Antibacterial and therapeutic effects of low energy shock waves on uropathogenic E. coli investigated by in vitro and in vivo cystitis rat model.

AIMS: Low-energy shock waves (LESWs) are known to alter cell-membrane permeability. This study aimed to investigate the effect of LESWs on Escherichia coli and E. coli-induced cystitis in rats. MAIN METHODS: Standardized suspensions of E. coli ATCC25922 were treated with or without LESWs (100 or 300 pulses; 0.12 mJ/mm2; 2 pulses/s) followed by bacterial counting, an antibiotic sensitivity test, and gene ontology analysis and gene-set enrichment analysis. Intravesical administration of saline or E. coli (0.5 mL with 108 CFU/mL) for 30 min was performed in female Sprague-Dawley rats. The rats were treated with or without LESWs (300 pulses; 0.12 mJ/mm2; 2 pulses/s) on days 4 and 5. The changes in inflammatory reactions, uroplakin IIIa staining, and correlation with urodynamic findings were assessed on day 8. KEY FINDINGS: LESW treatment induced a decrease in CFU and the autoaggregation rate and increased the inhibition zone sizes in a cefazolin-sensitivity study. These changes were associated with gene expression in regulation of cellular membrane components, biofilm formation, and the ATP-binding cassette transporter pathway. E. coli induced bladder hyperactivity and an inflammatory reaction as well as decreased uroplakin IIIa staining; these effects were partially reversed by LESW treatment. SIGNIFICANCE: The LESW antibacterial effect occurs by altering bacterial cell-membrane gene expression, enhancing antibiotic sensitivity, and inhibiting bladder inflammatory reaction and overactivity. These findings support the potential benefits of LESWs for treatment of recurrent or refractory bacterial cystitis.

Uroplakin-IIIb as a novel immunohistochemical marker for mesothelioma.

Distinguishing mesothelioma from non-small cell lung carcinoma often requires a battery of immunohistochemical stains, as many traditional markers used in mesothelioma lack sufficient specificity to allow them to be used alone. A recent large-scale TMA screen identified uroplakin-IIIb (UpIIIb; clone MSVA-736M) as a potentially specific marker for mesothelioma. We examined the performance of this antibody using tissue microarrays containing a panel of 48 epithelioid mesotheliomas, 26 sarcomatoid mesotheliomas, and 144 non-small cell lung carcinomas (NSCLCs). Here we show that UpIIIb has good sensitivity (37/47 evaluable cases positive, 79%) and excellent specificity for distinguishing epithelioid mesothelioma from NSCLC (0/140 evaluable cases positive). UPIIIb sensitivity for epithelioid mesotheliomas was only slightly inferior to the established highly specific mesothelioma marker HEG1 (41/46 evaluable cases positive on the same TMA, 89%). However, UpIIIb did not stain any sarcomatoid mesotheliomas (0/24 evaluable cases positive). We also found that UpIIIb stained a proportion of high-grade serous ovarian carcinomas, a perennial diagnostic confounder in the context of mesotheliomas. Taken together, our data suggest that UpIIIb can be used as a highly specific and sensitive mesothelial marker when the diagnostic question is epithelioid mesothelioma versus NSCLC; in particular, UpIIIb staining will pick up some number of epithelioid mesotheliomas that are HEG1 negative. Since UpIIIb is known to stain some proportion of urothelial carcinomas as well as gynecologic and a few pancreatic tumors, it should be used with caution in the peritoneal cavity or when the differential diagnosis includes carcinomas from these locations.

Properties of Pleural Mesothelial Cells in Idiopathic Pulmonary Fibrosis and Cryptogenic Organizing Pneumonia.

BACKGROUND: Profibrotic properties of pleural mesothelial cells may play an important role in the fibrosis activity in idiopathic pulmonary fibrosis (IPF). The purpose of this study was to compare the expression of pleural mesothelial cell markers in IPF and cryptogenic organizing pneumonia (COP), with an assumption that increased expression implies increase in fibrosis. METHODS: Twenty IPF lung samples were stained by immunohistochemistry for the pleural mesothelial cell markers: leucine rich repeat neuronal 4 (LRRN4), uroplakin 3B, CC-chemokine ligand 18, and laminin-5. Nine COP lung samples were used as controls. A semi-quantitative analysis was performed to compare markers expression in IPF and COP. RESULTS: LRRN4 expression was found in epithelial lining cells along the honeycombing and fibroblastic foci in IPF, but not in the fibrotic interstitial lesion and airspace filling fibrous tufts in COP. We found a significant decrease in baseline forced vital capacity when LRRN4 expression was increased in honeycombing epithelial cells and fibroblastic foci. CONCLUSION: LRRN4 expression patterns in IPF are distinct from those in COP. Our findings suggest that mesothelial cell profibrotic property may be an important player in IPF pathogenesis and may be a clue in the irreversibility of fibrosis in IPF.

Analysis of More than 16,000 Human Tumor and Normal Tissues Identifies Uroplakin 3B as a Useful Diagnostic Marker for Mesothelioma and Normal Mesothelial Cells.

Uroplakin 3B (Upk3b) is involved in stabilizing and strengthening the urothelial cell layer of the bladder. Based on RNA expression studies, Upk3b is expressed in a limited number of normal and tumor tissues. The potential use of Upk3b as a diagnostic or prognostic marker in tumor diagnosis has not yet been extensively investigated. A tissue microarray containing 17,693 samples from 151 different tumor types/subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. In normal tissues, Upk3b expression was largely limited to mesothelial cells, urothelial umbrella cells, and amnion cells. In tumor tissues, Upk3b was detectable in only 17 of 151 (11.3%) of tumor types. Upk3b expression was most frequent in mesotheliomas (82.1% of epithelioid and 30.8% of biphasic) and in urothelial tumors of the urinary bladder, where the positivity rate decreased from 61.9% in pTaG2 (low grade) to 58.0% in pTaG3 (high grade) and 14.6% in pT2-4 cancers. Among pT2-4 urothelial carcinomas, Upk3b staining was unrelated to tumor stage, lymph node status, and patient prognosis. Less commonly, Upk3b expression was also seen in Brenner tumors of the ovary (10.8%), as well as in four other subtypes of ovarian cancer (0.9-10.6%). Four additional tumor entities showed a weak to moderate Upk3b positivity in less than 5% of cases. In summary, Upk3b immunohistochemistry is a useful diagnostic tool for the distinction of mesotheliomas from other thoracic tumors and the visualization of normal mesothelial and umbrella cells.

Large-scale human tissue analysis identifies Uroplakin 1a as a putative diagnostic marker for urothelial cancer.

Uroplakin 1A (Upk1a) protein is relevant for stabilizing and strengthening urothelial cells and helps to prevent them from rupturing during bladder distension. Based on RNA expression data Upk1a is expressed in a limited number of normal tissues and tumors. To comprehensively evaluate the potential diagnostic and prognostic utility of Upk1a immunohistochemistry, a tissue microarray containing 6929 samples from 115 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed. Upk1a positivity was found in 34 (29.6 %) different tumor types including 9 (7.8 %) tumor types with at least one strongly positive case. The highest rates of Upk1a positivity were seen in various subtypes of urothelial neoplasms (42.6-98 %) including Brenner tumors of the ovary (64.9 %) followed by neoplasms of the thyroid (10.4-33.3 %). In urothelial tumors, Upk1a staining predominated at the cell membranes and staining intensity was often moderate to strong. In thyroidal neoplasms the staining was mostly purely cytoplasmic and of low to moderate intensity. Upk1a positivity was also seen in up to 15 % of cases in 25 additional tumor categories but the staining intensity was often cytoplasmic and the intensity was usually judged as weak and only rarely as moderate. Within non-invasive (pTa) tumors, the Upk1a positivity rate decreased from 94 % in pTa G2 (low grade) to 90.1 % in pTa G3 (p = 0.012) and was even lower in muscle-invasive carcinomas (41.5 %; p < 0.0001 vs pTaG3). Within muscle invasive carcinomas, Upk1a expression was unrelated to nodal metastasis (p > 0.05) and patient outcome (p > 0.05). In conclusion, Upk1a immunohistochemistry is a potentially useful and specific diagnostic marker for the distinction of urothelial carcinomas from other neoplasms. However, its sensitivity is less than 50 % in muscle-invasive cancers because Upk1a expression decreases during grade and stage progression.

Reduced urothelial expression of uroplakin-IIIa in cystitis areas in bladders of postmenopausal women with recurrent urinary tract infections: pilot study.

PURPOSE: To study human bladder biopsies to investigate urothelial response to UTI, expression of uroplakin, and urothelial response after healing from cystoscopy with electrofulguration (CEF) treatment for antibiotic-recalcitrant RUTI. METHODS: Following IRB approval, cold cup bladder biopsies from "no cystitis" and "cystitis" regions were obtained from women with antibiotic-recalcitrant rUTI undergoing CEF under anesthesia. "No cystitis" and "cystitis" biopsies from 14 patients (5 had prior CEF, 9 naïve) were analyzed by immunofluorescence (IF) confocal microscopy using antibodies against uroplakin-IIIa. For an additional 6 patients (2 prior CEF, 4 naïve), only "cystitis" area biopsies were taken and analyzed. Cytokeratin 5 (marker for squamous metaplasia) staining was performed on 14 patients. RESULTS: In healthy tissue, uroplakin-IIIa staining was observed as a contiguous line on the luminal surface of umbrella cells. In "cystitis" areas for 19/20 patients, there was either no uroplakin-IIIa staining observed or spotty (+) staining. The "cystitis" regions of all patients had less intense uroplakin-IIIa staining compared to the matched "no cystitis" area in the same patient. In patients post-CEF but requiring repeat EF for persistent RUTI lesions, healed areas served as control and in 3 of 7 patients no uroplakin-IIIa staining was observed. Squamous metaplasia was observed in 10 patients. CONCLUSION: In bladders of postmenopausal women with antibiotic-recalcitrant RUTI, areas with visible cystitis expressed less uroplakin-IIIa, supporting the model of urothelial exfoliation in response to UTI.

Uroplakin II as a single marker for luminal versus basal molecular subtypes in muscle invasive urothelial carcinoma.

Bladder cancer is a heterogeneous disease classified into two broad molecular subtype categories, basal and luminal, with critical treatment and prognostic implications. Recent studies have shown the utility of immunohistochemistry in predicting bladder cancer molecular subtypes, with a two-marker approach using GATA3 and CK5/6 showing over 80% reliability. In the current study, we calculated the accuracy of uroplakin II (UPII), a marker of urothelial differentiation, with different scores (0: <1%, 1+: 1-10%, 2+: 10-50%, 3+: >50%) to predict RNA-based luminal versus basal subtypes in a cohort of muscle-invasive bladder cancer-received neoadjuvant chemotherapy followed by radical cystectomy. The 1% cutoff of the UPII stain predicts the luminal subtype with the sensitivity and specificity of 95% and 56%, respectively. With a UPII cutoff of 10%, the sensitivity and specificity were 93% and 81%, respectively, and with a UPII cutoff of 50%, the sensitivity and specificity were 91% and 96%, respectively. The prediction performance of UPII was better than either GATA3 or CK5/6. There was no significant difference in prognoses between UPII 0-2+ and UPII 3+ patients in this cohort. The current study shows that evaluating the staining proportion score of UPII can accurately predict basal and luminal subtypes of muscle-invasive bladder cancer.

Large-scale human tissue analysis identifies Uroplakin 1b as a putative diagnostic marker in surgical pathology.

Uroplakin 1B (Upk1b) stabilizes epithelial cells lining the bladder lumen to prevent rupturing during bladder distension. Little is known about Upk1b expression in other normal and malignant tissues. To comprehensively evaluate the potential diagnostic and prognostic utility of Upk1b expression analysis, a tissue microarray containing 14,061 samples from 127 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Upk1b immunostaining was found in 61 (48%) different tumor types including 50 (39%) with at least one moderately positive and 39 tumor types (31%) with at least one strongly positive tumor. Highest positivity rates were found in urothelial neoplasms (58-95%), Brenner tumors of the ovary (92%), epithelioid mesothelioma (87%), serous carcinoma of the ovary (58%) and the endometrium (53%) as well as in squamous cell carcinoma of the head and neck (18-37%), lung (39%), and esophagus (26%). In urothelial carcinoma, low Upk1b expression was linked to high grade and invasive tumor growth (P < .0001 each) and nodal metastasis (P = .0006). Our data suggest diagnostic applications of Upk1b immunohistochemistry in panels for the distinction of malignant mesothelioma from adenocarcinoma of the lung, urothelial carcinoma from prostatic adenocarcinoma in the bladder, or pancreaticobiliary and gastroesophageal from colorectal adenocarcinoma.

Histological and immunohistochemical investigation of canine prostate carcinoma with identification of common intraductal carcinoma component.

A limited number of species, including men and dogs, spontaneously develop prostate cancer (PC). The histological and molecular relevance of canine PC as a model for the disease in men remains controversial. To address this challenge, this study aimed to assess the histomorphology and expression of basal cell, urothelial and neuroendocrine markers [p63, high molecular weight cytokeratin (HMWCK), Uroplakin 3 (UPIII), neuron-specific enolase (NSE)] in canine PC (n = 41). Based on histomorphology, 10/41 (24%), 21/41 (51%) and 9/41 (22%) were classified as adenocarcinoma (AC), urothelial carcinoma (UC), and mixed carcinoma, respectively. Tumour inflammation was common, frequently severe [20/41 (49%)], and associated with neutering (p < .02) and urothelial differentiation (p < .02). Most (36/40, 90%) cancers contained only rare cells with basal cell marker expression or were negative. The expression of UPIII was absent or weak in the majority (33/38, 87%) of tumours, with moderate to strong staining in the remaining cases. NSE expression in PC was rare and limited to 2/14 (14%) cases. Tumour extension into benign ducts and glands was a common finding with presence in 17/39 (44%) of carcinomas with and without urothelial differentiation. In conclusion, we confirm that canine PC is characterized by absent or weak expression of basal cell and urothelial markers. Although rare, NSE expression, potentially indicating neuroendocrine differentiation, is reported for the first time in canine PCa. Intraductal carcinoma of the prostate with concurrent invasive PCa (IDCP-inv) is a frequent, not previously described, finding in dogs with PC.

Initial Evaluation of Uroplakins UPIIIa and UPII in Selected Benign Urological Diseases.

BACKGROUND: Uroplakins (UPs) are glycoproteins that play a specific role in the structure and function of the urothelium. Disorders which affect the normal expression of UPs are associated with the pathogenesis of infections and neoplasms of the urinary tract, primary vesicoureteral reflux, hydronephrosis and renal dysfunction. The appearance of uroplakins in the urine and/or plasma may be of potential importance in the detection of urinary tract dysfunction. The aim of the present study was to investigate uroplakin IIIa (UPIIIa) and uroplakin II (UPII) expression in patients with selected urological diseases. METHODS: Plasma and urine from patients with benign prostatic hyperplasia (BPH), urethral stricture (US), urinary tract infection (UTI) and urolithiasis were compared to healthy people without urological disorders. UPs concentrations were measured by the immunoenzymatic method. RESULTS: In patients with BPH and UTI, concentrations of UPIIIa in urine and plasma, as well as UPII in urine, were statistically significantly higher than in the control groups. In the US group, only the plasma UPIIIa concentration differed significantly from the control. CONCLUSION: The conducted research shows that benign urological diseases may affect the state of the urothelium, as manifested by increased concentrations of both UPs in patients' urine and plasma, especially in BPH and UTI.

Expression of uroplakin II and GATA-3 in bladder cancer mimickers: caveats in the use of a limited panel to determine cell of origin in bladder lesions.

Antibodies targeting uroplakin II (UPII) are highly specific for urothelial cells and are frequently used to determine if a primary bladder lesion or a metastatic lesion originates from the urothelium. However, to date, no studies have tested the expression of UPII in histological mimickers of bladder cancer that are nonurothelial in origin. Given the potential risk of misdiagnosis, immunohistochemical markers are often used to better characterize these lesions. In the present study, we analyzed the immunohistochemical expression of UPII in a set of urothelial carcinoma mimickers that included conventional nephrogenic adenoma (n = 8), papillary nephrogenic adenoma (n = 6), endometriosis/endosalpingiosis (n = 5), inflammatory myofibroblastic tumor (n = 4), ectopic prostate tissue (n = 2), and malakoplakia (n = 2). We also examined the expression of GATA-3, another commonly used immunohistochemical marker in bladder cancer diagnosis, in the same lesions. Weak immunoreactivity for UPII was identified in 6 of 27 mimickers (22%), and GATA-3 was expressed in 16 of 27 mimickers (59%). Strong immunoreactivity for UPII appeared to be a specific marker for urothelial cell of origin, although weak staining was seen in a significant proportion of mimickers. GATA-3 immunostaining was present in a greater number and broader spectrum of mimickers; however, only one case of papillary nephrogenic adenoma showed dual positivity for UPII and GATA-3. These findings support the immunohistochemical panel-based approach in the diagnosis of bladder lesions, especially if nonurothelial bladder cancer mimickers are in the differential diagnosis. Additional larger studies would be of value to expand on these findings.

High HNRNPA3 expression is associated with lymph node metastasis and poor prognosis in patients treated with radical cystectomy.

OBJECTIVE: We sought to identify heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) expression in bladder cancer and its relationship to clinicopathological findings and prognosis. METHODS: Immunohistochemical staining for HNRNPA3 was performed on 122 archived radical cystectomy specimens, with immunoreactivity being stratified on a 0 to 3 scale. The percentage of HNRNPA3 expressing tumor cells was calculated and multiplied by the staining score over an average of 5 areas to obtain a semiquantitative H-score (maximum value: 300). HNRNPA3 expression was categorized as high (≥80) or low (<80). RESULTS: The patients' median age was 70 years, and the median follow-up period was 39.4 months. High HNRNPA3 expression was significantly associated with lymph node metastasis (P= 0.014) and S100A8, S100A9 and uroplakin III expression (P= 0.028, 0.002, and 0.047, respectively). Log-rank tests indicated that high HNRNPA3 expression was significantly associated with disease progression and cancer-specific death (P= 0.013 and 0.006, respectively). In the Cox proportional hazards regression analysis, only lymph node metastasis was associated with disease progression and cancer-specific survival. CONCLUSION: HNRNPA3 may be a new biomarker to predict biologically aggressive cancers and determine the appropriate treatment modality in patients after radical cystectomy.

Long Noncoding RNA Expression Profiling Reveals Upregulation of Uroplakin 1A and Uroplakin 1A Antisense RNA 1 under Hypoxic Conditions in Lung Cancer Cells.

Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1AAS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxiainducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxiainduced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.

Expression of NGF, MCP-1, uroplakin III, and NOS in bladder urothelium after partial urethral obstruction in rats.

INTRODUCTION: Although several cytokines, chemokines, and growth factors have been suggested to play a role in the development of bladder fibrosis and functional changes, the mechanisms that are effective in the pathogenesis of partial bladder outlet obstruction (pBOO)-induced bladder fibrosis are not well understood. OBJECTIVE: We investigated the expressions of nerve growth factor (NGF), monocyte chemoattractant protein-1 (MCP-1), uroplakin III (URPIII), inducible nitric oxide synthase (iNOS), and endothelial NOS (eNOS) that may be involved in fibrosis in rats with partial urethral obstruction for 1, 2 and 3 weeks, and the changes in the associated ischemic and inflammatory processes. After 1, 2, and 3 weeks of pBOO, blood samples were collected for assessment of renal function from the rats under anesthesia. The bladders were dissected for the tissue antioxidant enzyme activities and lipid peroxidation, including malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant status (TAS) and total oxidant status (TOS). The immunohistochemical studies were performed. Histopathologically, the number of urothelial layers was calculated and the thickness of the detrusor smooth muscle and lamina propria were quantitatively measured. Additionally, the edema and congestion in the submucosa were evaluated. STUDY DESIGN: Twenty-eight male Sprague-Dawley rats were used in this study. Three separate experimental groups of pBOO (1 week [n = 7], 2 weeks [n = 7], and 3 weeks [n = 7]) were created, with an additional sham-operated control group (n = 7). RESULTS: The MDA levels increased in pBOO groups. The SOD values were decreased in the pBOO group for 1 week, and higher in the 3-week pBOO group. The TAS levels were increased in the 3 week pBOO group. The TOS levels increased in the pBOO groups. The number of urothelial layers was decreased in pBOO groups. The lamina propria, the smooth muscle thickness, edema and congestion were increase in the 1 and 2 week pBOO groups. The NGF and MCP-1 expression was increased in the 1-week and 2-week pBOO groups. The expression of URPIII in the epithelium gradually increased in the pBOO groups. In the pBOO groups, iNOS expression in the epithelium cells was significantly elevated. However, the eNOS expression was also significantly increased in the 2 week pBOO group. CONCLUSION: Our study shows that overexpression of immunohistochemical parameters together with the negative effects of ischemic and inflammatory processes that subjected to pBOO for 1, 2 and 3 weeks may play a potential role in detrusor fibrosis in the rat bladders induced by pBOO. However, understanding of the immunohistochemical parameters investigated in this experimental study is limited, and further studies targeting their relationship to pBOO could help us develop new strategies.

Krt5+ urothelial cells are developmental and tissue repair progenitors in the kidney.

Congenital urinary tract obstruction (UTO) is the leading cause of chronic kidney disease in children; however, current management strategies do not safeguard against progression to end-stage renal disease, highlighting the need for interventions to limit or reverse obstructive nephropathy. Experimental UTO triggers renal urothelial remodeling that culminates in the redistribution of basal keratin 5-positive (Krt5+) renal urothelial cells (RUCs) and the generation of uroplakin-positive (Upk)+ RUCs that synthesize a protective apical urothelial plaque. The cellular source of Upk+ RUCs is currently unknown, limiting the development of strategies to promote renal urothelial remodeling as a therapeutic approach. In the present study, we traced the origins of adult Upk+ RUCs during normal development and in response to UTO. Fate mapping analysis demonstrated that adult Upk+ RUCs derive from embryonic and neonatal Krt5+ RUCs, whereas Krt5+ RUCs lose this progenitor capacity and become lineage restricted by postnatal day 14. However, in response to UTO, postnatal day 14-labeled adult Krt5+ RUCs break their lineage restriction and robustly differentiate into Upk+ RUCs. Thus, Krt5+ RUCs drive renal urothelial formation during normal ontogeny and after UTO by differentiating into Upk+ RUCs in a temporally restricted manner.

Differentiation of Urothelium from Mouse Embryonic Stem Cells in Chemically Defined Conditions.

The urothelium of the bladder and urethra are derived from the definitive endoderm during development. Cellular signaling molecules important to the developmental specification of the urothelium are also implicated in the dysregulation of the tissue repair mechanism characteristic of bladder disease. Hence, a complete understanding of the regulation of urothelium development is central to understanding the processes of bladder disease, and in development of simple chemically defined methods for use in regenerative medicine. Key to this is a suitable in vitro model that readily allows for the prosecution of biologically pertinent questions. Here a method for differentiating urothelium from mouse embryonic stem cells in chemically defined conditions is described. The method includes a description of flow cytometry and RT-PCR analysis of definitive endoderm markers Cxcr4, c-Kit, and FoxA2, and of terminally differentiated urothelial cell markers Upk1b and Upk2.

Successful Treatment of Metastatic Urothelial Carcinoma after Accurate Diagnosis by Immunohistochemistry.

Urothelial carcinoma usually presents with hematuria, but cases of multiple lymphadenopathy with elevated S-pancreas-1 antigen (SPan-1) levels have not been reported. A 62-year-old Japanese man with lymphadenopathies was diagnosed with an adenocarcinoma of unknown origin and transferred to our hospital for further diagnosis. Serum carbohydrate antigen 19-9 and SPan-1 levels were extremely elevated. Uroplakin III immunostaining was positive in the inguinal lymph node, and cystoscopy revealed the presence of invasive urothelial carcinoma. Treatment with cisplatin and gemcitabine promoted a complete metabolic response for > 4 years. The detection of uroplakin III and serum SPan-1 might help diagnose urothelial carcinoma.

[The pathological characters of urothelial barrier in prostatic ducts of elderly SD rats].

Objective: To investigate the pathological characters of urothelial barrier in prostatic ducts of elderly male SD rats. Methods: Ten elderly male SD rats were anesthetized with chloral hydrate and then took the bladder, prostatic urethra and prostate tissues for serial pathological sections and HE staining. Immunohistochemical method was used to detect the expression of the Cytokeratin 7 (CK7), Cytokeratin 20 (CK20), Uroplakin Ⅲ (UP Ⅲ), Zonula occludens-1 (ZO-1) and Occludin in issues, and to make densitometric analysis (IA) on immunohistochemical results of each antibody. Results: HE staining observed that urothelial umbrella cells exist in the bladder, prostatic urethra and proximal prostatic duct. Immunohistochemical method showed the CK7, UPⅢ, ZO-1 and Occludin were positive in bladder, prostatic urethra and prostatic duct, while CK20 was negative. CK7 and UPⅢ were positive in the proximal prostatic duct, and negative in the bottom of the ductal acinar lumen; ZO-1 and Occludin were positive in prostatic duct. The IOD/Area values of CK7 and UP Ⅲ, in bladder, prostatic urethra, proximal prostatic duct, the middle of prostatic duct and bottom of the ductal acinar lumen, were 0.16, 0.13, 0.06, 0.05, 0.00 and 0.17, 0.16, 0.08, 0.05, 0.00(P<0.05,respectively). The expression of ZO-1 and Occludin in bladder (0.14 and 0.13) were higher than those in other tissues (0.11-0.12 and 0.09-0.10, P<0.05); the expression of ZO-1 and Occludin, which in proximal prostatic duct to the middle of prostatic duct and bottom of the ductal acinar lumen, had no significant difference (0.12-0.11 and 0.09-0.09, P>0.05). The IA values of CK20 were extremely low (0.00-0.01, P>0.05). Conclusion: Urothelial barriers partially exist in the prostatic ducts of elderly male SD rats, and with the prostatic ducts gradually extending to the bottom of acinar lumen, the urothelial barriers disappear. The results lay a foundation for further study on the repair of urinary epithelial barrier after prostatectomy.

Membrane Microdomains as Platform to Study Membrane-Associated Events During Oogenesis, Meiotic Maturation, and Fertilization in Xenopus laevis.

Studies on the egg plasma membrane-associated tyrosine kinase Src have shed light on the identity of the molecular machinery that is responsible for gamete interaction and possibly fusion in African clawed frog Xenopus laevis. Here we describe our protocol for identifying and analyzing molecular and cellular machinery that contributes to a variety of biological processes in the course of oogenesis, oocyte maturation, egg fertilization, and early embryogenesis in Xenopus. Our current special interest is to evaluate the hypothesis that the oocyte/egg membrane microdomain (MD)-associated uroplakin III-Src system is responsible for mediating sperm-egg membrane interaction/fusion signal to the oocyte/egg cytoplasm to initiate embryonic and zygotic development in this species. Therefore, this chapter contains a brief introduction to biology of oocytes and eggs in Xenopus and addresses the following questions: (1) What is oocyte/egg MD? (2) Why do we study oocyte/egg MD? (3) How to manipulate oocyte/egg MD? (4) What has been achieved by oocyte/egg MD studies? (5) What are the next steps in oocyte/egg MD studies?

Cyclic hydrostatic pressure promotes uroplakin expression in human urothelial cells through activation of ERK1/2 signaling.

OBJECTIVES: To investigate the effect of cyclic hydrostatic pressure on the expression of uroplakins and the role of extracellular regulated protein kinases 1/2 (ERK1/2) in the hydrostatic pressure-induced uroplakin expression of human urothelial cells (UCs). METHODS: Human UCs were seeded into a cell culture flask and subjected to cyclic hydrodynamic pressures for 24 h. Pressure parameters were set as follows: static, 100 cm H2O, 200 cm H2O and 300 cm H2O pressure. Real-time polymerase chain reaction (RT-PCR) and western blot were used to detect the expression of uroplakins. The role of the ERK1/2 was investigated using ERK1/2 inhibitor. RESULTS: Compared with the 0 cm H2O control group, 200 cm H2O hydrostatic pressure significantly increased the expression of uroplakins, however, 100 cm and 300 cm pressures could not promote uroplakin expression. Hence, ERK1/2 expression was also detected under 200 cm H2O hydrostatic pressure. Western blot showed that 200 cm H2O pressure promoted the expression of ERK1/2. ERK1/2 inhibitor decreased the pressure-induced ERK1/2 activivation and uroplakin expression. CONCLUSIONS: Cyclic hydrostatic pressure increases the expression of uroplakins via activating ERK1/2 signaling pathway in human UCs, and 200 cm H2O pressure may be an optimal stress parameter to promote the uroplakin expression.