Loss of Janus Associated Kinase 1 Alters Urothelial Cell Function and Facilitates the Development of Bladder Cancer.

Inherited Primary Immunodeficiency (PID) disorders are associated with increased risk of malignancy that may relate to impaired antitumor immune responses or a direct role for PID germline mutations in tumorigenesis. We recently identified germline loss of function mutations in Janus Associated Kinase 1 ( causing primary immunodeficiency characterized by infections and associated with early onset, fatal high-grade bladder carcinoma. Somatic mutations in , required for immune cell signaling in response to interferon gamma (IFNγ), have been associated with several non-hematopoietic and hematopoietic cancer cell types but pathogenic mechanisms remain largely unexplored.

Hypoxic changes to the urothelium as a bystander of end-stage bladder disease.

Introduction: Urothelial cells harvested from benign diseased bladders have a compromised capacity to propagate or differentiate in vitro, potentially limiting their application in autologous tissue engineering approaches. The causative pathways behind this altered phenotype are unknown. The hypothesis is that hypoxic damage to the urothelium occurs as a bystander to chronic or recurrent episodes of infection and inflammation.

Differential transcription factor expression by human epithelial cells of buccal and urothelial derivation.

Identification of transcription factors expressed by differentiated cells is informative not only of tissue-specific pathways, but to help identify master regulators for cellular reprogramming. If applied, such an approach could generate healthy autologous tissue-specific cells for clinical use where cells from the homologous tissue are unavailable due to disease. Normal human epithelial cells of buccal and urothelial derivation maintained in identical culture conditions that lacked significant instructive or permissive signaling cues were found to display inherent similarities and differences of phenotype.

The human urothelial tight junction: claudin 3 and the ZO-1α switch.

Objective: Tight junctions are multicomponent structures, with claudin proteins defining paracellular permeability. Claudin 3 is a candidate for the exceptional "tightness" of human urothelium, being localised to the terminal tight junction (TJ) of superficial cells. Our aim was to determine whether claudin 3 plays an instigating and/or a functional role in the urothelial TJ.

Identification of ELF3 as an early transcriptional regulator of human urothelium.

Despite major advances in high-throughput and computational modelling techniques, understanding of the mechanisms regulating tissue specification and differentiation in higher eukaryotes, particularly man, remains limited. Microarray technology has been explored exhaustively in recent years and several standard approaches have been established to analyse the resultant datasets on a genome-wide scale. Gene expression time series offer a valuable opportunity to define temporal hierarchies and gain insight into the regulatory relationships of biological processes.

Nerve hyperplasia: a unique feature of ketamine cystitis.

Background: There is an emerging association between ketamine abuse and the development of urological symptoms including dysuria, frequency and urgency, which have a neurological component. In addition, extreme cases are associated with severe unresolving bladder pain in conjunction with a thickened, contracted bladder and an ulcerated/absent urothelium. Here we report on unusual neuropathological features seen by immunohistology in ketamine cystitis.

Use of donor bladder tissues for in vitro research.

Objectives: To evaluate deceased non-heart beating (DNHB) donors and deceased heart beating (DHB) brain-stem dead donors, as sources of viable urological tissue for use in biomedical research. To identify sources of viable human bladder tissue as an essential resource for cell biological research aimed at understanding human diseases of the bladder and for developing new tissue engineering and regenerative medicine strategies for bladder reconstruction. Typically, normal human urinary tract tissue is obtained from adult or paediatric surgical patients with benign urological conditions, but few surgical procedures yield useful quantities of healthy bladder tissue for research.

Tissue engineering potential of urothelial cells from diseased bladders.

Purpose: We examined the suitability of urothelium from patients with abnormal bladders for use in surgical reconstruction using a tissue engineering approach that would require autologous urothelium to be expanded by propagation in cell culture.

Materials And Methods: Resection specimens from 8 children (median age 9.8 years) with abnormal bladders (neuropathic in 4, posterior urethral valves in 2, epispadias in 1, nonneurogenic in 1) were collected with informed parental consent during planned urological procedures.

Cellular integration and vascularisation promoted by a resorbable, particulate-leached, cross-linked poly(ε-caprolactone) scaffold.

Flexible, strong scaffolds were created by crosslinking PCL with 1,6-hexamethylenediisocyanate, using paraffin beads as a porogen. Particulate leaching generated homogeneous scaffolds with interconnected spherical pores of 5-200 µm. Subcutaneous implantation in rats for 3 months resulted in minimal scaffold resorption and a non-inflammatory regenerative host response, with complete infiltration by alternatively-activated CD68(+) macrophages.

Transplantation of autologous differentiated urothelium in an experimental model of composite cystoplasty.

Background: Enterocystoplasty is associated with serious complications resulting from the chronic interaction between intestinal epithelium and urine. Composite cystoplasty is proposed as a means of overcoming these complications by substituting intestinal epithelium with tissue-engineered autologous urothelium.

Objective: To develop a robust surgical procedure for composite cystoplasty and to determine if outcome is improved by transplantation of a differentiated urothelium.

PPAR alpha and PPAR gamma coactivation rapidly induces Egr-1 in the nuclei of the dorsal and ventral urinary bladder and kidney pelvis urothelium of rats.

To facilitate studies of the rat bladder carcinogenicity of dual-acting PPAR alpha+gamma agonists, we previously identified the Egr-1 transcription factor as a candidate carcinogenicity biomarker and developed rat models based on coadministration of commercially available specific PPAR alpha and PPAR gamma agonists. Immunohistochemistry for Egr-1 with a rabbit monoclonal antibody demonstrated that male vehicle-treated rats exhibited minimal urothelial expression and specifically, no nuclear signal. In contrast, Egr-1 was induced in the nuclei of bladder, as well as kidney pelvis, urothelia within one day (2 doses) of oral dosing of rats with a combination of 8 mg/kg rosiglitazone and 200 mg/kg fenofibrate (specific PPAR gamma and PPAR alpha agonists, respectively).

Structurally diverse peroxisome proliferator-activated receptor agonists induce apoptosis in human uro-epithelial cells by a receptor-independent mechanism involving store-operated calcium channels.

Objectives: Peroxisome proliferator-activated receptors (PPARs) are implicated in epithelial cell proliferation and differentiation, but investigation has been confounded by potential off-target effects of some synthetic PPAR ligands. Our aim was to determine mechanisms underlying the pro-apoptotic effect of synthetic PPAR agonists in normal human bladder uro-epithelial (urothelial) cells and to reconcile this with the role of PPARs in urothelial cytodifferentiation.

Materials And Methods: Normal human urothelial (NHU) cells were grown as non-immortal lines in vitro and exposed to structurally diverse agonists ciglitazone, troglitazone, rosiglitazone (PPARgamma), ragaglitazar (PPARalpha/gamma), fenofibrate (PPARalpha) and L165041 (PPARbeta/delta).

Trans-species comparison of PPAR and RXR expression by rat and human urothelial tissues.

Because some investigational peroxisome proliferator-activated receptors (PPAR) agonists cause tumors in the lower urinary tract of rats, we compared normal human and rat urothelium in terms of PPAR and retinoid X receptor (RXR) expression and proliferation-associated phenotypes. In situ, few human but most rat urothelial cells were Ki67 positive, indicating fundamental differences in cell cycle control. Rat and human urothelia expressed all 3 PPAR and the RXRalpha and RXRbeta isoforms in a predominantly nuclear localization, indicating that they may be biologically active.

Differentiation potential of urothelium from patients with benign bladder dysfunction.

Objective: To develop a novel in vitro approach to test the hypothesis that failure of urothelial differentiation underlies the aetiopathology of interstitial cystitis (IC), where there is evidence of compromised urinary barrier function, as benign dysfunctional bladder disease encompass several poorly understood clinically defined conditions, including IC, idiopathic detrusor overactivity (IDO) and stress urinary incontinence (SUI).

Materials And Methods: Biopsy-derived urothelial cells from dysfunctional bladder biopsies were propagated as finite cell lines and examined for their capacity to differentiate in vitro, as assessed by the acquisition of a transitional cell morphology, a switch from a cytokeratin (CK)13(lo)/CK14(hi) to a CK13(hi)/CK14(lo) phenotype, expression of claudin 3, 4 and 5 proteins, and induction of uroplakin gene transcription.

Results: Two of 12 SUI cell lines showed early senescent changes in culture and were not characterized further; one of seven IC, one of five IDO and a further three SUI cell lines had some evidence of senescence at passage 3.

PPARgamma-regulated tight junction development during human urothelial cytodifferentiation.

Urothelial barrier function is maintained by apical membrane plaques and intercellular tight junctions (TJ). Little is known about the composition and regulation of TJ expression in human urothelium. In this study, we have characterised the expression of TJ components in situ and their regulation in an in vitro model of differentiating normal human urothelial (NHU) cells.