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JOBNAME: AUTHOR QUERIES PAGE: 1 SESS: 3 OUTPUT: Mon Sep 27 13:24:26 2010
/tapraid3/urlurl/urlurl/url99908/url7340w08z
AQ1 Please give temperature meant for T in the phrase 10 seconds at TC.
AQ2 Graphic for figures 1-4 was supplied as poor quality. Okay as is or supply new figures.
AQ3 Author affiliations will appear differently in the print and online versions of your paper.
The PDF shows how the affiliations will present following journal style, whereas the
searchable online version will present as follows in order to provide complete unabridged
affiliations. Please check the accuracy of the affiliation(s) of each author and make changes as
appropriate.
a
Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne,
United Kingdom
b
Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
c
Department of Urology, Newcastle Upon Tyne Hospitals National Health Service Foundation
Trust, Newcastle Upon Tyne, United Kingdom
AUTHOR QUERIES
AUTHOR PLEASE ANSWER ALL QUERIES 1
Basic and Translational Science
Tolerance of Bacteruria
After Urinary Diversion Is Linked
to Antimicrobial Peptide Activity
Claire L. Townes, Ased Ali, Wendy Robson, Robert Pickard, and Judith Hall
OBJECTIVES To compare the cationic antimicrobial peptide gene expression proles and urinary cationic
antimicrobial activities of patients after urinary diversion according to the urinary tract infection
(UTI) status. Ileal conduit urinary diversion joins the bacterial-tolerant ileal epithelium and
intolerant urothelium. After this procedure, one quarter of patients develop repeated symptom-
atic UTIs. Such development might reect the altered innate immune mechanisms centered on
epithelial expression and urinary activity of cationic antimicrobial peptides, such as defensins.
METHODS Ileal and ureteral biopsy specimens from ileal conduit subjects with (n 18) and without (n
18) recurrent symptomatic UTIs were assessed for cationic antimicrobial peptide gene expression
using quantitative reverse transcriptase polymerase chain reaction. Overnight urine collections
were analyzed for antimicrobial activity against a laboratory Escherichia coli strain, and infecting
organisms were isolated from individual subjects.
RESULTS Overall, the ureteral epithelium showed increased expression of human -defensin 5 and
decreased expression of the human -defensin 1 after urinary diversion (P .05). No signicant
changes were seen for the ileal epithelium. The expression levels of both defensins also did not
differ signicantly according to UTI status. Urinary cationic activity against infecting bacterial
isolates from the individual subjects was signicantly greater in those with symptomatic UTI
(P .001), and the activities against the laboratory E. coli strain were similar.
CONCLUSIONS The changes in the human -defensin 1 and human -defensin 5 expression proles and the link
between symptomatic infection and high urinary antimicrobial activity suggest that innate
mechanisms play signicant roles in balancing bacterial tolerance and killing after ileal conduit
urinary diversion. Future work needs to determine whether these changes can be therapeutically
modulated to benet the patients. UROLOGY xx: xxx, xxxx. 2010 Published by Elsevier Inc.
I
leal urinary conduit formation creates a functional
paradox in which enteric epithelium tolerant to com-
mensal bacteria is joined to urothelium, which seeks
to maintain a sterile environment. One consequence of
this surgery is persistent bacteriuria, typically with Esch-
erichia coli.
1
For most patients, this infection is asymp-
tomatic; however, approximately one quarter develop
repeated symptomatic urinary tract infections (UTIs)
that affect their well-being and require antibiotic treat-
ment.
1
The reasons for the differing individual suscepti-
bility to UTIs are unknown, but they might include
bacterial virulence, immune effectiveness, and adapta-
tion of the conjoined epithelia.
2-4
Synthesis of cationic antimicrobial peptides (CAMPs)
is a key element of epithelial protection.
5
In humans,
these molecules function as a rst-line antimicrobial de-
fense causing bacterial cell death by membrane disrup-
tion and inactivation of intracellular targets.
6
CAMPs,
particularly defensins, also enhance cytokine production
and chemotaxis of immune-competent cells, linking in-
nate and adaptive responses and accelerating bacterial
clearance.
7,8
Human -defensins, such as human de-
fensin 1 (hBD-1) and -defensins, such as human -de-
fensin 5 (HD-5) are constitutively expressed by enteric
epithelium,
9
where they maintain the epithelial barrier
and regulate ileal microora.
10
In contrast, the urinary
tract is normally kept sterile, despite frequent bacterial
contamination by bowel and genital ora from the peri-
neum. Emerging evidence has suggested that CAMP ac-
tivity helps maintain this state.
11,12
hBD-1 is expressed
by renal tubular epithelium,
13
and HD-5 has been local-
ized to the female genital tract
14
and male urethral se-
This study was supported by funding from the Newcastle Hospitals Healthcare Charity
and the Urostomy Association, UK. A Ali was supported by a Wellcome Trust Clinical
Training Fellowship.
From the Institute for Cell and Molecular Biosciences and Institute of Cellular
Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom; and Depart-
ment of Urology, Newcastle Upon Tyne Hospitals National Health Service Foundation
Trust, Newcastle Upon Tyne, United Kingdom
Reprint requests: Judith Hall, B.Sc., Ph.D., Institute for Cell and Molecular
Biosciences, Newcastle University Faculty of Medical Sciences, Framlington Place,
Newcastle Upon Tyne NE2 4HH, UK. E-mail: Judith.Hall@ncl.ac.uk
Submitted: May 10, 2010, received (with revisions): August 14, 2010
2010 Published by Elsevier Inc. 0090-4295/xx/$34.00 1.e1
doi:10.1016/j.urology.2010.08.019
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cretions,15 with evidence suggesting that both peptides
help eradicate local infection. HD-5 has also previously
been detected in urine immediately after bladder recon-
struction with bowel segments.
16
We hypothesized that the epithelial adaption balanc-
ing innate CAMP activity might determine whether a
patient develops symptomatic recurrent UTIs after diver-
sion. To address this, we rst identied and measured the
expression proles of constitutive CAMPs in the ileal
and ureteral epithelium and, second, compared the uri-
nary cationic antimicrobial activity in patients with and
without recurrent UTIs.
MATERIAL AND METHODS
Subject Proles
After ethical committee and institutional approval, 37 subjects
(21 women and 16 men), who had undergone ileal conduit
urinary diversion surgery at a median of 11 years previously,
were recruited to the present study. Of the 37 patients, 3
subsequently withdrew. Of the 34 patients completing the
study, 17 (median age 68 years, range 36-78) had had persistent
asymptomatic UTIs and 17 (median age 63 years, range 36-79)
had had recurrent symptomatic UTIs. Asymptomatic UTI was
dened as a pure growth of 2 uropathogens on 2 previous
occasions not accompanied by fever and symptomatic UTI was
dened as 2 previous episodes annually of a pure growth of 2
uropathogens accompanied by fever 38C. None of the sub-
jects had had clinical signs of UTI and were not taking antibi-
otics at specimen collection. Microbiologic analysis of the urine
obtained by aseptic catheterization of the conduit at recruit-
ment showed that 5 of the asymptomatic subjects (29%) and 3
of the symptomatic subjects (18%) had a pure culture of 2
uropathogens (10
4
colony-forming units/mL), characterized
as either E. coli (n 6), non-E. coli coliforms (n 2), Proteus
sp. (n 2), or Enterococcus faecalis (n 4) by color differen-
tiation on chromogenic agar and stored. The remaining 26
subjects had a mixed bacterial population of 2 organisms
(10
3
to 10
4
colony-forming units/mL), which were not
considered to represent active infection according to the Cen-
ters for Disease Control and Prevention criteria.
Urine and Tissue Samples
Multiple supercial biopsy specimens of the ileal conduit and
distal ureter were obtained using exible endoscopy with the
patient under local anesthesia and stored in a preserving solu-
tion (RNAlater, Ambion, Austin, TX). Overnight urine col-
lections (1 L) from each patient were stored at 80C until
processing. Biopsy segments were also taken from the distal
ureter and ileal segment at surgery from a separate group of 10
subjects (women and 4 men) with median age of 70 years (range
54-80) who had undergone cystectomy. The samples were fro-
zen at 80C until analysis.
Reverse Transcriptase-Polymerase Chain Reaction
RNA was extracted from homogenized tissues using Trizol
reagent (Invitrogen, Paisley, UK). RNase inhibitor (Promega,
Southampton, UK) was added to each sample (1 U/g of RNA)
before storage at 80C. Reverse transcriptase-polymerase
chain reaction (PCR) of DNase-treated samples was performed
as previously described.
17
The products were resolved by elec-
trophoresis and veried by DNA sequencing. Controls without
reverse transcriptase excluded genomic DNA contamination.
Quantitative Real-Time PCR
For hBD-1 gene quantication, a PCR mix of 1-L primer-
probe mix at a nal concentration of 1 M, 5 L of Lightcycler
480 probes master mix (Roche, Welwyn Garden City, UK), and
2.5 L of reverse transcriptase was used. For HD-5, 5 L of Sybr
green master mix (Roche) was used. The primers and annealing
temperatures were as follows: HBD-1, F: GTCAGCTCAGC-
CTCCAAAGGA, and R: CGCCGGTAGGAAGTTCT-
CATGGCG at 60C; and HD-5, F: GCCATCCTTGCTGC-
CATTC, and R: GATTTCACACACCCCGGAGA at 55C.
The amplication conditions included a preincubation step of 2
minutes at 50C, 2 minutes at 95C, and 45 cycles of 5 seconds
at 94C, 10 seconds at TC, and 10 seconds at 72C. The
transcripts were quantied relative to a standard curve derived
from dilutions of a cloned gene copy. The reactions were
performed in 96-well plates using Lightcycler 480 (Roche).
Negative controls without reverse transcriptase excluded DNA
contamination. The data were normalized to 18S ribosomal
RNA internal control, and all analyses were performed without
knowledge of the patients identity.
Immunocytochemistry
The ureter and ileum specimens were stored embedded in
parafn. Cut sections (4 m) were mounted on SuperFrost Plus
microscope slides (VWR International, Lutterworth, UK) and
allowed to dry at 56C. The sections were dewaxed in xylene,
rehydrated, and incubated for 10 minutes in 0.5% hydrogen
peroxide. Blocking with goat serum diluted 1:5 with phosphate-
buffered saline (PBS) for 10 minutes preceded overnight incu-
bation with anti-HD-5 at a dilution of 1:1000 (ileum) or 1:500
(ureter) or anti-hBD-1 at a dilution of 1:500 at 4C (Abcam,
Cambridge, UK). Primary antibody binding was detected using
biotinylated goat anti-mouse antibody (Dako, Cambridge, UK)
and visualized using diaminobenzidine. The slides were washed
using Tris-buffered saline at pH 7.6 between each step.
Urine Antimicrobial Activity
Patient urine samples from the overnight collection were
thawed, centrifuged at 7500g for 10 minutes at 4C to remove
mucus and cellular debris, and ltered using a 0.2-m vacuum
ltration system (VWR International). CAMPs were extracted
from 1 L of the ltered urine sample using C18 chromatography
columns (Thames Restek, Saunderton, UK) and 10% acetonitrile
in 0.15% triuoracetic acid buffer. The elutants were lyophilized
under nitrogen at 55C and resuspended in 200 L of PBS.
Time-Kill Antimicrobial Assays
The time-kill assay method, using the laboratory uropathogenic
E. coli (UPEC) strain NCTC10418 and clinical isolates from
the urine specimens taken from individual subjects at recruit-
ment, was adapted from a previous study.
17
A 20-L aliquot of
each culture was diluted to 2 mL in PBS. Samples (10 L) of
subjects urine extract or PBS were added to 90 L of this
diluted culture, vortexed, and incubated for 3 hours at 37C.
The suspensions were sequentially diluted to 10
4
in PBS, and
each dilution was plated onto LB agar. All plates were incu-
bated overnight at 37C and the colonies counted. Control
experiments with PBS substituted for urine extract were used to
standardize the bacterial numbers between the assays. The results
1.e2 UROLOGY xx (x), xxxx
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were normalized to the respective subjects urinary creatinine con-
centration. All urinary antimicrobial activity determinations were
conducted without knowledge of the patients identity.
Bacterial Invasion Frequency
The bladder epithelial cell line RT4
18
was maintained in cell
culture medium comprising Roswell Park Memorial Institute
1640 medium (HEPES modication), 2 mM glutamine, and
10% fetal calf serum (Sigma, Poole, UK). For the bacterial
challenge experiments, RT4 cells were seeded onto 30-mm
diameter, 6-well plates at a density of 10
4
cells/well, and cul-
tured in 2 mL medium at 37C and 5% carbon dioxide until
conuent. Before the challenge, the cells were washed in PBS
and incubated in 1 mL of medium for 1 hour at 37C with 5%
carbon dioxide. Bacterial cells were harvested from an over-
night culture, washed, and resuspended in PBS to 10
4
colony-
forming units/mL. Each well of RT4 cells was inoculated with
100-L bacteria, the plates centrifuged for 2 minutes at 250g
and incubated at 37C and 5% carbon dioxide for 90 minutes.
After washing with PBS, the cells were incubated for another
90 minutes in 1 mL of medium containing 200 g/mL genta-
micin, washed, and lysed using 0.1% Triton. Recovered intra-
cellular bacteria were serially diluted onto LB agar plates and
incubated overnight at 37C, and the colonies were counted.
The invasion frequency was calculated by the total output from
the cells divided by the total input and multiplied by 100.
Hemolysis Assay
Bacterial strains were cultured from glycerol stocks, streaked
onto LB agar plates containing 5% debrinated horse blood
(Oxoid, Basingstoke, UK), and incubated overnight at 37C.
Hemolysis was dened by the presence of a clear halo surround-
ing individual colonies.
Statistical Analysis
The comparative data were not normally distributed, and the
statistical analyses were performed using the Kruskal-Wallis test
followed by Dunns comparison. A difference with a P value
of .05 was considered statistically signicant, and values are
expressed as the median and interquartile range (IQR).
RESULTS
Identication of Constitutive
CAMPs Expressed in Both Ureter and Ileum
The paired ureter and ileal samples obtained from 4
patients at urinary diversion were screened for CAMP
gene expression using reverse transcriptase PCR. The
candidate genes were human cathelicidin (hCAP-18/LL-
37), hepcidin (LEAP-1), liver expressed antimicrobial
peptide 2 (LEAP-2), hBD-1 and hBD-2, and HD-5.
hBD-1 and HD-5 were constitutively expressed in the
ileum and ureter from all 4 patients and selected for
Figure 1. Ureteral and ileal CAMP gene expression and immunohistochemical localization. Endpoint PCR gene expression
proles obtained in paired samples of ureter and ileum from 4 patients. Expression panel consisted of marker (M),
cathelicidin (LL-37; hCAP-18), hepcidin (Hep; LEAP-1), liver expressed antimicrobial peptide-2 (LP-2; LEAP-2), human
-defensin 1 (BD1), human -defensin 2 (BD2), human -defensin 5 (HD5), and 18S ribosomal RNA internal control (18S)
and presented in (A) ileum and (B) ureter. Primers and annealing temperatures used were LL37, F: 5=CATGAAGACCCAAAGG-
GATG3=, R: 5=CACACTAGGACTCTGTCCTG3= (55C); Hep, F: GTCACCAGTGGCTCTGTTTTC, R: GTCTTGCAGCACATCCCACAC
(58C); LP-2, F: CAAGATGTGTGGCACCTCAAAC, R: GCATTGTCGGAGGTGACTG (58C); BD1, F: CCATGAGAACTTCCTACCTTC, R:
GTCACTCCCAGCTCACTTG (58C); BD2, F: GTGAAGCTCCCAGCCATCAG, R: GATTGCGTATCTTTGGACACC (58C); HD5, F:
GCCATCCTTGCTGCCATTC, R: GATTTCACACACCCCGGAGA (58C). (C) Table showing number of ileal and ureteral samples in
which expression of each gene was detected. (D-F) Immunohistochemical image of ileal and ureter mucosa. (Di) Localiza-
tion of human -defensin 5 (HD-5; 1:1000) to ileal crypt Paneth cells; (Dii) no primary antibody control seen. (Ei) Localization
of HD-5 (1:500) to ureteral epithelial cells; (Eii) no primary antibody control seen. (Fi) Localization of hBD-1 (1:500) to
ureteral epithelium cells; (Fii) no primary antibody control seen. All images at 400 magnication.
UROLOGY xx (x), xxxx 1.e3
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additional study (Fig. 1A-C). The expected presence of
hBD-1 within the ureteral epithelium and HD-5 in the
intestinal crypts of Lieberkhn was conrmed using im-
munocytochemistry. Identication of HD-5 gene expres-
sion in the ureteral epithelium was novel, and immuno-
histochemistry showed that the peptide was localized in
the supercial layers of the urothelium (Fig. 1D-F).
Changes in HD-5 and hBD-1
Expression After Urinary Diversion
Expression of HD-5 and hBD-1 was measured by quan-
titative real-time PCR in the ileal conduit and ureteral
biopsy specimens from the 34 patients who had under-
gone urinary diversion and compared with control tissue
obtained from the 10 patients just before diversion. The
median HD-5 gene expression in the ileal epithelium did
not change signicantly after diversion (median 4.2 AU,
IQR 0.9-11.8, n 9; vs median 6.9 AU, IQR 0.1-33.7,
n 30; P NS). In contrast, ureteral HD-5 gene expres-
sion was signicantly increased after urinary diversion (me-
dian 0 AU, IQR 0-0.4, n 9; vs median 5.7 AU, IQR
1.1-65.9, n 29; P .05; Fig. 2A). For hBD-1, again, no
signicant change was found in ileal expression after
urinary diversion (median 0.9, IQR 0.1-2.0, n 10; vs
median 2.7 AU, IQR 0.6-26.5, n 27; P NS); how-
ever, the expression had decreased signicantly in the ure-
teral epithelium (median 4.4 AU, IQR 1.0-48.1, n 9; vs
median 0.1 AU, IQR 0.03-1.8, n 27; P .05; Fig. 2B).
The individual gene expression data showed marked vari-
ability, and we excluded the outlying data values (n 0-6)
from the statistical analyses.
Differences in Expression of HD-5 and
hBD-1 According to UTI Status and Gender
We next compared the HD-5 and hBD-1 expression in
the ileal and ureteral tissue from the 34 subjects with an
existing urinary diversion according to whether they had
had recurrent symptomatic UTIs. The expression of both
genes showed high interindividual variability, and, although
trends toward greater expression of HD-5 were seen in both
tissues for those subjects with UTIs, the changes did not
reach statistical signicance (Fig. 3A,B). Regarding the gen-
der differences, a trend toward greater ileal and ureteral
HD-5 expression in the women was observed but, again, did
not reach statistical signicance (Fig. 3C,D).
Differences in Urinary
Antimicrobial Activity With UTI Status
The urinary cationic antimicrobial activity of the pa-
tients with and without a history of recurrent symptom-
atic UTIs was initially investigated using a time-kill
assay and the laboratory uropathogenic E. coli strain
NCTC10418, but no clear difference was found (Fig.
4A). We next measured the urinary antimicrobial activ-
ity in those subjects with pure growth isolates at urine
sampling against their respective infecting organisms.
Virulence testing of the 6 clinical E. coli isolates showed
that those isolated from the subjects with symptomatic
Figure 2. Differences in expression of HD-5 and hBD-1 before and after urinary diversion. Quantication of HD-5 and hBD-1
gene expression in samples of ileum and ureteral tissue obtained from separate groups of subjects recruited before and
after ileal conduit urinary diversion. (A) Expression of HD-5. (B) Expression of hBD-1. Vertical axes broken to accommodate
outlying data points. Respective summary statistics used for nonparametric testing tabulated below each graph, *Statis-
tically signicant (P .05).
1.e4 UROLOGY xx (x), xxxx
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UTIs (n 2) had greater invasion frequency and hemo-
lysis activity than those isolated from asymptomatic pa-
tients (n 4; Fig. 4B). The individual urinary CAMP
activity against the paired bacterial isolate (Fig. 4C)
was signicantly greater in the samples from the symp-
tomatic group than in to the urine samples from
asymptomatic patients (15.0% 5.3% vs 81.4%
4.0% bacterial survival; P .001). The urine samples
from the subgroup of patients with a history of symp-
tomatic UTIs and a current pure isolate also showed
greater activity against the UPEC NCTC10418 strain
than the urine samples from those with a history of
asymptomatic UTIs (12.8% 6.5% vs 70.2% 8.1%
bacterial survival; P .05; Fig. 4D).
Figure 3. Differences in expression of HD-5 and hBD-1 according to UTI status and gender in subjects with existing urinary
diversion. (A,B) HD-5 and hBD-1 gene expression according to UTI status (recurrent symptomatic UTI vs asymptomatic
bacteriuria). (C,D) HD-5 and hBD-1 gene expression according to gender. Respective summary statistics used for nonpara-
metric testing tabulated below each graph. Vertical axes broken to accommodate outlying data points.
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COMMENT
Increasing evidence has indicated that innate immunity
centered on cationic peptides is important as the initial
defense against infection in humans; however, the clin-
ical correlates concerning UTI are lacking.
12
The aim of
the present study was to investigate whether differences
in CAMP gene expression and urine cationic antimicro-
bial activity are associated with susceptibility to symp-
tomatic UTI after ileal conduit urinary diversion. We
found signicant upregulation of HD-5 and downregula-
tion of hBD-1 in ureteral epithelium after anastomosis to
the ileal segment. Markedly increased antimicrobial activ-
ity was present against the infecting organisms in the urine
samples from those with a history of symptomatic UTIs
compared with those with asymptomatic bacteriuria. Taken
together, these ndings suggest a more aggressive innate
immune response is linked to symptomatic UTI.
Reverse transcriptase PCR expression screening using
healthy tissue samples obtained just before diversion
showed hBD-1 was constitutively expressed by both ure-
teral and ileal epithelium, in line with previous re-
ports.
13,19,20
Localization of HD-5 to the ileal crypts of
Lieberkhn has also been previously well established;
however, our nding of consistent gene expression in
ureteral tissue and localization to the supercial urothe-
lium is novel and suggests a possible antimicrobial role for
this peptide in the urinary tract to add to those proposed
in the vagina and male urethra.
14,15
We did not explore
further other candidate CAMPs, given their inconsistent
expression; however, it is possible that some, particularly
hBD-2, might be induced by active infection and con-
tribute to defense.
21
The increased ureteral HD-5 expression after rerouting
into an ileal conduit is intriguing, and additional study is
required to identify the triggering factors. Previous his-
tologic studies have not suggested any structural changes
to the urothelium,
4
and although changes to urinary
solutes have been shown to alter CAMP activity,
22
no
evidence of any effect on gene expression is available.
Moreover, our clinical data showed little variation in the
electrolyte concentration or pH and no correlation of
these physical characteristics with HD-5 expression (data
not shown). Concerning the hormonal inuences, an
analysis of our quantitative expression data suggested a
trend toward greater HD-5 expression in women with uri-
nary diversion compared with men. Hormonal changes
have previously been implicated in the variation of HD-5
expression in the female genital tract
14
; however, the
precise mechanism is unknown and perhaps had a lower
inuence in our predominantly postmenopausal patient
group. The signicance of bacterial colonization and
HD-5 gene expression remains unclear. Although not
observed in the gut, HD-5 upregulation has been docu-
mented in women presenting with genital tract inam-
mation,
14
and HD-5 peptide concentrations have been
elevated in the vaginal secretions from women with
bacterial vaginosis,
23
supporting a role for bacteria in
inuencing HD-5 gene expression. Recent evidence has
implicated the Wnt signaling pathway in the control of
ileal HD-5 expression,
24
and it is possible that increased
ureteral Wnt activity related to greater epithelial turn-
Figure 4. Cationic antimicrobial activity of urine extracts. (A) UPEC survival according to patient infection history. (B)
Invasion and hemolytic properties of E. coli isolates puried from asymptomatic (patients 1 and 4-6) and symptomatic
(patients 2 and 3) patients. (C) Clinical isolate and (D) NCTC10418 survival after incubation with urine cationic peptide
extract from respective subjects with existing urinary diversion.
1.e6 UROLOGY xx (x), xxxx
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over is involved in the changes to urinary tract HD-5
expression identied by the present study.
The changes in hBD-1 expression were more complex,
with a nonsignicant increase in the ileum and signi-
cant downregulation in the ureter. hBD-1 expression has
been thought to be exclusively constitutive in many
epithelia,
25,26
although modest induction was seen in
gastritis
27
and in colonic epithelial cells challenged with
live bacteria.
28
In the urinary tract, increased hBD-1
expression and secretion has been reported in response to
either infection
29
or cytokine challenge
30
; thus, the im-
munologic advantage of reduced hBD-1 levels requires
explanation. We can only speculate that in transposed
tissues, the modications in hBD-1 and HD-5 expression
function to encourage bacterial tolerance in the sterile
ureteral epithelium and, in contrast, provide increased
surveillance in the more distal ileal mucosa, discouraging
bacterial overgrowth.
A comparison of urinary cationic antimicrobial activ-
ity between the subjects with a history of symptomatic
UTI and those with asymptomatic bacteriuria against a
standard laboratory UPEC strain showed no clear differ-
ence, probably reecting our sampling protocol, with
urine collected from subjects in different phases of the
infection cycle. This was supported by the results from
patients with clearly identiable infecting organisms. In
the subjects whose epithelia were exposed to a specic
bacterial challenge at urine sampling, a strong link was
found among a history of symptomatic infection, high
urinary antimicrobial activity, and bacterial virulence.
The urine samples from symptomatic subjects showed
efcient killing of both the specic infecting organisms
and the laboratory UPEC strain, and the urinary cationic
antimicrobial activity was limited in subjects with asymp-
tomatic bacteriuria. For this initial study, we chose not to
further characterize the urine that showed a mixed bac-
terial population. It would be useful in the future to
perform a more in-depth microbiologic study to separate
true colonizers from transient contaminants in such cul-
tures and then to test their individual sensitivity to
endogenous CAMP activity.
CONCLUSIONS
To explain our ndings, we propose that in the asymp-
tomatic group, the ureteral urothelium adapted to a bac-
teria-tolerant phenotype, typical of the ileum, after urinary
diversion, and thereby allowing bacterial colonization.
Those with recurrent symptomatic UTIs retained an ag-
gressive antimicrobial response typical of the normal
urinary tract and associated with the development of
virulence factors by the invading bacteria. This response
is highly efcient in terms of clearing the offending
organisms but leads to typical systemic infective symp-
toms. The trend toward greater ileal and ureteral HD-5
expression in subjects with symptomatic UTI is support-
ive of a novel role of this peptide in modulating this
response. Additional work will require comparative anal-
ysis of the HD-5 urinary content on a longitudinal basis
to relate the levels to the phase of infection and to dene
the potential therapeutic use in controlling such infec-
tions.
Acknowledgment. To John Perry, Freeman Hospital, New-
castle Upon Tyne, UK, who provided the clinical isolates;
Natasha Rigas, who provided the RT-4 cells; and Kieran
OToole, Northern Institute Cancer Research, who performed
the immunocytochemistry.
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