Stage- and subunit-specific functions of polycomb repressive complex 2 in bladder urothelial formation and regeneration.

Urothelium is the protective lining of the urinary tract. The mechanisms underlying urothelial formation and maintenance are largely unknown. Here, we report the stage-specific roles of PRC2 epigenetic regulators in embryonic and adult urothelial progenitors. Without Eed, the obligatory subunit of PRC2, embryonic urothelial progenitors demonstrate reduced proliferation with concomitant dysregulation of genes including Cdkn2a (p16), Cdkn2b (p15) and Shh. These mutants display premature differentiation of keratin 5-positive (Krt5+) basal cells and ectopic expression of squamous-like differentiation markers. Deletion of Ezh2, the major enzymatic component of PRC2, causes upregulation of Upk3a+ superficial cells. Unexpectedly, Eed and Eed/Ezh2 double mutants exhibit delayed superficial cell differentiation. Furthermore, Eed regulates the proliferative and regenerative capacity of adult urothelial progenitors and prevents precocious differentiation. Collectively, these findings uncover the epigenetic mechanism by which PRC2 controls urothelial progenitor cell fate and the timing of differentiation, and further suggest an epigenetic basis of urothelial maintenance and regeneration.

Sleeping beauty: awakening urothelium from its slumber.

The bladder urothelium is essentially quiescent but regenerates readily upon injury. The process of urothelial regeneration harkens back to the process of urothelial development whereby urothelial stem/progenitor cells must proliferate and terminally differentiate to establish all three urothelial layers. How the urothelium regulates the level of proliferation and the timing of differentiation to ensure the precise degree of regeneration is of significant interest in the field. Without a carefully-orchestrated process, urothelial regeneration may be inadequate, thereby exposing the host to toxins or pathogens. Alternatively, regeneration may be excessive, thereby setting the stage for tumor development. This review describes our current understanding of urothelial regeneration. The current controversies surrounding the identity and location of urothelial progenitor cells that mediate urothelial regeneration are discussed and evidence for each model is provided. We emphasize the factors that have been shown to be crucial for urothelial regeneration, including local growth factors that stimulate repair, and epithelial-mesenchymal cross talk, which ensures feedback regulation. Also highlighted is the emerging concept of epigenetic regulation of urothelial regeneration, which additionally fine tunes the process through transcriptional regulation of cell cycle genes and growth and differentiation factors. Finally, we emphasize how several of these pathways and/or programs are often dysregulated during malignant transformation, further corroborating their importance in directing normal urothelial regeneration. Together, evidence in the field suggests that any attempt to exploit regenerative programs for the purposes of enhanced urothelial repair or replacement must take into account this delicate balance.

Inflammatory response to Escherichia coli urinary tract infection in the neurogenic bladder of the spinal cord injured host.

PURPOSE: Urinary tract infections cause significant morbidity in patients with spinal cord injury. An in vivo spinal cord injured rat model of experimental Escherichia coli urinary tract infection mimics human disease with enhanced susceptibility to urinary tract infection compared to controls. We hypothesized that a dysregulated inflammatory response contributes to enhanced susceptibility to urinary tract infection. MATERIALS AND METHODS: Spinal cord injured and sham injured rats were inoculated transurethrally with E. coli. Transcript levels of 84 inflammatory pathway genes were measured in bladder tissue of each group before infection, 24 hours after infection and after 5 days of antibiotic therapy. RESULTS: Before infection quantitative polymerase chain reaction array revealed greater than twofold up-regulation in the proinflammatory factor transcripts slc11a1, ccl4 and il1ß, and down-regulation of the antimicrobial peptides lcn2 and mpo in spinal cord injured vs control bladders. At 24 hours after infection spinal cord injured bladders showed an attenuated innate immune response with decreased expression of il6, slc11a1, il1ß and lcn2, and decreased il10 and slpi expression compared to controls. Despite clearance of bacteriuria with antibiotics spinal cord injured rats had delayed induction of il6 transcription and a delayed anti-inflammatory response with decreased il10 and slpi transcript levels relative to controls. CONCLUSIONS: Spinal cord injured bladders fail to mount a characteristic inflammatory response to E. coli infection and cannot suppress inflammation after infection is eliminated. This may lead to increased susceptibility to urinary tract infection and persistent chronic inflammation through neural mediated pathways, which to our knowledge remain to be defined.

Enhanced susceptibility to urinary tract infection in the spinal cord-injured host with neurogenic bladder.

Neurogenic bladder predisposes to recurrent urinary tract infections (UTI) and renal failure, and susceptibility is commonly ascribed to urinary stasis from elevated residual urine volumes. Escherichia coli UTI was modeled in the spinal cord-injured (SCI) rat with the hypothesis that SCI animals would require fewer bacteria to establish infection, have an exaggerated inflammatory response, and have delayed clearance of infection compared to normal-voiding controls. T10 SCI rats and controls had median infectious doses (ID50) of 10(2) and 10(5) CFU, respectively. Mean residual volumes in the SCI animals did not correlate with susceptibility to initiation of UTI or outcome. In the acute infection, control and SCI rats developed acute cystitis and pyelitis without acute differences in histopathological scores of inflammation. However, in vivo imaging of infected animals revealed persistently higher levels of bacteria in the SCI urine and bladders than were seen for controls over 2 weeks. Likewise, at 2 weeks, acute and chronic inflammatory infiltrates persisted in the bladders and kidneys of SCI rats, whereas inflammation largely resolved within the controls. Together these data demonstrate that SCI rats exhibit delayed clearance of infection and exaggerated inflammatory responses in bladders and kidneys; however, the severity of residual volumes does not predict increased susceptibility to UTI. These studies suggest that host-dependent mechanisms that are discrete from alterations in bladder physiology influence UTI susceptibility with the SCI-neurogenic bladder. This model will allow elucidation of SCI-neurogenic bladder-mediated changes in host response that yield UTI susceptibility and may lead to new preventative and therapeutic options.

Experience with glycerin for antegrade continence enema in patients with neurogenic bowel.

PURPOSE: Malone antegrade continence enemas are used in the management of neurogenic bowel to attain fecal continence. Several different irrigation solutions have been described but glycerin, an osmotic laxative that promotes peristalsis, has rarely been mentioned or studied. We assessed clinical outcomes in our patients with a Malone antegrade continence enema using glycerin based irrigation. MATERIALS AND METHODS: We retrospectively reviewed patients with neurogenic bowel who underwent a Malone antegrade continence enema procedure between 1997 and 2011. Glycerin diluted with tap water followed by a tap water flush is our preferred irrigation protocol. Bowel regimen outcomes examined included fecal continence, emptying time, leakage from stoma, enema volume, frequency and independence. RESULTS: Of the 23 patients with followup greater than 6 months 19 used glycerin based irrigation. Average age at surgery was 8.8 years. Patients using glycerin instilled a median of 30 ml (mean 29) glycerin and 50 ml (131) tap water. Fecal continence rate was 95% and stoma leakage rate was 16%, and only 16% of patients required daily irrigation. CONCLUSIONS: Glycerin is a viable and effective alternative irrigant for antegrade enemas of neurogenic bowel, with an excellent fecal continence rate. The volume of irrigant needed is typically less than 90 ml, which is much less than in published reports using tap water alone.

Targeting the PRC2-dependent epigenetic program alleviates urinary tract infections.

Urinary tract infection (UTI) is a pervasive health problem worldwide. Patients with a history of UTIs suffer increased risk of recurrent infections, a major risk of antibiotic resistance. Here, we show that bladder infections induce expression of Ezh2 in bladder urothelial cells. Ezh2 is the methyltransferase of polycomb repressor complex 2 (PRC2)-a potent epigenetic regulator. Urothelium-specific inactivation of PRC2 results in reduced urine bacterial burden, muted inflammatory response, and decreased activity of the NF-κB signaling pathway. PRC2 inactivation also facilitates proper regeneration after urothelial damage from UTIs, by attenuating basal cell hyperplasia and increasing urothelial differentiation. In addition, treatment with Ezh2-specific small-molecule inhibitors improves outcomes of the chronic and severe bladder infections in mice. These findings collectively suggest that the PRC2-dependent epigenetic reprograming controls the amplitude of inflammation and severity of UTIs and that Ezh2 inhibitors may be a viable non-antibiotic strategy to manage chronic and severe UTIs.

An Enlarged and Infected Prostatic Utricle as a Rare Cause of Lower Urinary Tract Symptoms in Adolescent Males.

Dysuria with lower abdominal pain is a common presentation for a urinary tract infection (UTI), and diagnosis is based on symptoms together with a urinalysis and urine culture suggestive of infection. UTI is uncommon in circumcised males who are not sexually active. When urine culture is negative, alternate diagnoses including, but not limited to, gastroenteritis, severe constipation, appendicitis, or epididymitis need to be considered. In patients with a known urologic history of proximal hypospadias and/or disorders of sexual development, rarer diagnoses also need to be considered. This paper reports the case of a 13-year-old male with a remote history of proximal hypospadias repair, who presented with nonspecific lower urinary tract symptoms. Initially he was treated for UTI. However, urine cultures remained negative despite persistent urinary tract symptoms. On further workup, he was found to have an enlarged and infected prostatic utricle. This report illustrates the importance of considering an enlarged prostatic utricle in the differential diagnoses of patients with chronic lower urinary tract symptoms and a history of hypospadias. Additionally, this case highlights the utility of magnetic resonance imaging (MRI) in clarifying lower urinary tract anatomy in cases where ultrasound is inconclusive.

Risk Factors Associated With Recurrent Urinary Tract Infection in Neurogenic Bladders Managed by Clean Intermittent Catheterization.

OBJECTIVE: To identify risk factors for recurrent urinary tract infection (UTI) in patients who perform clean intermittent catheterization (CIC). METHODS: A 6-year retrospective chart review of patients with spina bifida or tethered cord who perform clean intermittent catheterization (8 months to 58 years) was conducted. A strict case definition for UTI was applied, and per-subject UTI events, demographic, and clinical data were abstracted. Data were compared between groups defined by no or infrequent UTI (≤1.0 UTI/study year) and frequent UTI (>1.0 UTI/study year). RESULTS: Of 194 total patients, 146 (75%) had no UTIs or infrequent UTIs, and 48 (25%) patients had frequent UTIs. On univariate analysis, only younger age and suprasacral cord lesions were associated with frequent UTIs (P = .002 and P = .007, respectively). Among the 128 patients with urodynamic studies, bladder capacity, compliance, detrusor overactivity, and detrusor leak point pressure were not associated with frequent UTI on univariate analysis. On multivariate analysis, increasing age was found to be associated with decreased odds of UTI by 7% per year (odds ratio 0.93, P = .016). CONCLUSION: The risk of UTI among individuals with spina bifida or tethered cord declines with increasing age. Bladder function based on urodynamic parameters did not correlate with frequent UTIs.

Congenital spigelian hernia and ipsilateral cryptorchidism: raising awareness among urologists.

Spigelian hernias (SHs) are rare in the pediatric population. Although pediatric general surgeons often treat this defect, the increased association between a congenital SH and an ipsilateral undescended testis suggests that urologists may be the first provider encountering this entity. Knowledge of this condition is therefore important. We report one such case of a male infant referred to urology for an undescended testicle. Further investigation revealed the testicle to be within a congenital SH sac. Herein, we additionally review the literature concerning SHs associated with ipsilateral undescended testicles.

Variation in use of nephron-sparing surgery among children with renal tumors.

OBJECTIVE: Given the negative long-term effects of renal insufficiency, nephron-sparing surgery (NSS) is increasingly discussed for the treatment of pediatric renal tumors. We sought to examine variation in practice patterns of NSS among children with renal tumors. MATERIALS AND METHODS: We performed a retrospective cohort analysis of claims data for pediatric inpatient admissions captured by the Kids Inpatient Database (1997-2009). We identified children with renal tumors who underwent surgery, including radical nephrectomy (RN) and NSS. We used multivariable logistic regression to assess the relationship between use of NSS and various clinical, demographic, and geographic predictors of interest. RESULTS: We identified 10,108 pediatric inpatient admissions for renal tumors. Of these, 1657 were surgical admissions, with 1501 patients (90.5%) undergoing RN and 156 (9.5%) undergoing NSS. On multivariable analysis, NSS was associated only with a concomitant diagnosis of renal insufficiency (relative ratio [RR] 3.37, p = 0.01) and surgery in the Northeastern USA (RR 3.07, p = 0.03). Race/ethnicity, age, payer type, procedure year, and other non-clinical factors were not significantly associated with NSS. CONCLUSION: In a large, nationwide pediatric cohort, RN remains the most common surgical intervention for renal tumors. NSS is significantly associated with a diagnosis of renal insufficiency, but not non-clinical factors such as patient gender or race. © 2014 Published by Elsevier Ltd on behalf of Journal of Pediatric Urology Company.

Chlamydia trachomatis-derived deubiquitinating enzymes in mammalian cells during infection.

Chlamydia trachomatis is an obligate intracellular bacterium that causes a variety of diseases in humans. C. trachomatis has a complex developmental cycle that depends on host cells for replication, during which gene expression is tightly regulated. Here we identify two C. trachomatis proteases that possess deubiquitinating and deneddylating activities. We have designated these proteins ChlaDub1 and ChlaDub2. The genes encoding ChlaDub1 and ChlaDub2 are present in all Chlamydia species except for Chlamydia pneumoniae, and their catalytic domains bear similarity to the catalytic domains of other eukaryotic ubiquitin-like proteases (Ulp). The C. trachomatis DUBs react with activity-based probes and hydrolyse ubiquitinated and neddylated substrates. ChlaDub1 and ChlaDub2 represent the first known bacterial DUBs that possess both deubiquitinating and deneddylating activities.

Developmental regulation of Chlamydia trachomatis class I accessible protein-1, a CD8+ T cell antigen.

In this report, we show that the Chlamydia trachomatis-specific CD8+ T cell antigen class I accessible protein-1 (Cap1) is expressed during the early stages of the C. trachomatis developmental cycle. We provide additional evidence suggesting that Cap1 may be important in early immune recognition of the organism. Understanding the temporal and spatial expression of CD8+ T cell antigens such as Cap1 may be beneficial in designing multisubunit vaccines to stimulate a vigorous immune response against C. trachomatis.

Chlamydia trachomatis infection induces cleavage of the mitotic cyclin B1.

The obligate intracellular pathogen Chlamydia trachomatis interferes with a number of host cell processes, including cytoskeletal organization, vesicular trafficking, and apoptosis. In this study we report that C. trachomatis-infected cells proliferate more slowly than uninfected cells, suggesting that C. trachomatis may also manipulate the eukaryotic cell cycle. We further demonstrate that C. trachomatis infection destabilizes specific cell cycle proteins involved in the G2/M transition. C. trachomatis-infected cells, compared to uninfected cells, have lower levels of cyclin-dependent kinase 1. Additionally, C. trachomatis infection induces an N-terminal truncation of the mitotic cyclin B1. Manipulation of the host cell cycle may represent a strategy used by C. trachomatis to ensure a stable environment conducive to bacterial growth and replication.

The p47 GTPases Igtp and Irgb10 map to the Chlamydia trachomatis susceptibility locus Ctrq-3 and mediate cellular resistance in mice.

Infections caused by the bacteria Chlamydia trachomatis contribute to diverse pathologies in a variety of human populations. We previously used a systemic model of C. trachomatis infection in mice to map three quantitative trait loci that influence in vivo susceptibility differences between the C57BL/6J and C3H/HeJ inbred strains of mouse. One of these quantitative trait loci, Ctrq-3, influences an IFN-gamma-dependent susceptibility difference in primary embryonic fibroblasts isolated from these strains. Here we use fine structure mapping in congenic fibroblasts carrying DNA from the susceptible parent to localize the effect of Ctrq-3 to a 1.2-megabase interval of genomic DNA that contains Irgb10 and Igtp, two members of the IFN-gamma-inducible p47 family of GTPases. This class of proteins has been widely implicated in resistance to intracellular pathogens in mice. We analyzed expression of Irgb10 and Igtp in parental and congenic embryonic fibroblasts treated with IFN-gamma and found that relatively resistant fibroblasts express more Irgb10 than relatively susceptible fibroblasts. However, we also found that abolishing the expression of either Irgb10 or Igtp increases susceptibility of embryonic fibroblasts to C. trachomatis. Thus, we conclude that, although a difference in Irgb10 expression is likely responsible for the effect of Ctrq-3 on susceptibility to C. trachomatis, both genes play a role in intracellular resistance to C. trachomatis.

Hematopoietic cells are required to initiate a Chlamydia trachomatis-specific CD8+ T cell response.

Chlamydia trachomatis is a global human pathogen causing diseases ranging from blinding trachoma to pelvic inflammatory disease. To explore how innate and adaptive immune responses cooperate to protect against systemic infection with C. trachomatis L2, we investigated the role of macrophages (Mphi) and dendritic cells (DCs) in the stimulation of C. trachomatis-specific CD8(+) T cells. We found that C. trachomatis infection of Mphi and DCs is far less productive than infection of nonprofessional APCs, the typical targets of infection. However, despite the limited replication of C. trachomatis within Mphi and DCs, infected Mphi and DCs process and present C. trachomatis CD8(+) T cell Ag in a proteasome-dependent manner. These findings suggest that although C. trachomatis is a vacuolar pathogen, some Ags expressed in infected Mphi and DCs are processed in the host cell cytosol for presentation to CD8(+) T cells. We also show that even though C. trachomatis replicates efficiently within nonprofessional APCs both in vitro and in vivo, Ag presentation by hematopoietic cells is essential for initial stimulation of C. trachomatis-specific CD8(+) T cells. However, when DCs infected with C. trachomatis ex vivo were adoptively transferred into naive mice, they failed to prime C. trachomatis-specific CD8(+) T cells. We propose a model for priming C. trachomatis-specific CD8(+) T cells whereby DCs acquire C. trachomatis Ag by engulfing productively infected nonprofessional APCs and then present the Ag to T cells via a mechanism of cross-presentation.