Normal Human Urothelium Research Articles

Serum-free cell culture of normal human urothelium

A simple and effective method is described for obtaining epithelial cell cultures from normal human urothelium (bladder or ureter epithelium) using a new serum-free medium, HMRI-2. Tissue explanted in HMRI-2 exclusively produced epithelial outgrowths that could be subcultured once or twice and cryopreserved in liquid nitrogen in the complete absence of serum.

EPIDERMAL-GROWTH-FACTOR RECEPTORS IN HUMAN BLADDER CANCER: COMPARISON OF INVASIVE AND SUPERFICIAL TUMOURS

The presence of epidermal-growth-factor (EGF) receptors in normal and neoplastic human urothelium was studied in 12 control patients and in 48 patients with transitional cell carcinoma of the bladder, 24 with invasive (pT3) and 24 with superficial tumours (9 pT1, 15 pTa). EGF receptors were identified on frozen sections by means of an indirect immunoperoxidase technique with a monoclonal antibody against the EGF receptor. Significantly more invasive tumours (21 of 24) than superficial (7 of 24) were stained positively for the EGF receptor (χ 2=14·49; p<0·001). Significantly more poorly differentiated tumours (18 of 21) than moderately differentiated tumours (10 of 27) were EGF-receptor positive (χ 2=9·6; p<0·01). No control sample stained positively for the EGF receptor. These findings suggest that the presence of a high intensity of staining for the EGF receptor in human bladder tumours is associated with poor differentiation and with invasion.

A modified immunoperoxidase method for the detection of ABH tissue isoantigens in patients with bladder carcinoma

Detectable ABH blood group antigens (BGAg) on tumor cells from transitional carcinoma of the urinary bladder, used for diagnosis and prognosis may be evidenced with the existing immunoperoxidase methods. In this study, a modification of the immunoperoxidase method was shown to enable the detection of BGAg in deparaffinized tissue section of normal and malignant human urothelium. To detect antigens A and B a peroxidase-conjugated rabbit anti-human IgM was used and antigen H was visualized by biotinylated lectin.

Immunohistochemical Demonstration of Blood Group Antigens in Neoplastic and Normal Human Urothelium: A Comparison with Standard Red Cell Adherence

The specific red cell adherence test as a method to detect blood group antigen deletion in urothelial malignancy has been reported to yield approximately 40 per cent false negative results in 0 blood group patients. Our study of multiple sections of 8 normal ureters from blood group 0 patients and more than 220 specimens of transitional cell cancer taken from 48 patients reveals that the immunoperoxidase technique is more specific than the specific red cell adherence method in predicting subsequent invasion in blood group O(H) patients presenting with superficial transitional cell carcinomas (71 compared to 29 per cent) but is no more specific for tumors containing A or B antigens. However, immunoperoxidase staining does improve discernment of underlying histologic detail and, thereby, facilitates recognition of false positive antigen testing associated with squamous and adenomatous metaplasia. Areas of squamous and adenomatous metaplasia in specimens we tested were frequently antigen positive in invasive tumors. Therefore, we believe that these areas must be disregarded in determining antigen deletion in transitional cell carcinomas.

Growth and characterization of normal human urothelium in vitro.

A method for initiating rapidly growing cultures of normal human transitional cells from ureter and embryonic bladder specimens has been developed and quantified. A new microdissection technique was used to nonenzymatically separate the urothelium. The use of enriched medium containing 10 micrograms/ml insulin, 5 micrograms/ml transferrin, and 1 microgram/ml hydrocortisone resulted in improved growth. The use of thin collagen gel substrates (0.6 ml/60 mm petri dish) resulted in 97% attachment of explants compared to 77% attachment on plastic. Explants grown on thicker collagen (2 ml/60 mm petri dish) showed, in addition to better attachment, enhanced growth of cells as determined both by measurements of colony size and cell density. Cultures of transitional cells that were initiated using explants could be passed three to five times using 0.1% EDTA for dispersion. Autoradiography of [3H]thymidine-labeled cells showed an initial phase of rapid cell division in primary explant cultures and restimulation of cell division in passaged cultures. Transmission electron microscopy showed that the cells growing out from the explants were continuous with the stratified urothelium maintained in the original explant. Stratification of transitional cells occurred in cultures of both ureter and embryonic bladder cells. Surface cells were joined near their apices by junctional complexes. Desmosomes and Golgi vesicles were present in all cells. Passage in culture did not alter the morphological characteristics of cells.

Immunological characterization of cell lines established from malignant and normal human urothelium

Cell lines from normal or malignant human urothelium, described elsewhere by morphological, cell kinetic and genotypical criteria, have been characterized further by their response to antibody or lymphocyte mediated cytotoxicity in vitro. Four groups of cell lines were included: (1) two fast proliferating cell lines from normal urothelium, (2) three fast proliferating cell lines from transitional cell carcinoma (TCC) together with two transformed sublines of normal derived cell lines, (3) six slowly proliferating TCC lines, and (4) a line from squamous cell carcinoma. HLA antigen expression was demonstrated in the cell lines of groups 1 and 3, but not in lines from group 2 or 4. The sensitivity to spontaneous lymphocyte mediated cytotoxicity (SLMC) of cells in group 1 and 2 exceeded that of cells from group 3 by a factor of 8. The TCC line, HU 456, was more susceptible to SLMC than T 24. Specifically increased cytotoxicity of lymphocytes from patients suffering from interstitial cystitis (IC) against HU 609 from normal urothelium indicated that this line expresses tissue specific antigens, and at the same time that an immune reaction may be part of the pathogenesis of IC. By a sensitive test for antibody-dependent, cell-mediated cytotoxicity (ADCC) antibody activity against HU 456 was found in only one of nine TCC patients and none of five clinical controls. A pronounced non-disease-related blocking serum activity, frequently found, may account for some of the negative findings. The SLMC against HU 456 could be inhibited but not abolished by Fab fragments of rabbit anti-human immunoglobulin, indicating an ADCC mechanism as part of the SLMC.

Scanning electron microscopy in the study of normal, inflamed and neoplastic human urothelium.

Seventy-two samples of human urothelium have been examined by scanning electron microscopy (SEM) and the appearances compared with those of conventional light microscopy. Six main SEM cell types were recognised. None was diagnostic of malignancy; in particular, the presence of surface pleomorphic microvilli was not pathognomonic. However, the relation of the SEM appearance to malignancy and the accepted histological grades and clinical stages of malignancy was such as to warrant further examination.

Titration of natural and disease-related cytotoxicity of lymphocytes from bladder cancer patients

A controlled investigation of lymphocyte-mediated cytotoxicity has been carried out in patients with transitional cell carcinoma (TCC) of the urinary bladder, three long-term established cell lines with comparable sensitivities to the natural cytotoxicity (NC) being used as targets, namely HU 456 derived from human TCC, HU 609 from normal human urothelium, and SAOS 2 from a human osteosarcoma. The 44-h incubation microcytotoxicity assay (MA) was used with ‘blinded’ semiautomatic visual counting of target cells. The cytotoxicity was determined by titration with a serial dilution of lymphocyte suspensions 1:2, five different concentrations being used in each experiment. Fifty tests were performed with lymphocytes from 48 TCC patients. As controls, 65 patients with diseases other than TCC were tested simultaneously. The average cytotoxicity of lymphocytes from TCC patients against HU 456 was only slightly and insignificantly higher than that of the control patients. However, a marked decrease of the NC against the control cells HU 609 and SAOS 2 was noted with TCC patient lymphocytes. After correction of the data for the depression of background NC, increased tumor-specific cytotoxicity (TSC) and tumor type-specific cytotoxicity (TTSC) were demonstrated in TCC patients with noninvasive malignant tumors (grade 2 or 3), whereas no increased specific cytotoxicity was found in TCC patients with invasive grade 2 or 3 tumors. Neither was any increased specific cytotoxicity found in patients with very well-differentiated grade tumors. The presence of specific reactivity in patients with noninvasive TCC in contrast to invasive TCC is supposed to indicate growth-controlling function of the cellular immune reaction. Tests were also performed with lymphocytes from 35 patients in the postoperative phase without any history of TCC. Surprisingly, this group revealed a significantly elevated TSC but not increased TTSC when compared with the 65 untreated control patients, thus indicating that serious reservations must be made in the interpretation of the cytotoxocity assay.

Scanning electron microscopy of normal and malignant human urothelium

Scanning electron microscopy (SEM) was used to evaluate and compare normal bladder mucosa to tumor epithelium as well as to normal-appearing mucosa in patients with diagnosed bladder tumor. Bladder urothelium adjacent to tumors exhibits a characteristic pattern of fine structural relief which is distinct from normal uroepithelium. These changes in microcontour may be guides to the identification of early malignancy, and thus the technique will be helpful in deciding how aggressive the treatment of bladder tumor should be.