Are dietary fiber-induced alterations in colonic epithelial cell proliferation predictive of fiber's effect on colon cancer?

Alterations in cell proliferation of the colon have been observed as a result of changes in amount and type of dietary fiber and in relation to risk of developing colon cancer. Although some human observational and intervention studies contribute to the database, most information results from experiments on rodents. Because of numerous contradictory reports linking dietary fiber, cell proliferation, and colon cancer, we undertook a critical review of existing methods in an attempt to explain the inconsistencies. Although there may be some individual types of dietary fiber that protect against chemically induced colon cancer, dietary fiber as a single entity does not appear to afford any consistent protection. Because of significant differences in experimental protocols among laboratories, it is not yet possible to state with certainty that increases in cell proliferation, induced by fiber consumption, are predictive of increased tumorigenesis. Much of what has been observed and interpreted as elevation of risk may simply be normal homeostatic changes in cell proliferation. Even though fermentation to short-chain fatty acids is a mechanistically attractive hypothesis to explain why fiber modulates cytokinetics, data do not consistently support short-chain fatty acids as biological intermediates in risk of colon cancer. The state of the art in this field has not yet progressed to the point where a clear effect of dietary fiber on cytokinetics and colon carcinogenesis can be assessed with any degree of certainty. Additional markers of apoptosis, differentiation, and cell-cell communication may be required for a more accurate analysis of the relation among fiber, cytokinetics, and colon cancer.

Validation of the galactose oxidase-Schiff's reagent sequence for early detection and prognosis in human colorectal adenocarcinoma.

Based on the multistage and multifocal nature of colorectal carcinogenesis, it is likely that reduction of cancer mortality through early detection and identification of new prognostic markers is an attainable goal. Well-documented changes occur in mucin glycoconjugates during neoplastic progression in the colon, and the nonneoplastic colonic mucosa in colon cancer patients is morphologically and histochemically abnormal. In this retrospective study, 152 archival colorectal tissues from 49 patients were studied for changes in mucin secretions as detected by the galactose oxidase-Schiff's (GOS) sequence. Intensity of the stain was evaluated in histological sections by semiquantitative analysis, and the area percentage of epithelium stained was quantified by image cytometry. The correlation between gender or tumor size, location and reactivity with peanut agglutinin and quantitative expression of GOS-reactive mucins was determined as well as intratumor and inter individual variability. Reactivity with GOS: (a) decreased during neoplastic progression and malignant conversion in the neoplasm; (b) increased in the normal colonic mucosa of patients with progressively more advanced disease; and (c) was of prognostic significance for patient survival or recurrence both in the normal colon of cancer patients and in invasive neoplasms. These data are consistent with the conclusion that GOS reactivity in the normal colonic mucosa is a dosimeter of exposure to environmental/lifestyle colorectal carcinogens rather than a marker for an oncodevelopmental cancer-associated antigen.

Aberrant crypt foci in the colonic mucosa of rats treated with a genotoxic and nongenotoxic colon carcinogen.

Aberrant crypt foci (ACFs) are putative preneoplastic lesions in the colonic mucosa identified by examining methylene blue-stained whole mounts of colon. ACFs have been previously described in rats treated with genotoxic colon carcinogens. This study determined whether or not a nongenotoxic colon carcinogen could induce ACFs and compared the morphology of these ACFs with those induced by a genotoxic colon carcinogen. Six-wk-old Fischer-344 rats were administered dextran sulfate (DSS, nongenotoxin) in the drinking water or azoxymethane (AOM, genotoxin) by single subcutaneous injection. Rats were sacrificed at 9 and 14 wk after study initiation. Colons were fixed and stained with methylene blue, and the mucosal surface of transilluminated whole mounts was examined with a microscope. The number of ACFs and number of crypts per focus (multiplicity) were recorded. Representative ACFs were processed into glycol methacrylate for hexosaminidase enzyme histochemistry and sections of the remaining colon containing ACFs were embedded in paraffin for morphologic evaluation. In whole mounts, ACFs from AOM- and DSS-treated rats had elongated slit-to-oval-shaped lumens surrounded by a thickened and intensely stained epithelium. DSS-induced aberrant crypts differed from those induced by AOM in that they were frequently larger, tended not to form discrete foci circumscribed by normal crypts, and were located adjacent to ulcers. Total ACFs and large foci (4 or more crypts/focus) were significantly more numerous in AOM-treated rats at both time points. Histologically, DSS-induced ACFs had segmental to diffuse loss of hexosaminidase activity, mucin depletion to increased prominence of goblet cells, and marked distortion of crypt architecture. AOM-induced ACFs had diffuse loss of hexosaminidase activity, variable depletion of mucin, and less distortion of crypt architecture. Variable degrees of epithelial dysplasia were seen in ACFs with both carcinogens, but dysplasia was more severe in DSS-induced ACFs. Colonic mucosal neoplasms were induced by both carcinogens. In subchronic studies, the ACF assay may be a useful method to improve the identification and characterization of xenobiotic-induced changes in colonic mucosal crypts.

Evaluation in rats of the dose-response relationship among colonic mucosal growth, colonic fermentation, and dietary fiber.

The dose-response relationship among dietary fiber, colonic fermentation, fecal weight, and mucosal growth were evaluated in this study. The morphometric parameter of total mucosal volume was used to assess diet-induced differences in colonic mucosal growth. Dietary fibers with a wide range of fermentability and that have previously been shown to inhibit the development of colonic neoplasia in rats were used. Sprague-Dawley rats were fed Purina Rodent Chow, AIN-76a fiber-free diet, or an AIN-76a diet supplemented with three different dietary fibers, (cellulose, guar gum, or wheat bran) at 2, 5, 10, or 15% of the diet. Diets were fed for 28 days. Total colonic mucosal volume was determined using stereologic principles and computerized image analysis; 48-hr fecal weight was measured; and the concentration of short-chain fatty acids (SCFA) in colonic contents was determined at study termination. Each type of fiber induced a dose-dependent increase in total mucosal volume of the colon and fecal weight. Mucosal volume and fecal weight were closely correlated (R2 > 0.95). Total mucosal volume was not correlated with the concentration of total SCFA or butyrate in the colon. These results indicate that diet-induced change in colonic mucosal growth, as measured by total mucosal volume, is positively correlated with fecal weight and not related to alterations in colonic fermentation. Enhanced colonic mucosal growth occurs in rats fed dietary fibers that have previously been shown to inhibit the development of genotoxin-induced colonic neoplasia in rats.

Alterations in pulmonary morphology and peripheral coagulation profiles caused by intratracheal inoculation of live and ultraviolet light-killed Pasteurella haemolytica A1 in calves.

Eighteen male Holstein calves were divided into groups of three and inoculated intratracheally with 5 x 10(9) logarithmic phase or ultraviolet light-killed Pasteurella haemolytica biotype A serotype 1. Serial coagulation profiles were done on one calf from each group during the first 24 hours after inoculation. One calf from each group was necropsied at 4, 12, and 24 hours after inoculation and lesions were characterized with light and transmission electron microscopy. We found that 1) the pulmonary intravascular macrophage may have an important role in the early intravascular inflammatory events; 2) there was morphologic evidence for local initiation of the coagulation cascade in the lung early in the disease process but it was not a consumptive process; and 3) killed-bacteria were capable of causing fibrin exudation, platelet aggregation and alveolar epithelial damage similar to live bacteria, but the degenerative changes in neutrophils, endothelial cells and intravascular fibrin formation that occur with live bacteria were not seen.

Altered expression of transforming growth factor-alpha in hereditary rat renal cell carcinoma.

A hereditary form of renal cell carcinoma exists in rats that results from a single gene mutation and is histologically similar to that described in humans. Cell lines derived from these rat tumors were shown to express abundant transforming growth factor-alpha (TGF-alpha) and epidermal growth factor (EGF)-receptor RNA transcripts, but no EGF mRNA. In contrast, normal kidney expressed EGF and EGF-receptor transcripts, but TGF-alpha transcripts were barely detectable. Other kidney epithelial cell lines examined (NRK 52E, MDCK, and LLCPK) were negative for expression of both TGF-alpha and EGF transcripts, but expressed EGF receptors. In addition, the renal cell carcinoma-derived lines secreted TGF-alpha into the media. Immunohistochemistry of normal kidney with a TGF-alpha specific antibody revealed a characteristic pattern of staining of collecting ducts and, to a lesser degree, proximal tubules. In the neoplastic kidney tissue, both the cystic and solid portions of the tumors displayed intense immunoreactivity, indicating that altered expression of this growth factor by the transformed cells occurred in situ. These results suggest that altered TGF-alpha expression is an important aspect of the neoplastic phenotype in rodent as well as human renal cell carcinoma, and support the use of this hereditary rodent tumor model for studying the pathogenesis of this disease.

Morphological and morphometrical analysis of the acute response of the bovine alveolar wall to Pasteurella haemolytica A1-derived endotoxin and leucotoxin.

Endotoxin or leucotoxin derived from Pasteurella haemolytica biotype A serotype 1 or saline was deposited by fibreoptic bronchoscopy into the caudal segment of the right anterior lung lobe of calves, and the lesions were characterized by light and transmission electron microscopy. Morphometric techniques were used to determine if changes in the arithmetic mean thickness of the alveolar wall occurred. Group 1 calves (n = 2) were inoculated with 6 ml saline, groups 2 calves (n = 3) received 6 ml of a partially purified leucotoxin preparation, group 3 calves (n = 3) received 96 micrograms of endotoxin in 6 ml of saline and group 4 calves (n = 3) received 2.5 mg of endotoxin in 6 ml of saline. Calves were killed 4 h after inoculation. Lesions in groups 2, 3 and 4 were similar and we found that (a) endotoxin alone is capable of initiating an inflammatory response in the bovine lung, (b) leucotoxin causes cytotoxic changes in alveolar macrophages but not in parenchymal cells of the lung, (c) neutrophil sequestration and platelet aggregation occur in alveolar capillaries in association with pulmonary intravascular macrophages, (d) neutrophils and fibrin were found in the alveolus in close association with alveolar macrophages, (e) disruption of the alveolar epithelial layer occurred in association with neutrophils and (f) there were no significant increases in the arithmetic mean thickness of the alveolar wall.

Ultrasonographic appearance of primary and metastatic canine hepatic tumors. A review of 48 cases.

Forty-eight cases of dogs with primary and metastatic hepatic neoplasia were reviewed to determine if a predictable relationship between sonographic appearance and neoplastic cell type could be found. A focal mass was almost always a hepatocellular carcinoma (14 of 15) and 71% of these were hyperechoic. Focal or multifocal hyperechoic masses were most likely to be carcinomas (14 of 15). Focal or multifocal mixed neoplasms were most likely to be carcinomas (6 of 7). Two distinct patterns of lymphosarcoma were found: diffuse, mildly hyperechoic (6/11) and multifocal, hypoechoic (5/11). No neoplastic cell-type predictions could be made for focal or multifocal hypoechoic lesions. Diffuse fine or coarse patterns with minimal architectural distortions could be mistaken for normal or degenerative processes. However, in the presence of increased serum liver enzyme values, these subtle sonographic changes would warrant a liver biopsy to differentiate neoplastic infiltrate from non-neoplastic infiltrative and degenerative processes.

Production and partial characterization of monoclonal antibodies to Pasteurella haemolytica A1 capsular polysaccharide and lipopolysaccharide.

Hybridoma-derived monoclonal antibodies (MAB) against the cell surface antigens of Pasteurella haemolytica serotype 1 were obtained by the fusion of murine myeloma cells (P3 X 63 - Ag 8.653) with splenocytes of BALB/c mice immunized with crude logarithmic growth-phase culture supernatant. Initial screening was performed, using an ELISA, with the same bacterial growth culture supernatant as coating antigens. Further selection was done, using a panel of purified antigens--either capsular polysaccharide or lipopolysaccharide--as the coating antigen in an ELISA, and then performing a leukotoxin-neutralization assay. Two MAB, designated IIB-6 and H-2, reacted specifically with the capsular polysaccharide and the other 3, designated IVG-3, IH-3, and IIC-2, reacted with the lipopolysaccharide. One MAB, designated IH-6, did not react with leukotoxin, capsular polysaccharide, or lipopolysaccharide. The MAB to the capsular polysaccharide (IIB-6 and H-2) were characterized further; both antibodies belonged to the IgM class and were agglutinating. In addition, they promoted neutrophil-mediated opsonophagocytosis and complement-mediated immune bacteriolysis of P haemolytica serotype 1. Results from 3 studies indicated that the MAB IIB-6 and H-2 were specific only to the capsular polysaccharide of serotype 1 of P haemolytica. The MAB to the lipopolysaccharide (IVG-3, IH-3, and IIC-2) were of the IgG1, IgG3, and IgM classes, respectively and were not characterized further. The availability of a MAB identifying a serotype-specific, surface-exposed determinant on the capsule of P haemolytica serotype 1 should facilitate and expand studies concerning the role of the capsular material and lipopolysaccharide in the pathogenicity of P haemolytica infection in cattle.