCR amplification using primers spanning a 246-basepair AT-rich region of the P-selectin promoter (including basepairs −542 to −521; “Promoter” in Upper Panel and all lanes in Lower Panel) or 200-basepair fragments spanning a coding region or upstream promoter region of P-selectin (“Exon” and “Upstream Promoter”, respectively in Upper Panel; see sequences in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#s2" target="_blank">Methods</a> section). PCR amplification was undertaken for immunoprecipitated DNA as well as for “input” DNA as an additional control for each condition. Quantitation of precipitated DNA relative to input DNA was undertaken using Quantity One 1-D Analysis Software (Bio-Rad). This experiment was performed two separate times. (*p<0.05, for increase in binding for Veh (HMGA1 Ab) compared with IgG control; **p<0.05, for reduction in binding for TNF-α/Dist compared with TNF-α/Vehicle).</p

Distamycin A disrupts binding of an inducible protein-DNA complex containing NF-κB to an AT-rich region of the P-selectin promoter.

<p><b>A.</b> As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#pone-0010656-g003" target="_blank">Fig. 3B</a>, lung tissue was harvested from wild type mice two hours following treatment with Vehicle (Veh), LPS/Vehicle, or LPS/Dist A (Dist), then subjected to RNA extraction. The same blot from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010656#pone-0010656-g003" target="_blank">Fig. 3B</a> was hybridized with a radiolabeled probe for HMGA1 (and an 18S probe as loading control). This experiment was repeated two separate times. <b>B.</b> Nuclear extracts from BAEC cells (Lanes 1–7) and primary murine lung endothelial cells (MLEC, Lanes 8–12) (“Nuc Ext”) with (Lanes 3–7, 9–12) or without (Lanes 2,8) TNF-α stimulation were subjected to electrophoretic mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521). Lane 1 represents the radiolabeled probe without addition of nuclear extract. TNF-α-treated nuclear extract was additionally incubated and electrophoresed with the radiolabeled probe and vehicle (V, lanes 3 and 10 (or in the presence of TNF-α without vehicle, Lane 9)) or increasing concentrations of Dist A (D₁ = 10 µM, D₂ = 20 µM, Lanes 4–5, 11–12) as well as with an identical competitor (IC, Lane 6) and a non-identical competitor (NIC, Lane 7). (* represents the inducible, specific complex seen following TNF-α treatment; “→” represents disruption of the TNF-α-inducible complex following addition of Dist A (Lanes 4–5 compared with Lane 3 and Lanes 11–12 compared with Lane 9–10). All of the binding studies were repeated at least two separate times. <b>C.</b> Nuclear extracts from BAEC cells (“Nuc Ext”) with (Lanes 3–7) or without (Lane 2) TNF-α stimulation were subjected to electrophoretic mobility shift assays (EMSA) using a radiolabeled probe spanning the AT-rich region of the P-selectin promoter (basepairs −542 to −521). Lane 1 represents the radiolabeled probe without addition of nuclear extract. TNF-α-treated nuclear extract was additionally incubated and electrophoresed with the radiolabeled probe and antibodies to the NF-κB family members p50 and p65 (lanes 4–5), or unrelated and control antibodies (Ets-1 and IgG control respectively, lanes 6–7). (* represents the inducible, specific complex seen following TNF-α treatment; “←” represents supershifted band/disruption of the TNF-α-inducible complex following addition of p50 and p65 antibodies (Lanes 4-5 compared with Lanes 6–7). All of the binding studies were repeated at least two separate times.</p

Distamycin A selectively decreases induction of P-selectin promoter activity and expression.

<p><b>A.</b> BAEC cells were transiently transfected with promoter-reporter constructs for P-selectin and E-selectin. Transfected cells were treated with Vehicle, TNF-α/Vehicle, or TNF-α/Dist A, then harvested twelve hours after treatment and assessed for luciferase activity (with normalization for β-galactosidase levels). Similar experiments were performed in which transfected cells were treated with Vehicle, LPS/Vehicle, or LPS/Dist A and harvested four hours after treatment. Fold change was assessed relative to normalized values of “1” for the Vehicle-treated condition for each construct. These experiments were repeated 3 separate times with duplicate wells for each condition. (*p<0.05 compared with Vehicle for each construct; **p<0.05 compared with TNF-α/Vehicle or LPS/Vehicle for P-selectin). <b>B.</b> Lung tissue was harvested from wild type mice two hours following treatment with Vehicle (Veh), LPS/Vehicle, or LPS/Dist A (Dist), then subjected to RNA extraction and Northern blotting using a radiolabeled probe for P-selectin or E-selectin (and an 18S probe as loading control). This experiment was repeated two separate times.</p