Jens Stahlschmidt

Role of PPARgamma and EGFR signalling in the urothelial terminal differentiation programme

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.

Activation of Peroxisome Proliferator-Activated Receptor-γ Reverses Squamous Metaplasia and Induces Transitional Differentiation in Normal Human Urothelial Cells

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.

Differentiation Potential of Urothelium from Patients with Benign Bladder Dysfunction

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).

Renal medullary carcinoma: Prolonged remission with chemotherapy, immunohistochemical characterisation and evidence of bcr/abl rearrangement

BACKGROUND Renal medullary carcinoma (RMC), an extremely rare tumour of the kidney, carries a dismal prognosis, with no reports to date of significant response to chemotherapy or radiotherapy. A case of this tumour in a male child, who showed a dramatic response to chemotherapy, is described. PROCEDURE A detailed histological evaluation of the tumour and cytogenetic analysis using fluorescent in situ hybridisation (FISH) was carried out. The child was treated with multiagent chemotherapy, followed by abdominal radiotherapy. RESULTS A detailed histopathological and immunohistochemical portrait of this tumour is described, and FISH studies confirmed the presence of a bcr/abl rearrangement. The child obtained complete radiological remission following chemotherapy, although he later relapsed and died of progressive disease despite further attempts at treatment with chemotherapy. CONCLUSIONS Although there are no previous reports of response of this tumour to chemotherapy, this case illustrates that treatment of this disease is justified. The responses of other cases to similar drug regimens would be of interest to confirm whether the encouraging response described for this case could be reproduced. Cytogenetic analysis of other cases of RMC may clarify whether the abnormalities seen in this case are typical.

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. Six patients had recurrent urinary tract infections and 7 underwent frequent intermittent catheterization. A representative sample was immunohistologically processed to assess urothelial proliferation and differentiation status, and the remaining 7 cases were processed for urothelial cell culture. Five normal adult urothelial samples were included as controls. RESULTS Immunohistological assessment indicated that 3 of 8 samples lacked urothelial differentiation associated expression of UPK3a or CK20. Four of 7 samples resulted in successful primary culture, with 1 sample lost to underlying infection and 2 not surviving in culture. All 4 cultures grew beyond passage 3 before senescence but all showed reduced proliferation capacity and a compromised ability to form a barrier urothelium compared to controls. CONCLUSIONS 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.

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. Design, setting, and participants Bladder 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. Measurements Urothelium 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 limitations Eleven 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. Conclusions The 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.

Nerve hyperplasia: a unique feature of ketamine cystitis

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.

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. In addition, scaffolds were populated extensively along microfractures by a stromal matrix, which was highly vascularised and contained a subset of stromal cells that expressed the anti-inflammatory CD163 antigen. Such microfractures may be an important physical feature for directing stromal integration and vascularisation events.

Histologic oddities at the porta hepatis in biliary atresia

PURPOSE Highly unusual histologic findings at the porta hepatis in 3 infants who underwent Kasai portoenterostomy for biliary atresia are reported. METHODS Portoenterostomy was performed using a standard operative technique. Serial transverse sections of the excised portal plate were examined by light microscopy along with sections from the distal extrahepatic biliary remnants, gallbladder, and liver biopsy. RESULTS Of 61 consecutive infants who underwent Kasai portoenterostomy for biliary atresia, 3 were found to have highly unusual histologic features at the porta hepatis. All had type 3 biliary atresia. Two had hilar biliary ductules lined in part by squamous epithelium, and the third had a focus of mature hyaline cartilage surrounded by perichondrium adjacent to biliary ductules. In each case, these unusual histologic features were localized to the porta hepatis in the region of the transected portal plate. CONCLUSIONS The presence of hyaline cartilage at the portal plate is likely to be an expression of defective morphogenesis, thus supporting the concept of disordered embryogenesis in the etiology of biliary atresia. Squamous epithelium within biliary ductules might also reflect a similar mechanism but could alternatively be an unusual metaplastic response to inflammation at this site.

Urinary schistosomiasis in children in the United Kingdom

We report on two patients with urinary schistosomiasis, who both presented within a fortnight to our hospital with similar symptoms of persistent painless haematuria. Ultrasound, cystoscopic biopsies and histology were used to confirm diagnosis. Treatment with praziquantel was given. Symptoms of urinary schistosomiasis can easily be missed in non-endemic areas and possibly confused with a more sinister pathology. A thorough history and awareness of disease can avoid interventional investigations.