Human urinary bladder carcinoma glycoconjugates expressing T-(Gal beta(1-3)GalNAc alpha 1-O-R) and T-like antigens: a comparative study using peanut agglutinin and poly- and monoclonal antibodies.

T- and T-like antigens on glycoproteins and glycolipids were examined in extracts of human urinary bladder tumors and normal tissue by Western blot analysis and reagent binding to thin layer chromatograms. Three different anti-T-reagents were used: peanut (Arachis hypogaea) lectin (PNA) and mono- and polyclonal antibodies specific for T-antigen (Gal beta(1-3)GalNAc alpha 1-O-R). Immunodetection with the T-specific reagents in nitrocellulose replicas of bladder tumor glycoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated tumor-specific T-antigen-bearing glycoproteins compared to normal urothelial glycoproteins. In addition, a remarkable difference in binding was found between the immunological reagents and PNA lectin. PNA showed major reactivity to a 28-kD glycoprotein extracted from tumors. Monoclonal anti-T-antibody (clone HH8) showed major reactivity with an M(r) 34,000 glycoprotein, and polyclonal anti-T-antibody showed major reactivity with an M(r) 36,000 glycoprotein. PNA agarose column affinity-purified tumor glycoproteins did not bind the antibodies. Glycoproteins, M(r) 28,000 and 34,000, were shown to be O-linked by stepwise deglycosylation. In solid phase monosaccharide inhibition tests, galactose followed by N-acetyl-galactosamine were the most potent monosaccharides inhibiting binding to immobilized bladder tumor glycoproteins. None of the anti-T-reagents reacted with glycolipids extracted from tumor tissue. It is concluded that PNA lectin, in addition to the T-disaccharide, reacts with other protein-anchored carbohydrate structures in carcinomas.

Lectinohistochemistry of human bladder cancer: loss of lectin binding structures in invasive carcinomas.

With the purpose of studying changes in the expression of glycoconjugate structures in urothelium, nine different lectins (PNA, WGA, VFA, GSA II, STA, UEA I, LCA, DBA and HPA) with specificity for mono- or oligo-saccharides were used on formalin-fixed, paraffin-embedded tissue sections from 47 patients who had undergone surgical resection for bladder tumors and on normal urothelial biopsies from 10 patients. The tumors were graded and a lectinohistochemical method using biotinylated lectins and avidin-biotin-peroxidase complex was used to demonstrate the lectin binding. Positive staining reactions of cells in cytoplasm and on membranes were evaluated in the basal, the intermediate, and the luminal cell layers, respectively. In both normal and atypical urothelium lectin binding predominated in the luminal cell layer and decreased towards the basal cell layer. In normal urothelium all lectins stained greater than 66% of the cells in the luminal cell layer in cytoplasm and between 5 and 100% of the cells on membranes depending on the lectin used. A gradual loss of lectin-binding structures was seen with increasing grade of atypia. The range of this decrease varied considerably from one lectin to another, but it was consistently found that the percentage of cells stained in cytoplasm and on membranes decreased. A significantly lower percentage of cells stained in cytoplasm was found in invasive tumor cell-islands compared to normal urothelium. In invasive tumor cell-islands staining of cells on membranes was completely absent, except for HPA lectin that stained less than 10% of the cells. In conclusion, we demonstrate a dramatic decrease in lectin-binding carbohydrate structures associated with urothelial malignant progression.

Cellular localization of PNA binding in colorectal adenomas: comparison with differentiation, nuclear:cell height ratio and effect of desialylation.

The lectin Arachis Hypogaea (Peanut Agglutinin, PNA) was used to study the cellular localization of the Thomsen-Friedenreich (T) disaccharide Gal-beta (1-3)-GalNAc alpha 1-R in 22 formalin-fixed paraplast-embedded colorectal adenomas of varying cellular dysplasia. An indirect immunoperoxidase method was used prior to and after neuraminidase treatment. Detailed information on the cellular localization of PNA binding was obtained. In addition, morphometric measurements of the nuclear: cell height ratios were performed on staining-filtered micrographs of crypts from all adenomas. We found 1) a statistically significant increase in the nuclear:cell height ratio with increasing grade of dedifferentiation (p less than 0.003), 2) a statistically significant smaller nuclear:cell height ratio in crypts that were PNA-positive in the Golgi region when these were compared to crypts that were PNA-positive on luminal cell membranes, 3) a decreasing number of crypts expressing PNA binding sites in the Golgi region with increasing dedifferentiation, leading to complete absence of PNA binding sites in Grade IV adenomas, 4) neuraminidase pretreatment increased the number of crypts expressing PNA binding sites in cytoplasm and on luminal membranes, whereas no changes were detected in crypts expressing PNA binding sites in the Golgi region. Our results confirm the general concept of accumulation of precursors of carbohydrate antigens in dedifferentiated cells. On the basis of the results presented, we conclude that the nuclear:cell height ratio shows a good correlation with the cellular localization of PNA binding, cellular differentiation and classic pathologic grading.

[Treatment and socioeconomic consequences of skiing injuries in the County of Vejle during the 1986/1987 season]. / Behandling og sociøkonomiske konsekvenser af skiskader i Vejle Amt saesonen 1986/1987.

In a prospective study conducted in the County of Vejle in the skiing-season 1986/1987, the hospitals were contacted by 129 persons who had skiing-injuries. Forty-one patients were primarily treated abroad and 35 patients were primarily examined or treated in Denmark after their return. Fifty-three patients who were injured in Denmark were primarily examined or treated in the casualty ward. The injuries the most often seen were knee and thumb-injuries. Twenty-three operations were undertaken, 15 on knees, and five on thumbs. A total of 139 bed-days were registered, 114 for injuries abroad and 25 for Danish injuries. Fifty-nine injured persons lost 1,481 days on account of sickness. The yearly nationwide costs for the health services in connection with skiing-injuries are calculated to be 9.2 million kroner (approximately 760,000 pounds). The expenses for loss from production are calculated to be 8.6 million kroner (approximately 716,000 pounds).

T-antigens in primary non-invasive and superficially invasive human urinary bladder tumors: the correlation to tumor recurrence and tumor progression. A mini-review.

As cellular carbohydrate structures are involved in multiple cellular functions, alterations in these structures have been studied in an effort to find markers and predictors of the clinical course of disease in human cancers. Special interest has been given to recurrence and progression of cancer disease. At present no such marker or predictor exists for the prediction of recurrence and progression of initially non-invasive (Ta) or superficially invasive (T1) human urinary bladder tumors. Over the years the T- (Thomsen-Friedenreich) antigen (the disaccharide Gal beta(1-3)GalNAc alpha(1-O)-R) has been investigated for its possible role as such a marker. An overview is given. It is concluded that there is a general, but not an individual correlation between T-antigen expression and recurrence or progression of initially non- or superficially invasive human bladder tumors.

A comparative study of peanut agglutinin and amaranthin binding to human urinary bladder tumor glycoproteins.

The reactivity of two T-antigen specific lectins, Amaranthin (ACA) and Peanut Agglutinin (PNA), was compared by immunohistochemical staining of serial sections of human bladder tumors and by Western Blot analysis of glycoproteins extracted from human bladder tumors prior to and after sialic acid removal. In low grade tumors ACA reacted prior to neuraminidase treatment. PNA on the other hand only showed positive reaction in these tumors after neuraminidase treatment. The two lectins showed identical staining patterns after neuraminidase treatment in tumors. In Western blottings both lectins detected a 28 kD glycoprotein. PNA also reacted with several other bands after neuraminidase treatment. The reactivity of ACA was not enhanced after neuraminidase treatment. In monosaccharide inhibition tests ACA- and PNA-binding to different T-antigens was most efficiently inhibited by GalNAc and Gal respectively. PNA binding was also inhibited by Glc, GlcNAc and GalNAc at higher concentrations, while the binding of ACA was only scarcely affected by Glc, GlcNAc and Gal.

DNA replication in experimental rat bladder tumours: immunohistochemical detection of bromodeoxyuridine labelled nuclei.

With the purpose of studying DNA replication activity and location in normal and malignant tissue, bladder tumours were induced in ten rats by the intravesical installation of N-Nitroso-N-Methyl-Urea. Ten rats were installed with the NMU solvent and served as controls. The animals were injected intraperitoneally with 1 mg bromodeoxyuridine/100 g weight one hour prior to sacrifice. An indirect immunoperoxidase method using mouse monoclonal anti-bromodeoxy-uridine antibody was used on tissue sections of normal urothelium and bladder tumours to visualize labelled nuclei. Labelling indices were calculated and correlated to the grade of atypia. We found that normal urothelium had very little proliferative activity. The labelling index of tumours increased with increasing grade of atypia, being highest in invasive tumours. Only cells located to the basal cell layer were labelled in normal and grade I urothelium. In grade II and III tumours labelled cells were found in the intermediate cell layer as well as in the basal cell layer. Areas of squamous metaplasia showed high labelling indices, however the proliferative cells were restricted to the basal cell layer. The bromodeoxyuridine labelling index thus increases with increasing grade of atypia.

Subcutaneous bleeding: first sign of prostate cancer.

We present here a case of subcutaneous bleeding being the first recognized sign of prostate cancer.

Lewis antigen expression in benign and malignant tissues from RBC Le(a-b-) cancer patients.

Eight red blood cell (RBC) Le(a-b-) individuals were selected from a series of patients with bladder or colon cancer. Defined by the presence or absence of alpha 1-4-L-fucosyltransferase activity in saliva, four of these patients were characterized as non-genuine Lewis negative [RBC Le(a-b-) with alpha 1-4-L-fucosyltransferase activity in saliva], and four as genuine Lewis negative [RBC Le(a-b-) with no alpha 1-4-L-fucosyltransferase activity in saliva]. Stainings of paraffin embedded formalin fixed tissue sections for Lea and Leb antigens were performed by means of an indirect immunohistochemical method on all malignant and benign tissue previously removed from these eight patients. Leb antigens were always expressed independently of both the Lewis and the secretor status of the individual. Lea antigens, on the other hand, showed a different staining pattern. Although primarily expressed in non-genuine Le(a-b-) individuals, Lea antigens were expressed in genuine Le(a-b-) individuals as well--to a limited extent, but still detectable. Thus, these findings seem to show that the Lewis antigen expression is tissue dependent, and it is not possible to predict tissue Lewis antigen expression by merely examining erythrocytes or saliva.

Binding of wheat and peanut lectins to human transitional cell carcinomas. Correlation with histopathologic grade, invasion, and DNA ploidy.

The binding of peanut (PNA) and wheat germ (WGA) lectins to tissue sections was examined in biopsy specimens from normal urothelium (ten patients) and from tumor tissue of noninvasive (17 patients) and invasive bladder (31 patients) carcinomas. The results were correlated to DNA content, histopathologic grade, and the presence or absence of invasion. Significant alterations in lectin binding associated with the development of cancer were found. A gradual loss of both PNA and WGA binding was found to correlate with higher grades of atypia (P less than 0.001). The loss of WGA binding was significantly correlated with both tumor aneuploidy (P less than 0.001) and the presence of invasion (P less than 0.05), whereas no significant correlation was found between loss of PNA binding and these variables. We concluded that the loss of WGA binding structures associated with bladder cancer shows a better correlation with known risk factors (aneuploidy and invasion) than the loss of PNA binding does.

O-linked mucin-type glycoproteins in normal and malignant colon mucosa: lack of T-antigen expression and accumulation of Tn and sialosyl-Tn antigens in carcinomas.

The expression of carbohydrate core-structures on O-linked glycoproteins was examined in fetal (n = 6), infantile (n = 2), normal adult (n = 15), and malignant (n = 22) colorectal tissue by means of monoclonal antibodies (MAbs) specific for Tn (GalNAc alpha 1-O-R), sialosyl-Tn (NeuAc alpha 2-6GalNAc alpha 1-O-R), and T (Gal beta 1-3GalNAc alpha 1-O-R) antigens. Immunolabelling of solubilized malignant tissue, separated by SDS-PAGE, showed expression of Tn and sialosyl-Tn antigens on 3 molecules of similar mw (230, 210, and 170 kDa), whereas no T antigens could be detected. Immunohistochemical techniques showed that fetal colon mucosa expressed Tn antigens but no sialosyl-Tn antigens. Infantile colon mucosa, however, expressed Tn as well as sialosyl-Tn antigens, and normal adult colon mucosa cells expressed no Tn antigens but sialosyl-Tn in 2 out of 6 biopsies from cecum, which indicates occurrence post partum of alpha-6-NeuAc-transferase. Endothelium in normal adult mucosa showed expression of both Tn and sialosyl-Tn antigens; 82% of carcinoma tissue sections expressed Tn antigens, and 73% expressed sialosyl-Tn antigens in mucin or cytoplasm, or on luminal cell membranes. T antigens could be detected neither in normal mucosa cells at any stage of development, nor in carcinomas. The possibility exists that this could be due to masking of T antigen. Mucin-type blood-group A antigens which contain an internal T-disaccharide were demonstrated in 4 out of 4 A1 tumors by means of MAb HH5. However, urea-containing SDS-PAGE analysis demonstrated an HH5 binding to molecules different from those binding anti-Tn. In remote morphologically normal and abnormal crypts in colons from carcinoma patients, both Tn and sialosyl-Tn antigens were expressed in secreted mucin in 40% of the cases. The data indicate an expression of O-linked Tn and sialosyl-Tn core structures in fetal and infantile colon and in colorectal carcinomas.

Surgical repair of vesicovaginal fistulae--a ten-year retrospective study.

OBJECTIVE: Vesicovaginal fistulae in the western world generally occur as complications to pelvic surgery or radiation therapy of pelvic cancers. We have reviewed our results of vesicovaginal fistula closure procedures over a 10-year period. PATIENTS AND METHODS: From 1985 to 1996, 55 patients were referred to our department due to vesicovaginal fistulae. Five patients had fistulae due to malignant recurrence and one patient was considered inoperable. Thus, 49 patients were operated on. Thirty patients had fistulae resulting from pelvic surgery. Nineteen of the 25 patients admitted with fistulae secondary to radiation therapy of pelvic cancers were operated on. RESULTS: Of the 30 patients with postoperative fistulae, 23 had an abdominal repair and 7 a vaginal repair. A success rate of 90% was achieved after a first closure procedure, as 3 patients within a month experienced a recurrence. These three recurrences were all successfully closed in a second operation, augmenting the success rate to 100% in this group of patients. In the group of patients with fistulae caused by irradiation, a urinary diversion was performed in 12 patients, and in 7 patients a primary attempt to close the fistula was made, either by an abdominal approach (2 patients) or by a vaginal approach (5 patients). The fistula recurred in 6 of these 7 patients. Despite several additional attempts to close the recurrent fistulae, only one patient was successfully operated on. CONCLUSION: It seems that vesicovaginal fistulae resulting from pelvic surgery, in our hands, can be managed successfully either by an abdominal or vaginal approach. For patients with vesicovaginal fistulae resulting from radiation therapy, a urinary diversion appears to be the method of choice.

Nuclear volume and expression of T-antigen, sialosyl-Tn-antigen, and Tn-antigen in carcinoma of the human bladder. Relation to tumor recurrence and progression.

The T-antigen system and the mean nuclear volume have been proposed as risk variables in bladder tumors. This study includes 34 patients with initially noninvasive (Ta) transitional cell carcinomas who experienced different courses of disease. Tissue specimens of primary tumors were analyzed for the expression of T-antigen, Tn-antigen, and sialosyl-Tn-antigen using monoclonal antibodies (MoAb) and the lectin peanut agglutinin (PNA) in an indirect immunoperoxidase method. In addition, the mean nuclear volume was estimated by morphometry. Tissue from 7 of 13 patients (54%) who had invasive disease during a follow-up period of 5 years expressed T-antigen, as defined by MoAb HH8 in the primary tumor, whereas tissue of only 3 of 21 patients who did not have invasive disease expressed the antigen (P less than 0.02). No association was found between tumor progression to invasion and the expression of Tn-antigen or sialosyl-Tn-antigen. Tn-antigen expression was partially lost in invasive tumors (P less than 0.03) when compared with the expression in primary noninvasive tumors. A high mean nuclear volume in tissue specimens of primary tumors correlated with a progression to invasive disease (P less than 0.01). A significantly (P less than 0.003) higher mean nuclear volume was found in tumor areas that were positive for PNA compared with areas that were negative for PNA in primary tumors. A significantly lower mean nuclear volume was found in Tn-antigen-positive invasive Grade 3 tumor areas than in Tn-antigen-negative areas (P less than 0.005). The combined use of T-antigen expression and mean nuclear volume is of potential clinical interest for determining patients who are at high risk of disease progression.

A two-site lectinoenzymatic assay for determination of tumour marker glycoproteins in rectal secretions.

A method is described for a titre-tray based two-site lectinoenzymatic assay of glycoproteins. WGA lectin, reacting with the core-part of glycans, was combined with lectins PNA and DBA, the latter two reacting with terminal parts of glycans. A standard curve was obtained with bovine submaxillary gland asialomucin, and measurements of human rectal secretion were calibrated against this curve. The assay showed an intra-assay reproducibility of 2.4-7.5%, and inter-assay reproducibility of 3.9-20.8%. Recovery tests showed a linearity close to predicted values. The selected standard was ideal as inhibition of lectin binding by monosaccharides showed similar inhibition profiles for human rectal secretion and for asialomucin standard. Neuraminidase treatment dramatically increased the PNA binding to human rectal secretion immobilized on WGA. Western blotting of human rectal secretion demonstrated a large range of lectin-reactive glycoproteins, the main fraction reacting with all lectins being approximately 250 kDa. The assay described is well suited for studies of the glycan part of tumour marker glycoproteins, and changes occurring in these. It has a high sensitivity by ignoring that the glycans may be present on different molecules. Examination of rectal secretions from various cancer patients showed significantly increased PNA binding, as well as an increased PNA/DBA binding ratio, in patients with colorectal cancer (p<3x10(-3)) and, unexpectedly, in patients with other cancers (p<5x10(-3)).

Immunohistochemistry and cytochemistry of experimental rat bladder cancer: binding of the lectins PNA and WGA and of a Le(Y) mouse monoclonal antibody.

Two lectins, peanut agglutinin (PNA) and wheat germ agglutinin (WGA), and a mouse monoclonal antibody against blood group Le(Y) were used to study the distribution of carbohydrate antigens in an experimental rat bladder cancer model. Neoplasia was induced in 28 rats by intravesical installation of N-nitroso-N-methyl-urea (NMU). Fifteen rats were installed with the NMU solvent and served as controls. Urothelial samples were taken from all animals, the atypia were graded and detailed data on the location of the lectin-binding structures and the binding of the monoclonal antibody were obtained by immunohistochemical methods. Urine samples were collected at different times during tumor development and data on the cytological location of the lectin-binding structures and the binding of the monoclonal antibody were obtained by immunocytochemical methods. Examination of the histological distribution of lectin binding structures and Le(Y) antigen showed a characteristic change in carbohydrate antigen expression associated with the development of urothelial atypia. In normal urothelium carbohydrate antigens were present in cytoplasm, whereas they became expressed on cell membranes in non-invasive carcinomas. Immunocytochemistry of urine specimens revealed a lack of correlation between the antigen expression found in exfoliated cells and that found in tissue. Morphologically normal cells from NMU-treated animals were found to express a carcinoma associated antigen pattern.

Frequency and mechanism of Lewis antigen expression in human urinary bladder and colon carcinoma patients.

Changes in the expression of Lewis antigens have been associated with cancer diseases, and recent results have pointed at a possible increased risk of cancer development among Lewis negative patients. The frequency of the erythrocyte Lewis phenotypes Le(a- b+), Le(a+ b-) and Le(a- b-) was analysed in patients suffering from urinary bladder cancer (82), colon cancer (21), and benign urological diseases (45). An increased frequency of Lewis negative individuals was found among colon cancer patients (P less than 0.004) and bladder cancer patients (P = 0.05). The Lewis negative phenotype was shown to be associated with unfavourable disease parameters: invasion (P less than 0.02) and high grade of atypia (P less than 0.01) in bladder cancer patients, and high Dukes stage (P less than 0.05) in colon cancer patients. alpha 1-4fucosyltransferase activity (Lewis transferase) was shown to be present in saliva from four out of eight erythrocyte Lewis negative cancer patients, indicating that some patients with advanced cancer disease may have converted from a Lewis positive to a Lewis negative phenotype.

Localization and identification of T-(Gal beta 1-3GalNAc alpha 1-O-R) and T-like antigens in experimental rat bladder cancer.

A mouse monoclonal antibody and a rabbit polyclonal antibody against the T-antigen (Gal beta 1-3GalNAc alpha 1-O-R) were used to study the distribution of T-antigens in an experimental rat bladder cancer model. Neoplasia was induced in 28 rats by intravesical installation of N-nitroso-N-methylurea (NMU) dissolved in acetate buffer. Fifteen rats were installed with acetate buffer, and served as controls. Urothelial samples were taken from all animals, the atypia was graded and detailed data on the location of the antibody binding structures were obtained by immunohistochemical methods. In addition, Western Blots of glycoproteins and thin-layer-chromatography (TLC) immunostainings of glycolipids extracted from normal and malignant tissue were performed to characterize the molecules presenting T-antigens. Examination of the histologic distribution of T-antigens showed that both the monoclonal and the polyclonal reagents reacted with atypical cells in proportion to the grade of atypia, but showed no reaction in invasive cells. These results confirm previously obtained data on the T-antigen using peanut (arachis hypogaea) agglutinin (PNA), and support the structure identity as being the classical O-linked mucin type T-antigen. Western blots of tumor glycoproteins showed that the monoclonal and the polyclonal antibody reacted with epitopes different from that of PNA, but all the probes correlated with atypia. In addition PNA, as the only anti-T reagent, bound to glycolipid. By using well characterized and highly specific immunological reagents the present study shows that the T-antigen is a highly selective marker of urothelial atypia.

Short-chain fatty acids stimulate mucosal cell proliferation in the closed human rectum after Hartmann's procedure.

Hartmann's procedure in humans results in a closed rectum deprived of its natural short-chain fatty acid source. This induces atrophy of the entire rectal wall. Ki-67 is a monoclonal antibody directed towards proteins in the cell nucleus that are present only during cell proliferation. This study investigated the effects of short-chain fatty acids on mucosal cell proliferation in the human rectum after Hartmann's procedure by means of Ki-67. Eight patients in whom Hartmann's procedure was performed were treated with placebo and then short-chain fatty acids for 14 days. Biopsies specimens were taken from the rectum before and after treatment; these were prepared with Ki-67 and labeling index was determined. The treatment was found significantly to increase proliferative activity in the rectal mucosa (P<0.01); the increase was principally in the middle (P<0.01) and upper crypt (P<0.05) compartments.