Microsatellite instability in thyroid cancer: hot spots, clinicopathological implications, and prognostic significance.

PURPOSE: To determine whether microsatellite instability (MSI) in particular loci has clinicopathological significance in thyroid cancer. EXPERIMENTAL DESIGN: Seventy-six cases of surgically resected thyroid cancer were screened for MSI at nine microsatellites: THRA1, TSHR, D2S123, D11S912, D2S115, D2S399, p53, RET, or BAT-26. Multivariate analysis was performed to test for links between MSI and the clinical parameters of gender, age, histology, stage, nodal involvement, and prognosis. RESULTS: THRA1, residing in the thyroid hormone receptor alpha gene, displayed the highest levels of MSI at 36.5%. MSI in TSHR, located within the thyroid-stimulating hormone receptor gene, was found to be linked to cancer in the elderly (>70 years of age) and with high-grade (N 3, 4) nodal involvement. In follicular cancer, MSI in D2S123 occurred at a frequency of 100% (7/7) with no (0%) occurrence of MSI at the nearby D2S115, D2S399, or BAT-26 loci. Regarding prognosis, patients with MSI-positive cancer showed better long-term survival. BAT-26, which is an important marker in colorectal cancer, displayed the lowest frequency of MSI in our panel of thyroid tumors. CONCLUSION: Whereas patients with MSI-positive cancer showed better long-term survival, as is the case for colorectal cancer, our finding of the low frequency of MSI in BAT-26 suggests that the biochemical defects governing the spectrum of MSI in thyroid and colorectal cancer are different. MSI in THRA1, TSHR, and D2S123 appears to be an integral part of thyroid carcinogenesis, as evidenced by the high frequency of MSI and significant correlation to clinical data.

Evaluation of phenotypic and genotypic methods for subtyping Campylobacter jejuni isolates from humans, poultry, and cattle.

Six methods for subtyping of Campylobacter jejuni were compared and evaluated with a collection of 90 isolates from poultry, cattle, and sporadic human clinical cases as well as from a waterborne outbreak. The applied methods were Penner heat-stable serotyping; automated ribotyping (RiboPrinting); random amplified polymorphic DNA typing (RAPD); pulsed-field gel electrophoresis (PFGE); restriction fragment length polymorphisms of the flagellin gene, flaA (fla-RFLP); and denaturing gradient gel electrophoresis of flaA (fla-DGGE). The methods were evaluated and compared on the basis of their abilities to identify isolates from one outbreak and discriminate between unrelated isolates and the agreement between methods in identifying clonal lines. All methods identified the outbreak strain. For a collection of 80 supposedly unrelated isolates, RAPD and PFGE were the most discriminatory methods, followed by fla-RFLP and RiboPrinting. fla-DGGE and serotyping were the least discriminative. All isolates included in this study were found to be typeable by each of the methods. Thirteen groups of potentially related isolates could be identified using a criterion that at least four of the methods agreed on clustering of isolates. None of the subtypes could be related to only one source; rather, these groups represented isolates from different sources. Furthermore, in two cases isolates from cattle and human patients were found to be identical according to all six methods.

Electrophoretic separation of biopolymers in a matrix of polyacrylamide covalently linked to agarose.

A new type of agarose polyacrylamide mixed-bed gel, obtained by simultaneous gelation of a novel type of allyl-activated agarose and its copolymerization with acrylamide, has pore sizes intermediate between those of polyacrylamide and agarose. The process used to activate the agarose chains enables the substitution to be controlled. As indicated by nuclear magnetic resonance (NMR), only one allyl group was inserted per agarose basic unit. Several formulations of mixed-bed gels, containing different percentages of acrylamide, were compared with conventional polyacrylamide or agarose gels. Resolution, migration distance and band sharpness of different molecular mass fragments were evaluated, with two types of gel run side-by-side in a vertical or horizontal system. The faster electrophoretic mobility of DNA in dilute mix-bed gels and the improved separation of the component of high molecular mass (1 to 6 kbp) of the 1 kbp ladder indicate that these matrices have larger porosity than any dilute polyacrylamide formulations. Sodium dodecyl sulfate (SDS)-protein complexes migrate in the mixed gels faster than in polyacrylamide gels of the same %T.

Subclinical affection of liver and kidney function and solvent exposure.

The role of solvents as hepato- and nephrotoxic agents under present-day exposure levels is still unclear. The purpose of this study involving 99 metal degreasers was to examine dose-response relationships between long-term exposure of mainly trichloroethylene and a battery of liver function tests and one nephrotubular enzyme test. Serum gamma-glutamyltransferase and urinary N-acetyl-beta-glucosaminidase were elevated by increasing solvent exposure at bivariate level. The significance of this relationship, however, was not able to withstand a multiple regression analysis, with age and alcohol abuse as confounding variables. The conclusion is that of a nonsignificant association between solvent exposure and tests screening for early liver and kidney dysfunction.

Impact of dietary protein and fat source on the development of insulin-dependent diabetes in the BB rat.

Epidemiological studies show a remarkable geographical difference in the prevalence of IDDM, suggesting a role for environmental factors such as diet, infection, or stress in the etiology of the disease. Dietary modification has already been shown to be effective in the prevention of autoimmune diabetes in the BB rat and NOD mouse. We studied the effect of protein and fat source in the prophylaxis of diabetes in the BB rat. Natural ingredient rat chow was consistently associated with a high expression of the disease, whereas a casein-based, defined diet significantly inhibited the development of diabetes. Substitution of casein with raw red lentils resulted in a markedly higher incidence. This is the first highly diabetogenic defined diet in the BB rat. Neither fish oil nor soy oil enhanced diabetes expression in the BB rat. Increased amounts of soy oil also did not influence the disease process. These results suggest a central role for dietary protein source in the pathogenesis of BB rat diabetes. We speculate that plant proteins containing anti-nutrients such as chemicals, lectins, enzyme inhibitors, and nonphysiologic amino acids may initiate or hasten the pathogenesis process via beta cell stress or immune response activation.

Dependence of antigen expression on functional state of beta-cells.

Antigen expression corresponding to anti-islet cell surface monoclonal antibodies IC2 and A2B5 was studied. IC2 is a rat-rat hybridoma autoantibody produced from the BB rat; among islet cells, IC2 is beta-cell specific. A2B5 is an anti-ganglioside antibody described as labeling beta-cells. Islets of Langerhans from Lewis rats were isolated and cultured for 18 h in RPMI-1640 with five different glucose concentrations (2.2, 3.3, 5.5, 11.1, and 18.3 mM). In some experiments, islets were precultured for 2 or 3 days. After isolation of islet cells and antibody labeling, the percent of IC2+ beta-cells in the different groups increased from 33.3, 34.5, 40.9, and 57.2 to 58.6% (P less than 10(-6). For A2B5, the percent of labeled islet cells increased from 37.4, 41.8, 46.7, and 53.8 to 56.2% (P less than 10(-4). Thus, increasing glucose concentration leading to higher beta-cell activity implies an increase in antigen expression. Neither A2B5 nor IC2 reacts with insulin, as shown by absorption experiments and immune electron microscopy of binding sites. Electron microscopy of IC2-gold-labeled islet cells substantiated the beta-cell specificity of IC2. In conclusion, expression of the corresponding antigens to IC2 and A2B5 depends on the functional state of the beta-cells; because this has been shown to be an important factor in the development of insulin-dependent diabetes, our findings may be of potential pathogenetic interest.

Antigen expression of the pancreatic beta-cells is dependent on their functional state, as shown by a specific, BB rat monoclonal autoantibody IC2.

Antigen expression is studied corresponding to a monoclonal autoantibody (IC2) derived from a hybridoma of rat myeloma Y3 cells and splenocytes of the diabetic BB rat. The selective reactivity of IC2 with islet cells has earlier been proven. We studied the possible specificity for beta islet cells, and the possible variation in autoantigen expression. Islet cells were isolated by cautious collagenase and dispase treatment. The cells were labelled with IC2 alone or together with anti-insulin immunoglobulin in double-labelling experiments. Extensive series of cells were examined by immunofluorescence microscopy, and some samples also by flow cytometry. In double-labelling examinations we found that only anti-insulin positive cells could bind the IC2 antibody, thus showing beta-cell selectivity. On the other hand, not all anti-insulin positive cells were IC2-positive. Since insulin treatment has been shown to decrease the incidence of diabetes in the BB rat, islet cells were examined after reduced beta-cell strain. Islet cells from Lewis and Wistar Furth rats display 21.4 +/- 1.4% IC2-positive cells, while islet cells from 24-hour fasting animals showed 7.0 +/- 1.4% (p less than 0.0001). Similar results were seen for BALB/c mice (25.0 +/- 1.8% vs. 13.7 +/- 2.3%, p less than 0.002). Also, after a week of insulin treatment, autoantigen expression was significantly decreased. Thus, the IC2 antibody is beta-cell-specific, and expression of the corresponding cell surface antigen depends on the functional state of the beta-cells.

Production and characterization of a monoclonal islet cell surface autoantibody from the BB rat.

Islet cell surface autoantibodies are present in the serum of the spontaneously diabetic BB rat. The availability in large quantities of such autoantibodies should help us understand their significance in vivo. Fusions between BB rat lymphocytes and rat myeloma cells were screened by cellular enzyme linked immunosorbent assay and indirect immunofluorescence on rat living cells. They resulted in a stable hybridoma, called IC2, secreting a monoclonal immunoglobulin M specific for the surface of rat islet cells. This monoclonal antibody was found to bind to the surface of 56% normal rat islet cells and 72% rat insulinoma cells. Protease treatment of rat islet cells resulted in a subsequent 72-100% binding inhibition of IC2 to the surface of these cells, suggesting that IC2 specific antigen is a protein.

Occupational exposure to chlorinated organic solvents and its effect on the renal excretion of N-acetyl-beta-D-glucosaminidase.

Organic chlorinated hydrocarbon solvents are known to be nephrotoxic. However, very little is known about renal integrity after occupational exposure to these solvents. Increased urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion caused by necrosis of renal tubular cells can be used as a marker for nephropathy. In this study trichloroethylene (TRI), trichloroethane and freon 113 exposed persons from metal industries have been clinically investigated, and urine samples analysed for NAG activity and for trichloroacetic acid concentration. In order to analyse for possible subclinical kidney damage a group of diabetic patients with subclinical nephropathy served as positive controls. A significant higher NAG activity (p less than 0.001) was found in the group of exposed workers as compared to the control group. About 10% of the exposed workers had an enhanced NAG value, corresponding to the level of diabetic patients with subclinical nephropathy. Increased NAG activity was observed in previously TRI-exposed persons, which might indicate induction of an autoimmune renal necrosis.

Partial purification and characterization of chicken immune-associated antigen in serum coded for by the major histocompatibility complex.

Chicken serum contains 3 molecular species of immune-associated (Ia) antigen coded for by the major histocompatibility complex (MHC). These molecules are named B-L alloantigens [1]. They vary in electrophoretic migration velocity and molecular size [2]. The aim of this study was to characterize one of the antigen species - the low molecular size form. Therefore, we performed a partial purification by: (i) affinity chromatography; and (ii) ammonium sulfate precipitation of serum B-L antigen from the chicken plasma. Since the MHC-antigens were known to be glycoproteins, the purification was based on lectin affinity chromatography, as previously used for the membrane-bound MHC-antigens [3]. Electro-immunochemical analysis using rabbit antibodies against the chicken lymphocyte plasma membrane and the partially purified antigen were employed to monitor the purification and to characterize the different molecular forms of the Ia molecules. The partially purified preparation was then analyzed to elucidate the biochemical structure of the serum B-L antigen. Finally, rabbit antiserum was raised against this preparation to evaluate its level of purity and to follow further purification of this molecule.

Separation of Fc-binding serum proteins by preparative flat-bed isotachophoresis.

Preparative flat-bed isotachophoresis with discrete spacers was applied as a single-step procedure to separate 2 Fc-dependent activities of normal chicken serum, i.e., (1) the ability to raise the titre of haemagglutinating allo-antisera which is due to a high molecular weight beta-globulin (HEF), (2) the ability to activate guinea pig complement components in mixed complement reaction. The results demonstrate that the 2 activities can be clearly separated, and HEF must therefore be different from the first complement factor in the chicken. Under the chosen conditions the molecule active in the mixed complement reaction is not stacked in contrast to other serum protein including HEF. The same technique with human serum shows that human Clq behaves in the same was as the chicken complement factor. This means that by selective unstacking, flat-bed isotachophoresis can be used as an efficient single-step purification method for human and chicken Clq.

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.