Ex vivo construction of a novel model of bioengineered bladder mucosa: A preliminary study.

OBJECTIVE: To generate and to evaluate ex vivo a novel model of bioengineered human bladder mucosa based on fibrin-agarose biomaterials. METHODS: We first established primary cultures of stromal and epithelial cells from small biopsies of the human bladder using enzymatic digestion and selective cell culture media. Then, a bioengineered substitute of the bladder lamina propria was generated using cultured stromal cells and fibrin-agarose scaffolds, and the epithelial cells were then subcultured on top to generate a complete bladder mucosa substitute. Evaluation of this substitute was carried out by cell viability and histological analyses, immunohistochemistry for key epithelial markers and transmission electron microscopy. RESULTS: The results show a well-configured stroma substitute with a single-layer epithelium on top. This substitute was equivalent to the control bladder mucosa. After 7 days of ex vivo development, the epithelial layer expressed pancytokeratin, and cytokeratins CK7, CK8 and CK13, as well as filaggrin and ZO-2, with negative expression of CK4 and uroplakin III. A reduction of the expression of CK8, filaggrin and ZO-2 was found at day 14 of development. An immature basement membrane was detected at the transition between the epithelium and the lamina propria, with the presence of epithelial hemidesmosomes, interdigitations and immature desmosomes. CONCLUSIONS: The present results suggest that this model of bioengineered human bladder mucosa shared structural and functional similarities with the native bladder mucosa, although the epithelial cells were not fully differentiated ex vivo. We hypothesize that this bladder mucosa substitute could have potential clinical usefulness after in vivo implantation.

Localized amyloidosis of the vulva with and without vulvar intraepithelial neoplasia: report of a series.

Localized primary cutaneous amyloidosis is uncommon in Europe and North America and is infrequently reported in the English literature. The constituents of such deposits have not been previously examined; this series characterizes amyloid deposits in localized vulvar amyloidosis and their association with vulvar intraepithelial neoplasia. All biopsies and excisions of vulva over 18 months were reviewed. Cases with suspected amyloidosis were retrieved after institutional review board approval. Twenty cases mimicking amyloidosis were selected as controls. All study and control cases were stained with Congo red. Four Congo red-positive study cases were studied by liquid chromatography-tandem mass spectrometry. Of 27 Congo red-positive study cases, 25 were then examined by immunohistochemical stains with antibodies to cytokeratin 5 (CK5) and cytokeratin 14 (CK14). Of 149 cases reviewed, 26 localized and 1 systemic vulvar amyloidosis were identified. Liquid chromatography-tandem mass spectrometry analysis of the deposits revealed unique peptide profile consistent with CK5 and CK14. Immunohistochemical staining with antibodies to CK5 and CK14 also detected these components in the deposits. The vulvar deposit of systemic amyloidosis consisted of amyloid light chain (λ)-type amyloid deposit. All control cases were negative for Congo red. Keratin-associated amyloid materials (CK5 and CK14) were found to be unique in localized vulvar amyloidosis. Leakage of keratins from the basal layer of the epithelium into the superficial dermis may have been the possible source of the deposits. It appears to be associated with both high-grade and low-grade vulvar intraepithelial neoplasias and, rarely, lichen sclerosus, seborrheic keratosis, and benign vulvar skin.

Keratin 17 in premalignant and malignant squamous lesions of the cervix: proteomic discovery and immunohistochemical validation as a diagnostic and prognostic biomarker.

Most previously described immunohistochemical markers of cervical high-grade squamous intraepithelial lesion (HSIL) and squamous cell carcinoma may help to improve diagnostic accuracy but have a minimal prognostic value. The goals of the current study were to identify and validate novel candidate biomarkers that could potentially improve diagnostic and prognostic accuracy for cervical HSIL and squamous cell carcinoma. Microdissected tissue sections from formalin-fixed paraffin-embedded normal ectocervical squamous mucosa, low-grade squamous intraepithelial lesion (LSIL), HSIL and squamous cell carcinoma sections were analyzed by mass spectrometry-based shotgun proteomics for biomarker discovery. The diagnostic specificity of candidate biomarkers was subsequently evaluated by immunohistochemical analysis of tissue microarrays. Among 1750 proteins identified by proteomic analyses, keratin 4 (KRT4) and keratin 17 (KRT17) showed reciprocal patterns of expression in the spectrum of cases ranging from normal ectocervical squamous mucosa to squamous cell carcinoma. Immunohistochemical studies confirmed that KRT4 expression was significantly decreased in squamous cell carcinoma compared with the other diagnostic categories. By contrast, KRT17 expression was significantly increased in HSIL and squamous cell carcinoma compared with normal ectocervical squamous mucosa and LSIL. KRT17 was also highly expressed in immature squamous metaplasia and in endocervical reserve cells but was generally not detected in mature squamous metaplasia. Furthermore, high levels of KRT17 expression were significantly associated with poor survival of squamous cell carcinoma patients (Hazard ratio=14.76, P=0.01). In summary, both KRT4 and KRT17 expressions are related to the histopathology of the cervical squamous mucosa; KRT17 is highly overexpressed in immature squamous metaplasia, in HSIL, and in squamous cell carcinoma and the level of KRT17 in squamous cell carcinoma may help to identify patients who are at greatest risk for cervical cancer mortality.

Reduced expression of cytokeratin 4 and 13 is a valuable marker for histologic grading of esophageal squamous intraepithelial neoplasia.

Histologic evaluation of low-grade or high-grade intraepithelial neoplasia (LG-IN or HG-IN) of the esophagus is important for estimating the risk of progression to invasive carcinoma. Discrimination between LG-IN and HG-IN, or neoplasia and non-neoplastic lesion (NNL), however, is occasionally difficult. This study was designed to evaluate whether cytokeratin expression can be used for discrimination of these lesions. Esophageal Iodine-unstained lesions (n=154), less than 10 mm, were classified into HG-IN, LG-IN, and NNL. These lesions together with 154 foci of normal esophageal epithelium (NEE) were examined by immunohistochemistry for cytokeratins (CK4 and CK13), p53 overexpression, and the MIB-1 labeling index. The ratios of CK4- and CK13-positive staining were scored from 1 to 3. The CK4- and CK13-positive staining ratios were decreased in NNL (73% and 78%), LG-IN (55% and 69%), and HG-IN (33% and 48%), compared to NEE (91% and 95%). The differences between LG-IN and HG-IN, neoplasia and NNL, and among these three lesions and NEE were statistically significant (p < 0.005). The cytokeratin scores correlated with the MIB-1 labeling index (both: p < 0.0001), but not with p53 overexpression. CK4 and CK13 immunohistochemistry could be an objective method for evaluating the risk for progression to invasive carcinoma.