ΔNp73 status in peritoneal and ovarian dissemination of appendicular adenocarcinoids (goblet cells).

INTRODUCTION: Goblet cell carcinoma (GCC) is an appendicular neoplasia representing less than 5% of all appendicular tumors, found in 0.3-0.9% of the appendectomies, 35-58% of all appendicular neoplasms, and less than 14% of malign appendix tumors. The most frequent clinical presentation is abdominal pain associated with a picture of acute appendicitis. MATERIALS AND METHODS: We present 3 clinical cases of appendix GCC, 2 subjected to cytoreductory surgery plus intraperitoneal hyperthermic chemotherapy and a third, who is currently receiving neoadjuvant treatment with a good response to chemotherapy and who will be offered the same treatment as the first two patients. Given the unpredictable behavior of these tumors, the use of molecular markers could help us to predict their behavior and prognosis. In this context, the TP73 gene would make an interesting putative marker. ∆Np73 has been described as overexpressed in a great variety of tumor types including colon cancer and this up-regulation is associated with a poor prognosis. To evidence its role in this malignancy, we evaluate here the status of ∆Np73 in the primary tumor and normal counterpart tissues, in the metastatic implants and in healthy areas of the peritoneum from the appendicular GCC patients. In addition, we checked the expression levels of this p73 variant in the tumor and normal tissue of 26 patients with colon cancer. RESULTS: Remarkably, 2 patients showed significant ∆Np73 down-regulation in both the primary tumor and the implants. Case 1 presented a fourfold decrease of levels in the primary tumor and 20-fold decrease in the implants. Case 2 showed a seven- and fourfold down-regulation in the primary tumor and implants, respectively. However, Case 3 showed an up-regulation of 53- and threefold in the primary tumor and implants, respectively. CONCLUSION: Goblet cell carcinoma of the appendix is very rate. It tends to seed throughout the peritoneum, making aggressive surgical cytoreduction and chemotherapy viable treatment options. Investigation into the molecular basis of these tumors may improve the diagnosis, prognosis and therapeutic decisions regarding these patients. ∆Np73 seems a good candidate for further analysis in longer series.

Goblet Cell Carcinoid/Carcinoma: An Update.

Goblet cell carcinoid (GCC) or goblet cell carcinoma is a unique mixed endocrine-exocrine neoplasm that is almost exclusively seen in the appendix. The hallmark of GCC is the concentric infiltration of the appendiceal wall by small tight clusters, nests or cords of tumor cells that exhibit a goblet cell morphology with a small compressed nucleus and conspicuous intracytoplasmic mucin. The coexistence of high-grade adenocarcinoma with GCC has been increasingly recognized as a common finding, which has been called adenocarcinoma ex GCC or mixed GCC-adenocarcinoma. A number of studies have shown that it is the high-grade adenocarcinomatous component that dictates the prognosis. Several histologic classification/grading systems have been proposed, which correlate with overall patient survival. Treatment options are primarily based on tumor stage and the presence or absence of a high-grade adenocarcinomatous component.

Goblet cell-produced retinoic acid suppresses CD86 expression and IL-12 production in bone marrow-derived cells.

Conjunctival goblet cell loss in ocular surface diseases is accompanied by increased number of interleukin-12 (IL-12)-producing antigen-presenting cells (APCs) and increased interferon-γ (IFN-γ) expression. This study tested the hypothesis that mouse conjunctival goblet cells produce biologically active retinoic acid (RA) that suppresses CD86 expression and IL-12 production by myeloid cells. We found that conditioned media from cultured conjunctival goblet cells (CjCM) suppressed stimulated CD86 expression, NF-κB p65 activation and IL-12 and IFN-γ production in unstimulated and lipopolysaccharide-stimulated cultured bone marrow-derived cells (BMDCs) containing a mixed population of APCs. Goblet cell-conditioned, ovalbumin-loaded APCs suppressed IFN-γ production and increased IL-13 production in co-cultured OTII cells. The goblet cell suppressive activity is due in part to their ability to synthesize RA from retinol. Conjunctival goblet cells had greater expression of aldehyde dehydrogenases Aldh1a1 and a3 and ALDEFLUOR activity than cornea epithelium lacking goblet cells. The conditioning activity was lost in goblet cells treated with an ALDH inhibitor, and a retinoid receptor alpha antagonist blocked the suppressive effects of CjCM on IL-12 production. Similar to RA, CjCM increased expression of suppressor of cytokine signaling 3 (SOCS3) in BMDCs. SOCS3 silencing reversed the IL-12-suppressive effects of CjCM. Our findings indicate that conjunctival goblet cells are capable of synthesizing RA from retinol secreted by the lacrimal gland into tears that can condition APCs. Evidence suggests goblet cell RA may function in maintaining conjunctival immune tolerance and loss of conjunctival goblet cells may contribute to increased Th1 priming in dry eye.

Mouse intestinal niche cells express a distinct α1,2-fucosylated glycan recognized by a lectin from Burkholderia cenocepacia.

In this study, we examined the distribution of fucosylated glycans in mouse intestines using a lectin, BC2LCN (N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia), as a probe. BC2LCN is specific for glycans with a terminal Fucα1,2Galß1,3-motif and it is a useful marker for discriminating the undifferentiated status of human induced/embryonic stem cells. Apparent BC2LCN reactivity was detected in the secretory granules of goblet cells in the ileum but not those in the colon. We also found distinctive reactivity in the crypt bottom, which is known as the stem cell zone, of the colon and the ileum. Other lectins for fucosylated glycans, including Ulex europaeus agglutinin-I, Pholiota squarrosa lectin and Aleuria aurantia lectin, did not exhibit similar reactivity in the crypt bottom. Remarkably, BC2LCN-positive epithelial cells could be labeled with a niche cell marker, c-Kit/CD117. Overall, our results indicate that intestinal niche cells express distinct fucosylated glycans recognized by BC2LCN. Increasing evidence suggests that the self-renewal and proliferation of stem cells depend on specific signals derived from niche cells. Our results highlight novel molecular properties of intestinal niche cells in terms of their glycosylation, which may help to understand the regulation of intestinal stem cells. The distinct expression of glycans may reflect the functional roles of niche cells. BC2LCN is a valuable tool for investigating the functional significance of protein glycosylation in stem cell regulation.

Penile Analogue of Stratified Mucin-Producing Intraepithelial Lesion of the Cervix: The First Described Case. A Diagnostic Pitfall.

The authors report a case where undifferentiated (classic) penile intraepithelial neoplasia was associated with the presence of goblet cells throughout the full epithelial thickness and which later progressed into an invasive carcinoma. The lesion evolved in three consecutive biopsies from only surface epithelium occupying numerous goblet cells in the first to variably sized solid nodules in the dermis composed of atypical squamous and/or basaloid cells intermixed with numerous goblet cells in the third biopsy. Both cellular components expressed CK7 and p16 protein. Human Papillomavirus (HPV) genotyping revealed high risk HPV type 16. To the best of our knowledge, this is the first description of such a lesion occurring on the penis, which can be considered the penile analogue of cervical stratified mucin-producing intraepithelial lesion (SMILE). The correct diagnosis was rendered retrospectively, after recognition of the existence of a vulvar lesion resembling cervical SMILE. The initial biopsy was misinterpreted as extramammary Paget disease, which also constitutes the main pitfall in the differential diagnosis. Another important differential diagnosis is penile/vulvar mucinous metaplasia. The finding of atypical squamous epithelial cells positive for p16 associated with mucinous cells present throughout the full epithelial thickness is a clue to the diagnosis of penile SMILE.

Endometrioid adenocarcinoma with simultaneous endocervical and intestinal-type mucinous differentiation: report of a rare phenomenon and the immunohistochemical profile.

Intestinal differentiation in the endometrium is rare with only case reports in the international literature. We describe a case of simultaneous endocervical and intestinal-type mucinous differentiation with goblet cells arising in a FIGO grade 1 endometrioid adenocarcinoma. The patient had no involvement of the myometrium, cervix, or extra-uterine sites. There were no intestinal metaplastic changes of the endocervical canal. The etiology of this change is unknown, although recent reports suggest an association with hyperestrogenism. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1209512176931698.

Distribution and morphology of Clara cells in common marmosets (Callithrix jacchus).

BACKGROUND: The objective of this investigation was to define the phenotype and spatial distribution of Clara cells within the respiratory tract of common marmosets and to distinguish them from other non-ciliated cells (goblet cells, mixed type secretory cells). METHODS: Non-ciliated cells were identified immunohistochemically using antibodies against Clara cell secretory protein and mucin 5AC. Transmission electron microscopy and scanning electron microscopy were performed to characterize Clara cells ultrastructurally. RESULTS: Clara cells were present throughout the tracheobronchial tree, with lowest numbers in the trachea and highest numbers in bronchioles. Goblet cells and mixed type cells were scarce in the upper conducting airways and virtually absent within bronchioles. Ultrastructurally, Clara cells showed typical apical electron-dense granules and a prominent granular endoplasmatic reticulum. CONCLUSIONS: Clara cells of common marmosets have species-specific morphological characteristics, which suggest grouping the common marmoset phenotypically between primates and rodents.

Mucin granule-associated proteins in human bronchial epithelial cells: the airway goblet cell "granulome".

BACKGROUND: Excess mucus in the airways leads to obstruction in diseases such as chronic bronchitis, asthma, and cystic fibrosis. Mucins, the highly glycosolated protein components of mucus, are stored in membrane-bound granules housed in the cytoplasm of airway epithelial "goblet" cells until they are secreted into the airway lumen via an exocytotic process. Precise mechanism(s) of mucin secretion, including the specific proteins involved in the process, have yet to be elucidated. Previously, we have shown that the Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein regulates mucin secretion by orchestrating translocation of mucin granules from the cytosol to the plasma membrane, where the granules dock, fuse and release their contents into the airway lumen. Associated with MARCKS in this process are chaperone (Heat Shock Protein 70 [HSP70], Cysteine string protein [CSP]) and cytoskeletal (actin, myosin) proteins. However, additional granule-associated proteins that may be involved in secretion have not yet been elucidated. METHODS: Here, we isolated mucin granules and granule membranes from primary cultures of well differentiated human bronchial epithelial cells utilizing a novel technique of immuno-isolation, based on the presence of the calcium activated chloride channel hCLCA1 (the human ortholog of murine Gob-5) on the granule membranes, and verified via Western blotting and co-immunoprecipitation that MARCKS, HSP70, CSP and hCLCA1 were present on the granule membranes and associated with each other. We then subjected the isolated granules/membranes to liquid chromatography mass spectrometry (LC-MS/MS) to identify other granule associated proteins. RESULTS: A number of additional cytoskeletal (e.g. Myosin Vc) and regulatory proteins (e.g. Protein phosphatase 4) associated with the granules and could play a role in secretion were discovered. This is the first description of the airway goblet cell "granulome."

Osteopontin expression and relation to disease severity in human asthma.

Recent studies have associated osteopontin (OPN) with allergic inflammation; however, its role in human asthma remains unclear. The aim of this study was to measure OPN levels in the serum, bronchoalveolar lavage fluid (BALF) and bronchial tissue of healthy controls and asthmatics, identify cellular sources of OPN and examine possible correlations between OPN expression, disease severity and airway remodelling. Serum samples were obtained from 35 mild-to-moderate asthmatics, 19 severe asthmatics and 17 healthy controls in the steady state and in cases of exacerbation. Of these subjects, 29 asthmatics and nine controls underwent bronchoscopy with endobronchial biopsy and BALF collection. OPN expression was determined by ELISA and immunohistochemistry/immunofluorescence. Reticular basement membrane thickness and goblet cell hyperplasia were also determined. Serum and BALF OPN levels were significantly increased in all asthmatics in the steady state, whereas serum levels decreased during exacerbations. OPN was upregulated in the bronchial tissue of all patients, and expressed by epithelial, airway and vascular smooth muscle cells, myofibroblasts, T-lymphocytes and mast cells. OPN expression correlated with reticular basement membrane thickness and was more prominent in subepithelial inflammatory cells in severe compared to mild-to-moderate asthma. OPN expression is upregulated in human asthma and associated with remodelling changes, and its subepithelial expression correlates with disease severity.

Ciliated muconodular papillary tumour of the lung: a newly defined low-grade malignant tumour.

We present two cases of ciliated muconodular papillary tumour (CMPT) in this report. CMPT is a newly defined low-grade malignant tumour with ciliated columnar epithelial cells, occurring in the peripheral lung. Both patients underwent pulmonary resection due to an enlarged solitary pulmonary nodule. Pathological findings in both cases confirmed a papillary tumour with a mixture of ciliated columnar and goblet cells. The tumours were rich in mucous and had spread along the alveolar walls, as observed in bronchioloalveolar carcinoma. Nuclear atypia was mild, and no mitotic activity was observed. Immunohistochemically, tumour cells stained positive for carcinoembryonic antigen, thyroid transcription factor-1 and cytokeratin 7 but not for cytokeratin 20. The immunohistochemical staining patterns were almost identical to those of pulmonary adenocarcinoma. We definitively diagnosed as CMPT. Both patients remained relapse-free.

Combined classical carcinoid and goblet cell carcinoid tumor: a new morphologic variant of carcinoid tumor of the appendix.

Carcinoid tumors are the most common neoplasms of the appendix. Histologically they have been categorized as classical, tubular, or goblet cell types. Goblet cell carcinoid has been regarded as a distinctive tumor type, not related to classic carcinoids, and to our knowledge combinations of these 2 tumor types have not been described in detail. In this report, we describe 5 cases of combined classical carcinoid and goblet cell carcinoid (GCC) tumors of the appendix. Four men and 1 woman, (mean age 53.4 y) presented with acute appendicitis (4 cases), whereas 1 presented with a pelvic mass owing to widespread pelvic disseminated disease. The tumors (0.6 to 6.0 cm) were located in the mid-portion and the tip of the appendix. Four patients were treated with right hemicolectomies (the patient with disseminated pelvic and ovarian metastases also had a pelvic exenteration), and 1 was treated with an appendectomy only. Four patients are alive and asymptomatic, whereas the patient with disseminated pelvic disease died 6 months after surgery. All 5 appendiceal tumors had microscopic features of both classical carcinoid and GCC, either intimately admixed or separate but closely apposed. The extent of the 2 components varied, with classical carcinoid representing 60% to 90% of the tumor. Both components stained for the general neuroendocrine markers, however, staining in the classic component was greater. The Mib-1 proliferation index varied from 1-15%, again with higher Mib-1 indices seen in the GCC component of all 5 cases. The pelvic soft tissue and ovarian metastases in case 4 consisted predominantly of a signet ring cell carcinoma with a minor component of goblet cells and was interpreted as an adenocarcinoma ex-GCC. In view of the fact that these combined carcinoid tumors appear to behave more as goblet cell carcinoids, detailed microscopic examination of classical carcinoid tumors of the appendix is suggested and larger series with longer follow-up is required to ascertain the true biologic potential of this unique form of combined carcinoid tumor of the appendix. The occurrence of both carcinoid types in the same appendices suggests a closer histogenetic relationship than previously believed, although the possibility that the 2 components represent separate, independent primaries ("collision tumors") can also be considered.

Confocal endomicroscopy for phenotypic diagnosis of gastric cancer.

BACKGROUND AND AIM: Relationships between mucin phenotype and malignant potential in gastric cancers have attracted attention. We attempted to assess the possibility of obtaining phenotypic diagnoses by confocal endomicroscopy. METHODS: Confocal images of target lesions were obtained in 29 of 40 patients with gastric cancer. Appearances of the brush border, goblet cells, and gastric foveolar epithelium were investigated with immunohistochemical staining using CD10, MUC2, and human gastric mucin to evaluate phenotypic expression in gastric carcinomas. Confocal images were compared with immunohistochemical findings for goblet cells and brush borders. RESULTS: Both the endoscopists and the pathologist obtained high accuracy rates for differential diagnosis. Sensitivity and specificity for goblet cells were 85.7% and 92.3% (Endoscopist A), and 85.7% and 88.5% (Endoscopist B). The kappa-value for correspondence between two endoscopists for the diagnosis of goblet cells in confocal images was 0.73. Sensitivity and specificity for the brush border were 93.8% and 91.7% (Endoscopist A), and 81.3% and 91.7% (Endoscopist B). The kappa-value for correspondence between two endoscopists for diagnosis of the brush border in confocal images was 0.79. Intestinal phenotypic gastric cancers show a brush border, goblet cells, or both. Sensitivity and specificity for the intestinal phenotype in confocal endomicroscopy were 90.9% and 77.8% (Endoscopist A), and 86.4% and 83.3% (Endoscopist B). CONCLUSION: The confocal endomicroscopic diagnosis of the mucin phenotype in gastric cancers was limited to intestinal and mixed phenotypes, but may be useful for the diagnosis of mucin phenotype and differential diagnosis.

Editorial: Defining a bad Barrett's segment: is it dependent on goblet cells?

The identification of patients with "bad Barrett's" who are at risk of neoplastic progression is a vital and pressing task. The view that specialized intestinal metaplasia with goblet cells is the predominant preneoplastic phenotype has had wide, but by no means universal, acceptance. An important caveat is presented in the thoughtful paper by Liu et al. in this issue. Of 68 patients with columnar metaplasia, 22 patients had no goblet cells identified and 46 had goblet cells in their biopsies. In both groups there were chromosomal and DNA content abnormalities and genetic instability. However, there were no significant differences between these cellular DNA abnormalities in the two groups. Both were significantly different from gastric controls. Thus, all metaplasia in the columnar-lined esophagus may already be on the path to cancer.

Metaplastic esophageal columnar epithelium without goblet cells shows DNA content abnormalities similar to goblet cell-containing epithelium.

OBJECTIVES: The mucosa of patients with columnar-lined esophagus recognized on endoscopy usually shows epithelium with and without goblet cells. Columnar epithelium with goblet cells ("Barrett's esophagus") is generally believed to represent a premalignant lesion and has been shown to contain DNA abnormalities. However, the biological properties of non-goblet columnar epithelium remain unknown. The purpose of this study was to determine the DNA content properties of non-goblet epithelium in patients with metaplastic columnar epithelium of the esophagus. METHODS: Mucosal biopsies of the esophagus from 68 patients with columnar metaplasia of the esophagus (22 without goblet cells and 46 with goblet cells) and 19 patients with normal gastric mucosa (controls) were histologically evaluated for the density of goblet cells. The latter group was divided into low-density, high-density, and very high-density goblet cell subgroups. Tissue sections of non-goblet epithelium and goblet cell epithelium (where present) were evaluated by image cytometry, and high-fidelity DNA histograms were created to indicate the G0/G1 peak DNA index (DI), DNA content heterogeneity index (HI), and the percentage of cells with DNA exceeding 5N (5N-EC). G0/G1 peaks with DI>1.1 were considered aneuploid. RESULTS: Normal gastric controls showed a mean peak DI of 1.02+/-0.03 and an HI of 11.6+/-0.7. None of the controls revealed aneuploidy or 5N-EC. Patients with metaplastic columnar epithelium with goblet cells showed a DI of 1.15+/-0.12, HI of 18.2+/-2.1, mild aneuploidy in 54% of the cases, and 5N-EC in 15% of the cases, all of which were significantly higher than in controls. Patients with metaplastic columnar epithelium without goblet cells showed DNA content results statistically similar to those of patients with metaplastic columnar epithelium with goblet cells, and also revealed significantly higher values compared with those of controls. Furthermore, there were no significant differences in any of the key DNA content abnormalities between non-goblet and goblet cell-containing epithelium in patients with metaplastic columnar epithelium with goblet cells, or between these two types of epithelium according to the density of goblet cells. CONCLUSIONS: DNA content abnormalities occur with equal frequency and extent in metaplastic columnar epithelium of the esophagus without goblet cells compared with metaplastic columnar epithelium with goblet cells. These findings suggest that metaplastic non-goblet columnar epithelium of the esophagus may have neoplastic potential.

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae).

Glycoproteins (GPs) were visualised histochemically in the secretory cells - the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.

Asthma induction in mice leads to appearance of alpha2-3- and alpha2-6-linked sialic acid residues in respiratory goblet-like cells.

Allergic asthmatic inflammation in mice was induced by sensitization with ovalbumin and lipopolysaccharide from Escherichia coli and visualized in the airways of asthmatic mice by spatial and temporal changes of carbohydrates containing sialic acid residues. Immunohistochemistry was used to demonstrate binding of lectins and antibodies that detect alpha2-3- and alpha2-6-linked sialic acid residues. After sensitization and challenge, the histology of the lung changed markedly, and goblet-like cells appeared, most likely caused by Clara cell metaplasia. Normal Clara cells showed no reaction after incubation with the sialic acid detecting agents, while the goblet-like cells expressed both alpha2-3- and alpha2-6-linked sialic acid residues in the asthmatic animals. The lectins but not the antibodies reacted with intestinal goblet cells. Instead, an antibody recognizing a disialoganglioside, stained large mononuclear cells in the submucosa, indicating a difference in sialylation between goblet cells in the intestine and goblet-like cells developed from Clara cells.

Distribution of mucins and antimicrobial substances lysozyme and lactoferrin in the laryngeal subglottic region.

The subglottic region of the larynx is of high clinical relevance with regard to infections and malignancies. Little is known about the distribution of mucins and antimicrobial substances in this area. In this study, we have investigated the mucin distribution in the normal subglottis of the larynx. Moreover, we analysed the expression of lysozyme and lactoferrin in this area. Therefore, the subglottic region of 34 larynges was investigated immunohistochemically with different antibodies to mucins and antimicrobial substances. The epithelium reacted positive with antibodies to mucins MUC1 (34/34), 5AC (26/34), 5B (10/34), 7 (8/34), 8 (10/34) and 16 (19/34); submucosal glands were positive to mucins MUC1 (34/34), 5B (10/34), 7 (8/34), and 16 (19/34); high columnar epithelial cells and serous parts of subepithelial seromucous glands were also positive for lysozyme (34/34) and lactoferrin (34/34). The results show that human subglottic epithelium and subepithelial submucosal glands produce a broad spectrum of mucins that is almost comparable with that in other areas of the respiratory tract. We hypothesize that the mucin diversity of the subglottis has an impact on positive functional consequences during vocal production and antimicrobial defence. This antimicrobial defence is supported by synthesis and secretion of antimicrobial substances such as lysozyme and lactoferrin. Moreover, knowledge of the observed distribution pattern of mucins in the subglottis can be a useful tool for a classification of subglottic laryngeal carcinomas.

Differences in the expression of mucins in various forms of cystitis glandularis.

A wide spectrum of glandular epithelial metaplastic changes may be seen in the bladder. Cystitis glandularis (CG) is a well-known metaplastic lesion occurring in the presence of chronic inflammation, but there are a few data about mucin expression in its two subtypes (typical and intestinal). The purpose of the present study was to determine the expression of mucin core proteins and CD10 in the different types of CG. For this examination, we used a panel of monoclonal-specific antibodies for MUC1, MUC2, MUC5AC, and MUC6. CG of the intestinal type expressed MUC5AC both in goblet and columnar cells, and strongly expressed intestinal mucin MUC2 only in goblet cells in all cases. There was no expression of MUC1, MUC6, and CD10 in the metaplastic cells. CG of the typical type showed an expression of MUC1 similar to normal urothelium, but the CD10 expression was more intensive than in the control. The mucin expression profile in the different types of CG allows the identification of "gastric mucin" (MUC5AC) together with intestinal mucin (MUC2), while typical CG (CGTP) retains MUC1. Different and contrasting immunoprofiles were evident in various forms of CG. The absence of CD 10 in CG of the intestinal type is a finding that points towards an incomplete form of urinary bladder metaplasia.