ASB strains differentially attenuate UTI visceral pain.

<p>Modulation of UTI visceral pain by a panel of 16 ASB <i>E. coli</i> strains. A) Mice were infected with NU14 and then instilled with saline or ASB <i>E. coli</i> at PID1, and allodynia was quantified through PID7 (n = 6 for all groups). For clarity, timecourses of allodynia for all 17 conditions were divided among 3 panels. All 16 ASB strains attenuated NU14-induced pelvic pain compared to the saline treated group. B) The relative analgesic activity of ASB strains was arbitrary grouped according to the magnitude of analgesia: strains with analgesic relative to saline but increased allodynia from PID1 (black bars), those that exhibited no increase in allodynia from PID1 to less than 75% reduction in visceral pain from PID1 (white bars), and those that exhibited greater than 75% reduction (gray bars). C) Representative ASB strains from each group in (B) were used in serial infections at two-week intervals. Serial infection did not induce allodynia. Data are reported as the mean ± SEM.</p

<i>E. coli</i> 83972 attenuates NU14 bacteriuria.

<p>A) Experimental scheme for assessing efficacy of a single administration of ASB therapy relative to 3-day course of ciprofloxacin (n = 5 for all groups). B) Mice infected with NU14 exhibited a significant decrease in NU14 bacteriuria 24 hours after initiation of ciprofloxacin (group C, back triangles, PID2, 4, 10), relative to saline-treated mice (group S, white triangles, *P<0.05). A single instillation of 83972 (group ASB, grey inverted triangles) also resulted in a significant decrease in NU14 bacteriuria after 24 hours (PID2, 4, 10, *P<0.05). C) Urinary 83972 for all three groups during the experiment. D and E) Bladder colonization on PID20 was not significantly different between the groups tested. Dashed lines represent limits of detection.</p

ASB <i>E. coli</i> isolate 2–12 rapidly attenuates UTI visceral pain of diverse uropathogens.

<p>A) Allodynia was quantified daily in groups of sham– or NU14-infected mice that were then treated at PID1 with saline, a three-day course of ciprofloxacin, or a single dose of intravesical or intravaginal 2–12 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109321#pone-0109321-g002" target="_blank">Figure 2A</a> (n = 8 in all groups except the 2–12 intravaginal group where n = 6). NU14-infected mice exhibited a significant decrease in pelvic pain 24 hours following treatment with 2–12 via the bladder or vaginal introitus (P<0.05). Allodynia was quantified in mice infected via transureathral catheter with <i>Proteus mirabilis</i> (PM1), <i>Enterocccus faecalis</i> (EF1) and <i>Klebsiella pneumoniae</i> (KP1) and then treated with a three-day course of ciprofloxacin initiated at PID1 or a single intravesical dose of 2–12 (n = 5). B) PM1 induced pelvic allodynia that was significantly attenuated on PID3 and PID4 by 2–12 treatment, relative to the ciprofloxacin or saline groups (P<0.05). C) EF1 induced allodynia that was significantly attenuated on PID2-PID4 by 2–12 treatment, relative to ciprofloxacin or saline groups (P<0.05). Allodynia was also significantly reduced on PID5–6 compared to the saline group (P<0.05). D) KP1 induced allodynia that was significantly attenuated on PID2 by 2–12 treatment, relative to ciprofloxacin or saline groups (P<0.05).</p

<i>E. coli</i> 83972 attenuates NU14-induced bacteriuria and pelvic pain.

<p>A) Referred visceral hyperalgesia was measured as responses to mechanical stimulation of the pelvic region with von Frey filaments of 5 intensities. Responses were quantified at baseline, PID1 following NU14 infection, and 24 hours following of saline (n = 5) or 83972 (n = 10) instillation (PID2). B) NU14-infected mice exhibited a significant decrease in allodynia 24 hours following 83972 instillation. C) Percent increase in allodynia from PID1 was 146.2% in saline-treated mice, but decreased by over 68.9% in 83972-treated mice (P<0.01). D) Mice treated with 83972 exhibited a significantly less NU14 bacteriuria compared to saline-treated mice (P<0.01). Dashed line represents limit of detection. E) At PID1, NU14-infected mice were instilled with saline into the bladder or 2% lidocaine in the bladder, vaginal introitus or colon. Allodynia was significantly reduced at 1 h after lidocaine into any compartment, relative to bladder saline (*P<0.05; n = 9 saline, n = 10 bladder lidocaine, n = 11 colon lidocaine, n = 12 vaginal lidocaine). F) NU14-infected mice exhibited increased allodynia 24 hours after bladder instillation of saline, relative to PID1 (n = 10). Allodynia was significantly decreased at 24 hours following 83972 instillation into the bladder, colon or vaginal introitus (*P<0.05, n = 10 all groups). Data are reported as the mean ± SEM (A–C, E & F).</p

Rate of wound healing is significantly reduced in the presence of ERK 1/2 inhibition.

<p>Upper Panel: Rate of wounding recovery in immortalized urothelial cells from an IC/PBS patient bladder (trace 1) was decreased in the presence of SB203580 (1 µM, trace 2), PD95089 (2 µM, trace 3) and a combination of PD98059 and SB203580 (trace 4). Lower panel: Rate of wound healing is significantly decreased when immortalized urothelial cells are exposed to PD98059 (2 µM) with or without SB203580 (1 µM). Data shown are mean+SEM for results from 4 different cell cultures from 4 cell isolations from normal or IC/PBS patients. *p<0.05, **p<0.01 when compared to wound healing in the absence of inhibitor.</p

Inhibition of MAP kinases does not affect urothelial cell iPLA₂ activity.

<p>Pretreatment of immortalized urothelial cells from normal or IC/PBS bladders with PD98059 (5 µM, 10 min, open bars) or SB203580 (1 µM, 10 min, grey bars) had no effect on tryptase-stimulated (20 ng/ml, 5 mins) calcium-independent phospholipase A₂ (iPLA₂) activity (black bars). Data shown are mean±SEM for results from 3 different experiments using cell isolations from 4 separate patients or donors. **p<0.01 when compared to corresponding unstimulated values.</p

Phosphorylation of ERK1/2 in urothelial cells following tryptase stimulation.

<p>Upper panel: Representative immunoblots of phosphorylated and total ERK 1/2 in normal and IC/PBS urothelial cells stimulated with tryptase (20 ng/ml). ERK 1/2 activity was significantly increased in tryptase-stimulated immortalized cells from normal (filled circles) and IC/PBS bladders (filled squares). ERK 1/2 activity is expressed as a fold increase over unstimulated values. *p<0.05, **p<0.01 when compared to unstimulated activity. Data shown are mean±SEM for results from 4 different experiments using cell isolations from 4 separate patients or donors.</p

p38 MAP Kinase activation within immortalized urothelial cells following tryptase stimulation.

<p>Upper panel: Representative immunoblots of p38 MAP kinase measured as ATF-2 phosphorylation in tryptase-stimulated (20 ng/ml) human urothelial cells (HUC) and immortalized cells from normal and IC/PBS patients. Lower panel: p38 MAP kinase activity was significantly increased in tryptase-stimulated HUC (X) and immortalized cells from normal (filled circles) and IC/PBS bladders (filled squares). p38 MAP kinase activity is expressed as a fold increase over unstimulated values and was determined from 25 µg cytosolic protein. Data shown are mean±SEM for results from 4 different experiments using cell isolations from 3 (HUC) donors or 4 separate patients (immortalized cells from normal and IC/PBS bladder).</p

Incubation of urothelial cells with tryptase results in increased rate of urothelial cell wound healing.

<p>Upper Panel: Wound healing of immortalized urothelial cells from a normal bladder (8A, line 1) and from an IC/PBS bladder (8B, line 1) is accelerated in the presence of tryptase (20 ng/ml, Panels A and B, line 2). Lower Panel: Time to recover impedance to 80% of pre-wounding values is consistently reduced in the presence of tryptase (20 ng/ml) in normal and IC/PBS urothelial cells. Data shown are mean±SEM for results from 4 different cell cultures from 4 cell isolations from normal or IC/PBS patients.</p

Cytokeratin expression of immortalized urothelial cells.

<p><b>Row A:</b> A.1.HUC line. A.2–A.4. Representative immortalized cell lines from normal patients. <b>Row B:</b> B.1–B.3. Representative IC/PBS patient-derived immortalized cell lines. B.4. Representative background. Magnification 60×. Magnification Scale Bar = 20 µm.</p