Uroscopy in the 21st century: high-field NMR spectroscopy

From the experiments described, it can be seen that there are different research approaches that can be taken and these are summarized in Table 1. Whereas much scientific research is principally hypothesis led, there remains, nevertheless, an important place for exploratory research. High resolution NMR can measure, directly and simultaneously, a wide range of endogenous metabolites in biological fluids and has the unique capability of providing structural information on the metabolites detected. It has proved to be a powerful research tool with which to study inherited metabolic diseases, renal disease, drug metabolism, and toxicity, and can be used to monitor the effects of drug therapy. For instance, by using a library of experimental toxins one can map the metabolic profile of site-specific nephron injury. With this approach in man one could eventually take an unknown disease such as Balkan nephropathy and predict the initial site of tubular injury, the mode of injury and therefore the kind of toxin capable of producing that injury. NMR spectroscopic techniques are still advancing rapidly, with ever increasing sensitivity and sophistication of NMR pulse sequences to enhance structural elucidation in complex mixtures. Given the advances in directly coupled HPLC-NMR and even HPLC-NMR-mass spectroscopy it is likely that these technologies in conjunction with pattern recognition will make major contribution to our understanding of renal processes and provide new diagnostic insights in the 21st century.      

Sucrase-isomaltase: a marker associated with the progression of adenomatous polyps to adenocarcinomas.

Adenocarcinomas of the colon arise from adenomatous polyps. We hypothesized that sucrase-isomaltase (SI), a glycoprotein hydrolase, found in normal small intestine, fetal colon, and colon carcinomas is a marker associated with progression of adenomatous polyps with dysplasia to adenocarcinomas. To examine this hypothesis, we performed immunostaining using a polyclonal antihuman SI antibody in 32 adenomatous polyps with varying degrees of dysplasia. In addition, sucrase enzyme activity was determined in three sets of simultaneously harvested polyps, cancer, and adjacent normal mucosa from the same patient. All severely dysplastic polyps (6/6) exhibited SI staining. Most polyps (85%) with 3+ staining (i.e., greater than 10% of polyp positive for SI) had severe dysplasia, whereas those with mild dysplasia had either 1% to 5% staining or no staining in 95% of the cases. These data indicate that the extent of SI immunostaining in polyps correlates with the degree of dysplasia (p = 0.0001). Sucrase-isomaltase activity in the polyps was 18.1 +/- 1.8 mU/mg (mean +/- SD); in adjacent carcinoma SI activity was 29.1 +/- 1.8 mU/mg. Adjacent mucosa showed no activity in all cases. In summary, our results suggest that SI expression correlates with the progression of dysplastic adenomatous polyps to carcinoma. Sucrase-isomaltase expression may be useful as a clinical marker to improve our prognostic capabilities in patients with dysplastic lesions of the colon, that is, inflammatory bowel disease.     

Sialyltransferase activity and hepatic tumor growth in a nude mouse model of colorectal cancer metastases.

Sialyltransferase activity (EC 2.4.99.6) was measured in the microsomal fraction of colorectal cancer cell lines using an assay based on the incorporation of [14C]CMP-sialic acid into asialofetuin. In the poorly differentiated lines MIP101 and Clone A, sialyltransferase activity had a Vmax of 0.36 and 0.31 nmol/mg protein/h, respectively, while the moderately differentiated to well-differentiated cell lines HT-29, CCL188, and CX-1 had Vmaxs of 2.46, 1.05, and 1.24 nmol/mg protein/h, respectively. All cell lines tested had a Km of 15.4 (+/- 0.7)(SD) mumol/liter. The better differentiated cells had higher levels of sialyltransferase activity, which correlated with their higher levels of sialic acid and their enhanced ability to form liver metastases in the nude mouse following intrasplenic injection compared to the poorly differentiated cell lines. Treatment of the cell lines with KI-8110, a CMP-sialic acid derivative which prevents incorporation of sialic acid into glycoconjugates, resulted in reduced formation of hepatic metastases by the colorectal carcinoma cell lines in the nude mouse model. It is suggested that reduced sialylation of adhesion molecules such as carcinoembryonic antigen may change the biology of the tumor cell, one consequence of which is the prevention of implantation of the cells into distant sites, resulting in a reduced incidence of metastases.     

Laminin expression in colorectal carcinomas varying in degree of differentiation

We are studying the ability of colorectal carcinomas, which vary in degree of differentiation, to assemble a basement membrane and the relationship between differences in this ability and perturbations in laminin expression. For these studies, we are using human colorectal carcinoma cells grown both in vitro and in nude mice as well as tumors obtained at surgery. Immunoperoxidase staining of human tumors indicates that laminin is present in a defined basement membrane in moderately to well-differentiated tumors. This staining pattern is absent in poorly differentiated tumors. In these tumors, staining is discontinuous and sometimes observed intracellularly. The laminin synthesized by in vitro cells was immunoprecipitated and analyzed by acrylamide electrophoresis. Neither poorly nor well-differentiated carcinoma cells exhibit marked differences in the rate of synthesis of laminin. Differences are present in the rate at which newly synthesized laminin is secreted. These differences may result from alterations in posttranslational processing. Such alterations may contribute, along with other factors, to the inability of poorly differentiated tumors to make a basement membrane.     

Vitamin D3, gamma interferon, and control of proliferation of Mycobacterium tuberculosis by human monocytes.

Previous studies have shown that recombinant interferon gamma (IFN-gamma), crude T cell supernatants, or appropriate T-cell lines can cause total inhibition of the growth of M. tuberculosis inside murine peritoneal macrophages. In similar experiments with human monocytes much smaller effects are seen. This could be due to the relative immaturity of these cells. Because dihydroxy vitamin D3 (1,25-(OH)2 D3) can cause phenotypic differentiation of immature leukemic lines into macrophage-like cells, we have explored the possibility that exposure to cholecalciferol metabolites in vitro might increase the ability of monocytes to control proliferation of M. tuberculosis, or cause monocytes to mature into cells able to respond appropriately to IFN-gamma. Incubation of monocytes with three cholecalciferol metabolites induced anti-tuberculosis activity to an extent that correlated with their binding affinities to the intracellular receptor protein for the derivatives. 1,25-(OH)2 D3 also primed monocytes for phorbol myristate acetate-triggered reduction of nitroblue tetrazolium. The effects were additive rather than synergistic with those of IFN-gamma. Monocytes incubated with IFN-gamma developed 25-OH D3 1-hydroxylase activity, detected by conversion of tritiated 25-(OH) D3 to a more polar metabolite which coeluted with 1,25-(OH)2 D3 on straight and reverse-phase HPLC. The latter is a more active form in vivo. These findings help to explain claims for the efficacy of vitamin D in the treatment of some forms of tuberculosis, and also the occasional finding of raised serum calcium, and disturbed vitamin D metabolism in these patients.     

Leukocyte survival in cerebrospinal fluid.

Delays in the laboratory examination of cerebrospinal fluid are commonly encountered in clinical medicine. The present studies were designed to evaluate changes in cerebrospinal fluid leukocyte counts relative to time elapsed before analysis. Neutrophil counts decreased most rapidly, being 68 +/- 10% (standard error of the mean) and 50 +/- 12% of initial values at 1 and 2 h, respectively. Lymphocyte and monocyte numbers were not significantly altered until 3 h.     

Graves'' disease and Down''s syndrome

A case is described of severe thyrotoxicosis occurring in a patient with Down's syndrome, in which behavioural disturbance was a marked but reversible phenomenon. In addition, we report that antibodies capable of stimulating the thyroid were detectable in the sputum of both the patient and his mother.     

Lack of chemopreventive efficacy of DL-selenomethionine in colon carcinogenesis

Epidemiologic observations and laboratory research have suggested that dietary selenium reduces the risk of colon cancer. Selenium-enriched brewer's yeast as a dietary supplement reduces the incidence of and mortality from cancer of the colon in humans. It is not clear whether the observed inhibitory effect is due to selenomethionine, or to other forms of selenium, or to a mixture of the selenium compounds present in selenium-enriched brewer's yeast. Therefore, bioassay described in this study examined the chemopreventive efficacy of 10 and 15 ppm selenomethionine, equivalent to 3.6 and 5.4 ppm as selenium, against azoxymethane (AOM)-induced colon carcinogenesis. At five weeks of age, groups of male F344 rats were fed diets containing 0 (control diet), 10 or 15 ppm selenomethionine. At seven and eight weeks of age, all rats except those in vehicle-treated groups received s.c. injections of AOM at a dose rate of 15 mg/kg body wt. The rats were maintained on their respective diets for 52 weeks and were then sacrificed. Colon tumors were processed and evaluated histopathologically. Colon tumor incidence and multiplicity were analyzed statistically. No obvious toxic effects were observed following dietary administration of 10 or 15 ppm selenomethionine as indicated by body weight gain. Administration of 10 or 15 ppm selenomethionine had no significant effect on colon tumor incidence and multiplicity. This study suggests that i) selenomethionine lacks chemopreventive efficacy against AOM-induced colon carcinogenesis and ii) other forms of selenium or a mixture of selenium compounds present in selenium-enriched brewer's yeast need to be evaluated for their chemopreventive efficacy.