Björn Wullt

Toll-Like Receptor 4 Expression and Cytokine Responses in the Human Urinary Tract Mucosa

ABSTRACT Mucosal pathogens trigger a local innate host response by activating epithelial cells. Bacterial adherence and Toll-like receptor 4 (TLR4) signaling have been implicated as key events in this process. This study addressed the molecular basis of the epithelial response to gram-negative infection in the human urinary tract. Mucosal biopsies were obtained from kidneys, ureters, and bladders of patients undergoing urinary tract surgery, and epithelial TLR4 and CD14 expression was examined by immunohistochemistry. TLR4 was detected in epithelial cells lining the entire urinary tract and in the renal tubular epithelium. CD14, in contrast, was completely absent from the epithelial tissue. The response of the epithelial cells to infection was studied by in vitro challenge of the biopsies with uropathogenic Escherichia coli bacteria. A rapid cytokine response was observed, with production of interleukin-1β (IL-1β), IL-6, and IL-8 but not of IL-4 or gamma interferon. Adhering, P- or type 1-fimbriated E. coli activated IL-6 and IL-8 production more efficiently than the nonfimbriated control, as shown by cellular staining and analysis of secreted cytokines. The results demonstrate that human uroepithelial cells possess the molecular machinery needed to respond to uropathogenic E. coli. This includes recognition receptors for fimbriae and TLR4 for transmembrane signaling. We speculate that the lack of membrane-bound CD14 allows the epithelium to regulate its sensitivity to lipopolysaccharide and to discriminate between more-virulent and less-virulent strains.

Infective Complications After Prostate Biopsy: Outcome of the Global Prevalence Study of Infections in Urology (GPIU) 2010 and 2011, A Prospective Multinational Multicentre Prostate Biopsy Study

BACKGROUND Infection is a serious adverse effect of prostate biopsy (P-Bx), and recent reports suggest an increasing incidence. OBJECTIVE The aim of this multinational multicentre study was to evaluate prospectively the incidence of infective complications after P-Bx and identify risk factors. DESIGN, SETTING, AND PARTICIPANTS The study was performed as an adjunct to the Global Prevalence Study of Infections in Urology (GPIU) during 2010 and 2011. Men undergoing P-Bx in participating centres during the 2-wk period commencing on the GPIU study census day were eligible. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Baseline data were collected and men were questioned regarding infective complications at 2 wk following their biopsy. The Fisher exact test, Student t test, Mann-Whitney U test, and multivariate regression analysis were used for data analysis. RESULTS AND LIMITATIONS A total of 702 men from 84 GPIU participating centres worldwide were included. Antibiotic prophylaxis was administered prior to biopsy in 98.2% of men predominantly using a fluoroquinolone (92.5%). Outcome data were available for 521 men (74%). Symptomatic urinary tract infection (UTI) was seen in 27 men (5.2%), which was febrile in 18 (3.5%) and required hospitalisation in 16 (3.1%). Multivariate analysis did not identify any patient subgroups at a significantly higher risk of infection after P-Bx. Causative organisms were isolated in 10 cases (37%) with 6 resistant to fluoroquinolones. The small sample size per participating site and in compared with other studies may have limited the conclusions from our study. CONCLUSIONS Infective complications after transrectal P-Bx are important because of the associated patient morbidity. Despite antibiotic prophylaxis, 5% of men will experience an infective complication, but none of the possible factors we examined appeared to increase this risk. Our study confirms a high incidence of fluoroquinolone resistance in causative bacteria.

Host Imprints on Bacterial Genomes-Rapid, Divergent Evolution in Individual Patients

Bacteria lose or gain genetic material and through selection, new variants become fixed in the population. Here we provide the first, genome-wide example of a single bacterial strains evolution in different deliberately colonized patients and the surprising insight that hosts appear to personalize their microflora. By first obtaining the complete genome sequence of the prototype asymptomatic bacteriuria strain E. coli 83972 and then resequencing its descendants after therapeutic bladder colonization of different patients, we identified 34 mutations, which affected metabolic and virulence-related genes. Further transcriptome and proteome analysis proved that these genome changes altered bacterial gene expression resulting in unique adaptation patterns in each patient. Our results provide evidence that, in addition to stochastic events, adaptive bacterial evolution is driven by individual host environments. Ongoing loss of gene function supports the hypothesis that evolution towards commensalism rather than virulence is favored during asymptomatic bladder colonization.

Bladder cancers respond to intravesical instillation of HAMLET (human alpha-lactalbumin made lethal to tumor cells).

We studied if bladder cancers respond to HAMLET (human α‐lactalbumin made lethal to tumor cells) to establish if intravesical HAMLET application might be used to selectively remove cancer cells in vivo. Patients with nonmuscle invasive transitional cell carcinomas were included. Nine patients received 5 daily intravesical instillations of HAMLET (25 mg/ml) during the week before scheduled surgery. HAMLET stimulated a rapid increase in the shedding of tumor cells into the urine, daily, during the 5 days of instillation. The effect was specific for HAMLET, as intravesical instillation of NaCl, PBS or native α‐lactalbumin did not increase cell shedding. Most of the shed cells were dead and an apoptotic response was detected in 6 of 9 patients, using the TUNEL assay. At surgery, morphological changes in the exophytic tumors were documented by endoscopic photography and a reduction in tumor size or change in tumor character was detected in 8 of 9 patients. TUNEL staining was positive in biopsies from the remaining tumor in 4 patients but adjacent healthy tissue showed no evidence of apoptosis and no toxic response. The results suggest that HAMLET exerts a direct and selective effect on bladder cancer tissue in vivo and that local HAMLET administration might be of value in the future treatment of bladder cancers.

Molecular Basis of Commensalism in the Urinary Tract: Low Virulence or Virulence Attenuation?

ABSTRACT In some patients, Escherichia coli strains establish significant bacteriuria without causing symptoms of urinary tract infection (UTI). These asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence factors than the uropathogenic E. coli (UPEC) strains that cause severe, symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence genes, and the molecular basis of their low virulence therefore remains unclear. This study examined whether ABU strains might evolve from UPEC by genome loss and virulence gene attenuation. The presence of conserved E. coli K-12 genes was examined using an E. coli K-12 strain MG1655-specific DNA array and the distribution of UPEC virulence-related genes was examined with the E. coli pathoarray. Two groups of strains could be distinguished. Several ABU strains were shown by multilocus sequence typing and by comparative genomic analyses to be related to UPEC but to have smaller genome sizes. There were significant alterations in essential virulence genes, including reductive evolution by point mutations, DNA rearrangements, and deletions. Other strains were unrelated to UPEC and lacked most of the virulence-associated genes. The results suggest that some ABU strains arise from virulent strains by attenuation of virulence genes while others are nonvirulent and resemble commensal strains. We propose that virulence attenuation might constitute a general mechanism for mucosal pathogens to evolve toward commensalism.

P-fimbriae trigger mucosal responses to Escherichia coli in the human urinary tract.

Uropathogenic Escherichia coli elicit a host response that determines the severity of urinary tract infection (UTI). Specific adherence mechanisms allow the bacteria to initiate this process by targeting epithelial cells in the urinary tract mucosa. Epidemiological studies show a strong association of P‐fimbriae with disease severity, suggesting that adherence mediated by these organelles has a direct effect on mucosal inflammation in vivo. The present study examined the ability of P‐fimbriae to induce inflammation in the human urinary tract. Patients were subjected to intravesical inoculation with a non‐fimbriated E. coli strain or transformants of this strain expressing P‐fimbriae. The inflammatory response was analysed as a function of P‐fimbrial expression. The P‐fimbriated transformants invariably caused higher interleukin (IL)‐8, IL‐6 and neutrophil responses in the urinary tract than the ABU strain. Furthermore, loss of P‐fimbrial expression in vivo was accompanied by a return to background levels of neutrophils, IL‐6 and IL‐8 in individual patients. The results demonstrate that the pap sequences confer on a non‐fimbriated, avirulent strain the ability to induce a host response in the human urinary tract. P‐fimbriae thus fulfil the ‘molecular Koch–Henle postulates’ linking a single virulence factor to host response induction.

Escherichia coli 83972 bacteriuria protects against recurrent lower urinary tract infections in patients with incomplete bladder emptying.

PURPOSE We determined if the deliberate establishment of asymptomatic bacteriuria with Escherichia coli 83972 in patients with incomplete bladder emptying and recurrent urinary tract infection protects against recurrence. MATERIALS AND METHODS In phase 1 of the study the patients were randomized to blinded inoculations with E. coli 83972 or saline. Crossover occurred after monitoring for 12 months or after a urinary tract infection. The outcome was the time to the first urinary tract infection in patients with and without E. coli 83972 bacteriuria. In phase 2 patients were subjected to additional blinded inoculations to extend periods with and without E. coli 83972 bacteriuria. The outcome was the number of urinary tract infections during 12 months with and 12 months without E. coli 83972 bacteriuria. RESULTS A total of 20 patients completed the study. In phase 1 the time to the first urinary tract infection was longer with than without E. coli 83972 bacteriuria (median 11.3 vs 5.7 months, sign test p = 0.0129). Phase 2 was analyzed after patients had spent a total of 202 months with and 168 months without E. coli 83972 bacteriuria. There were fewer reported urinary tract infection episodes with vs without E. coli 83972 bacteriuria (13 vs 35 episodes, paired t test p = 0.009, CI 0.31-1.89). There was no febrile urinary tract infection episode in either of the study arms and no significant side effects of intravesical bacterial inoculation were reported. CONCLUSIONS Deliberately induced E. coli 83972 bacteriuria protected patients with incomplete bladder emptying who are prone to urinary tract infection from recurrent urinary tract infection as demonstrated by the delay in time to urinary tract infection and the decrease in number of urinary tract infection episodes.

PapG-Dependent Adherence Breaks Mucosal Inertia and Triggers the Innate Host Response

Mucosal pathogens differ from normal flora constituents in that they provoke a host response that upsets mucosal integrity. We investigated whether the elaboration of discrete adherence factors is sufficient to break the inertia of the mucosal barrier. PapG-mediated adherence was selected as an example, because P fimbrial expression characterizes uropathogenic Escherichia coli and because adherence starts the attack on the mucosal barrier. Patients were inoculated intravesically with transformed nonvirulent E. coli strains expressing functional P fimbriae (E. coli pap(+)) or mutant fimbriae lacking the adhesin (E. coli Delta papG). E. coli pap(+) was shown to activate the innate host response, and adherent gfp(+) bacteria were observed on excreted uroepithelial cells. E. coli Delta papG failed to trigger a response and was nonadhesive. We conclude that PapG-mediated adherence breaks mucosal inertia in the human urinary tract by triggering innate immunity and propose that this activation step differentiates asymptomatic carriage from infection.

Toll-like receptor 4 promoter polymorphisms: common TLR4 variants may protect against severe urinary tract infection.

Background Polymorphisms affecting Toll-like receptor (TLR) structure appear to be rare, as would be expected due to their essential coordinator role in innate immunity. Here, we assess variation in TLR4 expression, rather than structure, as a mechanism to diversify innate immune responses. Methodology/Principal Findings We sequenced the TLR4 promoter (4,3 kb) in Swedish blood donors. Since TLR4 plays a vital role in susceptibility to urinary tract infection (UTI), promoter sequences were obtained from children with mild or severe disease. We performed a case-control study of pediatric patients with asymptomatic bacteriuria (ABU) or those prone to recurrent acute pyelonephritis (APN). Promoter activity of the single SNPs or multiple allelic changes corresponding to the genotype patterns (GPs) was tested. We then conducted a replication study in an independent cohort of adult patients with a history of childhood APN. Last, in vivo effects of the different GPs were examined after therapeutic intravesical inoculation of 19 patients with Escherichia coli 83972. We identified in total eight TLR4 promoter sequence variants in the Swedish control population, forming 19 haplotypes and 29 genotype patterns, some with effects on promoter activity. Compared to symptomatic patients and healthy controls, ABU patients had fewer genotype patterns, and their promoter sequence variants reduced TLR4 expression in response to infection. The ABU associated GPs also reduced innate immune responses in patients who were subjected to therapeutic urinary E. coli tract inoculation. Conclusions The results suggest that genetic variation in the TLR4 promoter may be an essential, largely overlooked mechanism to influence TLR4 expression and UTI susceptibility.

The role of P fimbriae for Escherichia coli establishment and mucosal inflammation in the human urinary tract.

Bacterial adhesion to the bladder mucosa is a critical step for the establishment of Escherichia coli bacteriuria. The P-fimbriae, encoded by the pap gene cluster, are considered as virulence factors but the mechanisms have been debated. This study defined the roles for P fimbriation during the early colonization of the human urinary tract. Patients with recurrent UTI were first subjected to deliberate colonization with the non-fimbriated ABU strain E. coli 83972. Bacteriuria was established long term (1-4 years) in patients with dysfunctional bladders, but not in the patients with normal bladder function. Super-infections were transient and asymptomatic. P fimbriated transformants of the ABU strain (E. coli 83972pap+/prs+) reached 105 CFU/ml more rapidly than E. coli 83972 and the vector control. This was demonstrated by group wise and intra-individual analysis in patients colonized on different occasions with E. coli 83972 or the P fimbriated transformants. Higher neutrophil numbers and IL-8 and IL-6 concentrations in urine were obtained after colonization with the P fimbriated transformants. These results demonstrated that transformation of E. coli 83972 with the pap sequences is sufficient to convert it to a more potent host response inducer. The P fimbriae were shown to lower the significant bacteriuria threshold. The P fimbriated transformants needed lower bacterial numbers (103-4 CFU/ml) to predict a positive second urine culture with a >80% accuracy and to trigger a significant host response. These studies show that P fimbriae fulfil the Koch Henles molecular postulates for bacterial establishment and host response induction in the human urinary tract.