MMR assays identify patients with a low risk of recurrence. KRAS mutational analysis provides useful additional risk stratification to guide use of chemotherapy.

Recently, considerable interest has focused on the ability of activated peroxisome proliferator-activated receptor γ (PPARγ) to promote cytodifferentiation in adipocytes and some carcinoma cells; however, the role of PPARγ in normal epithelial cytodifferentiation is unknown. Using uroplakin (UP) gene expression as a specific correlate of terminal urothelial cytodifferentiation, we investigated the differentiation-inducing effects of PPARγ activation in normal human urothelial (NHU) cells grown as finite cell lines in monoculture. Two high-affinity activators of PPARγ, troglitazone (TZ) and rosiglitazone (RZ) induced the expression of mRNA for UPII and UPIb and, to a lesser extent, UPIa. The specificity of the effect was shown by pretreating cells with a PPARγ antagonist, GW9662, which attenuated the TZ-induced response in a dose-specific manner. The PPARγ-mediated effect on UP gene expression was maximal when there was concurrent inhibition of autocrine-activated epidermal growth factor receptor (EGFR) signalling through either the phosphatidylinositol 3-kinase or extracellular signal-regulated kinase (ERK) pathways. The use of a specific EGFR tyrosine kinase inhibitor, PD153035, correlated with PPARγ dephosphorylation and translocation to the nucleus, indicating a mechanism for regulating the balance between proliferation and differentiation. This is the first identification of specific factors involved in regulating differentiation-associated gene changes in urothelium and the first unambiguous evidence of a role for PPARγ signalling in the terminal differentiation programme of a normal epithelium.

We observed that in urothelium, both cornifying and noncornifying forms of squamous metaplasia are accompanied by changes in the localization of the nuclear hormone receptors, peroxisome proliferator activated receptor gamma (PPAR-gamma) and retinoid X receptor (RXR-alpha). To obtain objective evidence for a role for PPAR-gamma-mediated signaling in urothelial differentiation, we examined expression of the cytokeratin isotypes CK13, CK20, and CK14 as indicators of transitional, terminal transitional, and squamous differentiation, respectively, in cultures of normal human urothelial cells. In control culture conditions, normal human urothelial cells showed evidence of squamous differentiation (CK14+, CK13-, CK20-). Treatment with the high-affinity PPAR-gamma agonist, troglitazone (TZ), resulted in gain of CK13 and loss of CK14 protein expression. The effect of TZ was significantly augmented when the autocrine-stimulated epidermal growth factor receptor pathway was inhibited and this resulted in induction of CK20 expression. The RXR-specific inhibitors PA452, HX531, and HX603 inhibited the TZ-induced CK13 expression, supporting a role for RXR in the induction of CK13 expression. Thus, signaling through PPAR-gamma can mediate transitional differentiation of urothelial cells and this is modulated by growth regulatory programs.

LKT and IE contributed equally assessed by the acquisition of a transitional cell morphology, a switch from a cytokeratin (CK)13 lo /CK14hi to a CK13 hi /CK14 lo phenotype, expression of claudin 3, 4 and 5 proteins, and induction of uroplakin gene transcription. RESULTSTwo 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. Of the seven IC-derived cell lines, four showed a near normal range of differentiationassociated responses, but the remainder showed little or no response. Most IDO cell lines (four of five) showed a normal differentiation response, but at least three of the 10 SUI cell lines showed some compromise of differentiation potential. CONCLUSIONThis study supports the existence of a subset of patients with IC in whom a failure of urothelial cytodifferentiation might contribute to the disease, and provides a novel platform for investigating the cell biology of urothelium from SUI and other benign dysfunctional conditions.

A child of consanguineous parents of Pakistani origin developed jaundice at 5 weeks and then, at 3 months, irritability, a prolonged prothrombin time, a low albumin, and episodes of hypoglycaemia. Investigation showed an elevated alanine aminotransferase with a normal γ-glutamyl-transpeptidase. Analysis of urine by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) showed that the major peaks were m/z 480 (taurine-conjugated 3β-hydroxy-5-cholenoic acid) and m/z 453 (sulphated 3β-hydroxy-5-cholenoic acid). Analysis of plasma by gas chromatography-mass spectrometry (GC-MS) showed increased concentrations of 3β-hydroxy-5-cholenoic acid, 3β-hydroxy-5-cholestenoic acid and 27-hydroxycholesterol, indicating oxysterol 7 α-hydroxylase deficiency. The patient was homozygous for a mutation (c.1249C>T) in CYP7B1 that alters a highly conserved residue in oxysterol 7 α-hydroxylase (p.R417C) - previously reported in a family with hereditary spastic paraplegia type 5. On treatment with ursodeoxycholic acid (UDCA), his condition was worsening, but on chenodeoxycholic acid (CDCA), 15 mg/kg/d, he improved rapidly. A biopsy (after 2 weeks on CDCA), showed a giant cell hepatitis, an evolving micronodular cirrhosis, and steatosis. The improvement in liver function on CDCA was associated with a drop in the plasma concentrations and urinary excretions of the 3β-hydroxy-Δ5 bile acids which are considered hepatotoxic. At age 5 years (on CDCA, 6 mg/kg/d), he was thriving with normal liver function. Neurological development was normal apart from a tendency to trip. Examination revealed pes cavus but no upper motor neuron signs. The findings in this case suggest that CDCA can reduce the activity of cholesterol 27-hydroxylase - the first step in the acidic pathway for bile acid synthesis.

BackgroundEnterocystoplasty 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.ObjectiveTo develop a robust surgical procedure for composite cystoplasty and to determine if outcome is improved by transplantation of a differentiated urothelium.Design, setting, and participantsBladder augmentation with in vitro–generated autologous tissues was performed in 11 female Large-White hybrid pigs in a well-equipped biomedical centre with operating facilities. Participants were a team comprising scientists, urologists, a veterinary surgeon, and a histopathologist.MeasurementsUrothelium harvested by open biopsy was expanded in culture and used to develop sheets of nondifferentiated or differentiated urothelium. The sheets were transplanted onto a vascularised, de-epithelialised, seromuscular colonic segment at the time of bladder augmentation. After removal of catheters and balloon at two weeks, voiding behaviour was monitored and animals were sacrificed at 3 months for immunohistology.Results and limitationsEleven pigs underwent augmentation, but four were lost to complications. Voiding behaviour was normal in the remainder. At autopsy, reconstructed bladders were healthy, lined by confluent urothelium, and showed no fibrosis, mucus, calculi, or colonic regrowth. Urothelial morphology was transitional with variable columnar attributes consistent between native and augmented segments. Bladders reconstructed with differentiated cell sheets had fewer lymphocytes infiltrating the lamina propria, indicating more effective urinary barrier function.ConclusionsThe study endorses the potential for composite cystoplasty by (1) successfully developing reliable techniques for transplanting urothelium onto a prepared, vascularised, smooth muscle segment and (2) creating a functional urothelium-lined augmentation to overcome the complications of conventional enterocystoplasty.

BackgroundThere 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.ResultsIn all cases, the lamina propria was replete with fine neurofilament protein (NFP+) nerve fibres and in most patients (20/21), there was prominent peripheral nerve fascicle hyperplasia that showed particular resemblance to Morton’s neuroma. The nerve fascicles, which were positive for NFP, S100 and the p75 low-affinity nerve growth factor receptor (NGFR), were generally associated with a well-developed and in places, prominent, epithelial membrane antigen+/NGFR+ perineurium. This peripheral nerve fascicle hyperplasia is likely to account for the extreme pain experienced by ketamine cystitis patients. Urothelial damage was a notable feature of all ketamine cystitis specimens and where urothelium remained, increased NGFR expression was observed, with expansion from a basal-restricted normal pattern of expression into the suprabasal urothelium.ConclusionsThe histological findings were distinguishing features of ketamine cystitis and were not present in other painful bladder conditions. Ketamine cystitis afflicts predominantly young patients, with unknown long-term consequences, and requires a strategy to control severe bladder pain in order to remove a dependency on the causative agent. Our study indicates that the development of pain in ketamine cystitis is mediated through a specific neurogenic mechanism that may also implicate the urothelium.

While normal human urothelium is highly regenerative and derived cells are highly proliferative in culture, our results with urothelium from abnormal pediatric bladders indicate a reduced capacity for proliferation and differentiation in vitro. This finding may indicate a need to identify alternative cell sources for engineered bladder reconstruction.