Bladder inflammation (cystitis) underlies numerous bladder pathologies and is elicited by a plethora of agents such as urinary tract infections, bladder outlet obstruction, chemotherapies, and catheters. Pattern recognition receptors [Toll-like receptors (TLRs) and Nod-like receptors (NLRs)] that recognize pathogen- and/or damage-associated molecular patterns (PAMPs and/or DAMPs, respectively) are key components of the innate immune system that coordinates the production (TLRs) and maturation (NLRs) of proinflammatory IL-1β. Despite multiple studies of TLRs in the bladder, none have investigated NLRs beyond one small survey. We now demonstrate that NLRP3 and NLRC4, and their binding partners apoptosis-associated speck-like protein containing a COOH-terminal caspase recruitment domain (ASC) and NLR family apoptosis inhibitory protein (NAIP), are expressed in the bladder and localized predominantly to the urothelia. Activated NLRs form inflammasomes that activate caspase-1. Placement of a NLRP3- or NLRC4-activating PAMP or NLRP3-activating DAMPs into the lumen of the bladder stimulated caspase-1 activity. To investigate inflammasomes in vivo, we induced cystitis with cyclophosphamide (CP, 150 mg/kg ip) in the presence or absence of the inflammasome inhibitor glyburide. Glyburide completely blocked CP-induced activation of caspase-1 and the production of IL-1β at 4 h. At 24 h, glyburide reduced two markers of inflammation by 30-50% and reversed much of the inflammatory morphology. Furthermore, glyburide reversed changes in bladder physiology (cystometry) induced by CP. In conclusion, NLRs/inflammasomes are present in the bladder urothelia and respond to DAMPs and PAMPs, whereas NLRP3 inhibition blocks bladder dysfunction in the CP model. The coordinated response of NLRs and TLRs in the urothelia represents a first-line innate defense that may provide an important target for pharmacological intervention.

Purpose While bladder outlet obstruction (BOO) is well-established to elicit an inflammatory reaction in the bladder that leads to overactive bladder and fibrosis, little is known about the mechanism by which this is initiated. Nod-Like Receptors (NLRs) and the structures they form (inflammasomes) have been identified as sensors of cellular damage (including pressure-induced damage) and triggers of inflammation. Recently, we identified these structures in the urothelium. In this study we assess the role of the NLRP3 inflammasome in the bladder dysfunction resulting from BOO. Materials and Methods BOO was created in female rats by insertion of a 1 mm (o.d.) transurethral catheter, tying a silk ligature around the urethra and removing the catheter. Untreated and sham-operated rats served as controls. BOO rats were given vehicle (10% ethanol) or 10 mg/kg of glyburide (an NLRP3 inhibitor; daily for 12 days, p.o.). Inflammasome activity, bladder hypertrophy, inflammation and bladder function (urodynamics) were assessed. Results: BOO increased urothelial inflammasome activity, bladder hypertrophy, and inflammation and reduced voiding volume. Glyburide blocked inflammasome activation, reduced hypertrophy and prevented inflammation. The reduction in void volume was also attenuated by glyburide, mechanistically by an increase in the duration of detrusor contraction and voiding period. Conclusion The results suggest the importance of the NLRP3 inflammasome in the induction of inflammation and bladder dysfunction secondary to BOO. Arresting these processes with NLRP3 inhibitors may prove useful in treating the symptoms they produce.

Urinary tract infections (UTIs) typically evoke prompt and vigorous innate bladder immune responses including extensive exfoliation of the epithelium. To explain the basis for the extraordinarily high recurrence rates of UTIs, we examined adaptive immune responses in mouse bladders. We found that following each bladder infection, a highly T H 2 skewed immune response directed at bladder re-epithelization is observed with limited capacity to clear infection. Initiating this response is a distinct subset of CD301b + OX40L + dendritic cells, which migrate into the bladder epithelium after infection before trafficking to lymph nodes to preferentially activate T H 2 cells. The bladder epithelial repair response is cumulative and aberrant, as after multiple infections, the epithelium was markedly thickened and bladder capacity was reduced relative to controls. Thus, recurrence of UTIs and associated bladder dysfunction are the outcome of the preferential focus of the adaptive immune response on epithelial repair at the expense of bacterial clearance.

Direct force measurements, surface plasmon resonance spectroscopy, and genetic manipulations were used to investigate the impact of the orientation of immobilized cytochrome b5 (cyt b5) on its interactions with cytochrome c (cyt c). In this work, we used two cyt b5 orientational variants that present different regions of the protein surface when immobilized. Direct force measurements demonstrated that the two orientations generate a small difference in the electrostatic surface potential of the protein monolayers, in agreement with the calculated electrostatic potential distribution across the protein surface. This difference did not result in any differences in the electrostatic force between cyt c and the cyt b5 variants, however. The measured equilibrium binding constant for the cyt c interaction with cyt b5 also did not depend on the orientation of the latter. These results suggest that, at large separations, cyt c initially interacts relatively nonspecifically with a large patch of negative charge on the cyt b5. At short separations, it then adopts the optimum relative orientation for electron transfer. The force measurements not only elucidated the molecular basis of the equilibrium binding behavior, but also the possible molecular mechanisms that govern the interactions between these proteins in solution.

M.P.S and F.M.H contributed equally and share the first authorship. M.E.S and T.M.K contributed equally and share the senior authorship. ObjectivesTo develop a urodynamic model incorporating external urethral sphincter (EUS) electromyography (EMG) in awake rats. Materials and MethodsBladder catheters and EUS EMG electrodes were implanted in female Sprague Dawley rats. Assessments were performed in awake, lightly restrained rats on postoperative day 12-14. Measurements were repeated in the same rat on day 16 under urethane anaesthesia. Urodynamics and EUS EMG were performed simultaneously. In addition, serum creatinine and bladder histology was assessed. ResultsNo significant differences in urodynamic parameters were found between bladder catheter only vs bladder catheter and EUS EMG electrode groups. Urethane anaesthesia evoked prominent changes in both urodynamic parameters and EUS EMG. Serum creatinine was within the normal limits in all rats. Bladder weight and bladder wall thickness were significantly increased in both the bladder catheter only and the bladder catheter and EUS EMG group compared with controls. ConclusionsOur novel urodynamic model allows repetitive measurements of both bladder and EUS function at different time points in the same rat under fully awake conditions and opens promising avenues to investigate lower urinary tract dysfunction in a translational approach.

Utilizing a recently identified Sox10 distal enhancer directing Cre expression, we report S4F:Cre, a transgenic mouse line capable of inducing recombination in oligodendroglia and all examined neural crest derived tissues. Assayed using R26R:LacZ reporter mice expression was detected in neural crest derived tissues including the forming facial skeleton, dorsal root ganglia, sympathetic ganglia, enteric nervous system, aortae and melanoblasts, consistent with Sox10 expression. LacZ reporter expression was also detected in non-neural crest derived tissues including the oligodendrocytes and the ventral neural tube. This line provides appreciable differences in Cre expression pattern from other transgenic mouse lines that mark neural crest populations, including additional populations defined by the expression of other SoxE proteins. The S4F:Cre transgenic line will thus serve as a powerful tool for lineage tracing, gene function characterization and genome manipulation in these populations.The neural crest (NC) is a transient migratory collective of cell populations that arise at the dorsal aspect of the neural tube as it closes. This emigration occurs along the length of the anterior-posterior axis and gives rise to a myriad of structures including, but not limited to, melanocytes, the sympathetic nervous system, the enteric (parasympathetic) nervous system (ENS), connective tissue of the face and neck and peripheral myelinating glia (Schwann cells) (Douarin and Kalcheim, 1999). SOX10 (SRY-box containing gene 10) encodes a critical transcription factor in neural crest development (Britsch et al., 2001;Southard-Smith et al., 1998) and is expressed in most, if not all, NC-derived lineages. Loss-of-function SOX10 mutations result in Waardenburg-Shah Syndrome in humans (WS4; OMIM Accession No. 277580) (Mollaaghababa and Pavan, 2003;Pingault et al., 1998) which is

The advent of inexpensive, clinical exome sequencing (ES) has led to the accumulation of genetic data from thousands of samples from individuals affected with a wide range of diseases, but for whom the underlying genetic and molecular etiology of their clinical phenotype remains unknown. In many cases, detailed phenotypes are unavailable or poorly recorded and there is little family history to guide study. To accelerate discovery, we integrated ES data from 18,696 individuals referred for suspected Mendelian disease, together with relatives, in an Apache Hadoop data lake (Hadoop Architecture Lake of Exomes [HARLEE]) and implemented a genocentric analysis that rapidly identified 154 genes harboring variants suspected to cause Mendelian disorders. The approach did not rely on case-specific phenotypic classifications but was driven by optimization of gene-and variant-level filter parameters utilizing historical Mendelian disease-gene association discovery data. Variants in 19 of the 154 candidate genes were subsequently reported as causative of a Mendelian trait and additional data support the association of all other candidate genes with disease endpoints.

A delayed approach to gonadectomy and vaginal reconstruction respects patient autonomy and allows a more mature patient to handle the psychological and physical trauma of surgery and rehabilitation. Since we recognize the small number of patients in this series, strict guidelines cannot be proposed.