Biomedical subjects
Publications and source records attributed to W R Brown.
Carter-Wallace conducted a detailed audit and evaluation of the data available from the carcinogenicity studies with iodinated glycerol conducted in the B6C3F1 mouse and the F344/N rat by the National Toxicology Program (NTP). We conclude that there is no evidence of carcinogenicity of the compound in either the B6C3F1 mouse or the F344/N rat.
A reliable test for premalignant lesions in the development of colonic cancer in chronic ulcerative colitis has been needed. Thus, we designed this cytochemical study, using a model of experimental colitis and colonic tumors induced in Wistar male rats by the feeding of dextran sulfate sodium. The colitis had histologic similarities to ulcerative colitis in man. The percent frequency of polypoid lesions (dysplasia or dysplasia with carcinoma in situ) in the cecum and ascending colon was about 25% at three months and 90% at six months of dextran sulfate feeding. The cytochemical findings by high-iron diamine-Alcian blue staining and Ulex europeus agglutinin binding were chronologically paralleled by histological changes in the colonic mucosa, and the binding pattern of peanut agglutinin was not different between normal and dextran-treated animals. Moreover, some cytochemical changes that occurred during the inflammatory responses were not present in dysplastic or malignant lesions. Thus, the histochemical tests were not useful for detecting of premalignant lesions earlier than by conventional histology. Nevertheless, the dextran sulfate model of colitis in the rat appears suitable for study of cancer development in ulcerative colitis.
Quantitative data are presented showing that the method most commonly used to immobilize antibodies in microtiter immunoassays functionally inactivates most of the antibodies. These results were collected using five affinity purified polyclonal antibodies (pAbs) and six monoclonal antibodies (mAbs) specific for fluorescein (FLU) as capture antibodies (CAbs). These CAbs were tested for their ability to capture FLU4.2-BSA after immobilization by passive adsorption, the Protein-Avidin-Biotin-Capture (PABC) system or using previously adsorbed anti-globulins. Results indicate that under optimal conditions, < 10% of monoclonal capture antibody equivalents (CAbeqv) and congruent to 22% of polyclonal CAbeqv remain functional after passive adsorption. Immobilization via the PABC system improved the performance of mAbs by more than five-fold but had less than a two-fold effect on pAbs. Many CAbs immobilized using an anti-globulin retained full activity including the ability to bind two molecules of FLU4.2-BSA/molecule of CAb. The latter result is not necessarily a recommendation for the use of anti-globulin immobilization, since the number of functional CAbeqv per well is not significantly greater than that which can be achieved using passive adsorption.
We tested whether pilus proteins of rabbit diarrhoeagenic Escherichia coli (RDEC-1), incorporated into biodegradable microspheres, could function as safe and effective oral immunogens in the rabbit diarrhoea model. The RDEC-1 adhesin, AF/R1, incorporated into poly(D,L-lactide-co-glycolide) microspheres, was administered intraduodenally. Vaccinated and unvaccinated rabbits were challenged with RDEC-1 and killed 1 week later. Vaccination with AF/R1 in microspheres did not cause diarrhoea or weight loss. After challenge, rabbits given AF/R1 in microspheres, in contrast to unvaccinated animals, remained in good health. RDEC-1 attachment to caecal epithelium of vaccinated rabbits was reduced (p = 0.02), whereas numbers of RDEC-1 in intestinal fluids were little affected. Also, in vaccinated animals, biliary anti-AF/R1 IgA levels were increased, and AF/R1-induced blast-cell transformation was vigorous in spleen cell cultures. We conclude that vaccination with AF/R1 in microspheres was safe and protected rabbits against RDEC-1 disease, probably by interfering with adherence of the bacteria to the intestinal mucosa. The interference might have been due to the presence of specific antibodies secreted in bile.
Calcitonin, the serum calcium-lowering hormone, has been used in the treatment of hypercalcemia of malignancy and postmenopausal osteoporosis in humans for several years without any adverse effects. Recent studies in rats have indicated that calcitonin may be associated with morphologic effects on the pituitary. A large study was performed on 2 strains of rats, Sprague-Dawley (SD) and Fischer-344 (F-344), with 2 types of calcitonin, salmon-derived (sCT) and porcine-derived (pCT) calcitonin to evaluate possible effects on the pituitary. Sixteen groups of 42 male and 42 female SD or F-344 rats were given 0 (vehicle control), 1.25, 5.0, or 80.0 IU/kg/day of sCT or pCT, once daily, subcutaneously, for 1 yr. An increased incidence of adenomas of the adenohypophysis was observed in male SD rats at all dose levels of sCT, female SD rats given 80 IU/kg/day of sCT, male SD rats at the high dose level of pCT, and male F-344 rats at the high dose level of sCT. Also, an increased incidence of total proliferative lesions, due mostly to an increased incidence of focal hyperplasia of the pars distalis, occurred in female F-344 rats given the high dose of sCT. These pituitary proliferations were histologically similar to those that occur spontaneously, and the incidences observed were comparable to those that could occur in rats on 2-yr or lifetime studies, indicating that the injection of calcitonin had decreased the latency period.
Six monoclonal and two polyclonal antibodies to fluorescein (FLU) were affinity purified and immobilized on Immulon 2 polystyrene as capture antibodies (CAbs): (a) by passive adsorption at pH 9.6, (b) via a streptavidin bridge to a biotinylated carrier molecule, and (c) via an antiglobulin which had been previously adsorbed passively to the polystyrene. Data show that less than 3.0% of the binding sites of monoclonal CAbs and approximately 5-10% of those of polyclonal CAbs were capable of capturing antigen (FLU4.2-BSA) after passive adsorption. Immobilization of CAbs via an antiglobulin or a streptavidin bridge, resulted in the preservation of antibody binding sites to greater than 70% for some monoclonals although immobilization via the streptavidin bridge resulted in the highest number of functional sites/well. The data presented are consistent with studies on other adsorbed proteins which demonstrate that passive adsorption on polystyrene results in the loss of protein function. Furthermore, these data show that generally less than half of the binding sites of antibodies available in solution are available after solid-phase immobilization even when non-adsorptive methods are employed. Some polyclonal anti-FLU also have lower average avidity following passive adsorption compared with CAbs immobilization via a streptavidin bridge. Immunochemical studies revealed that adsorbed polyclonal-CAbs performed like monoclonals when tested with multivalent antigens (FLU10-IgA) but in an expected heterogeneous manner in Scatchard plots when tested using univalent FLU-insulin. This observation implied cross-linking of immobilized CAbs by the multivalent antigen. Because only 5-10% of adsorbed polyclonal CAbs are active, the survivors must be non-randomly distributed in clusters to explain the cross-linking. This was confirmed by scanning electron microscopy which gave rise to the hypothesis that antibodies which retain activity after adsorption, are those present in clusters, i.e., the functional adsorbed CAb is an antibody cluster. Data presented in this report on the behavior of adsorbed CAbs, and reviewed from the work of others for various adsorbed proteins, indicate that the method of passive adsorption at pH 9.6, which is widely used in popular microtiter ELISAs, and which has in many ways revolutionized immunoassay, is a method of protein denaturation. Assayists that utilize passive adsorption of proteins on hydrophobic supports as part of their research need to be cognizant of this phenomenon, while inventors of immunoassay should develop alternative methods of immobilization which do not destroy 90% of the functional activity of solid-phase reactant.
Serum concentrations of asialoglycoproteins are increased in cirrhosis. We hypothesized that this increase results from abnormalities in the asialoglycoprotein receptor, which is located on the sinusoidal and lateral membrane of hepatocytes. Therefore we searched for morphological alterations in the distribution of the asialoglycoprotein receptor in human liver, using a light microscopic immunoperoxidase method in autopsy livers. In 24 of 25 (96%) of patients without liver disease, the asialoglycoprotein receptor was located on the sinusoidal and, less prominently, the lateral surface of hepatocytes but not the canalicular surface. In contrast, in 12 of 18 (67%) patients with cirrhosis of various causes, the receptor also was localized strikingly along the canalicular surface, with a corresponding decrease on the sinusoidal and lateral surfaces. We conclude that an abnormal cell-surface distribution of the asialoglycoprotein receptor commonly occurs in cirrhosis. This abnormality might result in impaired clearance of desialylated glycoproteins from plasma.
Expression of proliferating cell nuclear antigen/cyclin (PCNA/cyclin) in skin tissue specimens and cultured keratinocytes was studied using a monospecific antibody, obtained from a patient with systemic lupus erythematosus, and a monoclonal antibody. Indirect immunofluorescent staining revealed that cultured keratinocytes obtained from human foreskins expressed PCNA/cyclin as variable nuclear patterns in 15-30% of the cells. In normal human skin tissue specimens, PCNA/cyclin was demonstrated in only a few basal cells. Interestingly, PCNA/cyclin was expressed strongly in almost all the cells of the lowest layer of the epidermis adjacent to squamous cell carcinomas, whereas the tumor aggregates themselves had no positive staining. In contrast, no such characteristic staining was demonstrated in specimens of basal cell carcinoma. The staining pattern of PCNA/cyclin was different from that of Ki-67 in the skin tissue specimens. Our results suggest that PCNA/cyclin could be a useful marker of cell proliferation.
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A mammalian artificial chromosome would enable analysis of the cis-acting DNA sequences necessary for mammalian chromosome function and would allow large numbers of genes in a defined sequence environment to be introduced into experimental animals, agricultural livestock or human cells. Recent technical progress suggests that a route to mammalian artificial chromosome construction is now open.
Novel approaches to the structural and functional analysis of mammalian chromosomes would be possible if the gross structure of the chromosomes in living cells could be engineered. Controlled modifications can be engineered by conventional targeting techniques based on homologous recombination. Large but uncontrolled modifications can be made by the integration of cloned human telomeric DNA. We describe here the combined use of gene targeting and telomere-mediated chromosome breakage to generate a defined truncation of a human chromosome. Telomeric DNA was targeted to the 6-16 gene on the short arm of chromosome 1 in a human cell line. Molecular and cytogenetic analyses showed that, of eight targeted clones that were isolated, one clone had the predicted truncation of chromosome 1.
Infection of rabbits with Escherichia coli RDEC-1 is a useful model for diarrheal disease caused by mucosally attaching E. coli. Understanding of the protective immunity induced by RDEC-1 infection in rabbits should provide information useful in the design of vaccines for protection against this infection and other mucosally attaching organisms as well. Thus, to define the time course and location of specific immunoglobulin A secretion in relation to bacterial colonization during primary RDEC-1 infection, we infected rabbits with RDEC-1, which express AF/R1 adherence pili, and compared sites of anti-AF/R1 antibody-containing cells in the intestinal mucosa with the sites of luminal colonization and mucosal attachment of RDEC-1. Also, anti-AF/R1 antibodies in intestinal fluids and bile were measured by enzyme-linked immunosorbent assay, and attachment sites of RDEC-1 to the intestinal epithelium were determined by immunohistochemical examination. Anti-AF/R1 pilus antibody-containing cells were most numerous in the proximal intestine (duodenum and jejunum). In contrast, both luminal colonization and attachment of RDEC-1 to epithelial cells were densest in the distal intestine (cecum and colon). Anti-AF/R1 antibodies were present in approximately equal amounts in fluids collected from all levels of the gut after week 1 postinfection. Anti-AF/R1 antibody levels in undiluted bile exceeded those in gut flushes by at least 2 orders of magnitude. Loss of RDEC-1 attachment to epithelial cells preceded resolution of diarrheal illness despite the presence of large numbers of organisms in the intestinal lumen. Our studies indicate that during RDEC-1 infection (i) sites of greatest mucosal anti-AF/R1 antibody secretion are proximal to sites of maximal RDEC-1 luminal colonization and attachment, (ii) bile is a major source of specific antibodies in the intestinal lumen, and (iii) interference with RDEC-1 attachment to epithelial cells may permit resolution of disease.
We have completed a long-range restriction map of the terminal region of the short arm of human chromosome 16 (16p13.3) by physically linking a distal genetic locus (alpha-globin) with two recently isolated probes to telomere-associated repeats (TelBam3.4 and TelBam-11). Comparison of 47 chromosomes has revealed major polymorphic length variation in this region: we have identified three alleles in which the alpha-globin genes lie 170 kb, 350 kb, or 430 kb from the telemere. The two most common alleles contain different terminal segments, starting 145 kb distal to the alpha-globin genes. Beyond this boundary these alleles are nonhomologous, yet each contains sequences related to other (different) chromosome termini. This chromosome size polymorphism has probably arisen by occasional exchanges between the subtelomeric regions of nonhomologous chromosomes; analogous length variation is likely to be present at other human telomeres.
Previously, we discovered a binding site for the Fc region of IgG in human small intestinal and colonic mucosa. The binding site (Fc gamma IBS) appeared to be primarily associated with goblet cells, to consist of greater than 200,000 Da and 78,000 Da components, and to be distinct from leukocyte FcR. In the present work, we used mAb made to colonocyte IgG-binding material to more accurately define the molecular structure and cellular locations of the Fc gamma IBS. In immunoblot and fast protein liquid chromatography analysis, the mAb revealed that the Fc gamma IBS consists of a 110,000- to 140,000-Da component in addition to the two components previously recognized. The greater than 200,000 component may be the critical component for IgG binding, inasmuch as mAb to it but not to the other two components inhibited binding of IgG to colonic sections in vitro. Used in immunoelectron microscopy, the mAb documented that the Fc gamma IBS is present in the endoplasmic reticulum of goblet cells, in the cytoplasmic matrix separating secretory granules of goblet cells, and within the granules themselves; occasionally it has the appearance of being secreted into the intestinal lumen with mucus. The Fc gamma IBS could not be solubilized from colonocyte homogenates by three different detergents, which suggests that it exists in complex with cytoskeletal elements. We speculate that the Fc gamma IBS aids in immunologic protection of the intestine by facilitating interaction between intestinal mucus and antigenic material in the lumen.
The data from published studies of circadian rhythms in epidermal cell proliferation in mice, rats, and humans were reanalyzed. They were calculated as the percent difference from the mean at six timepoints over 24 h. Composite circadian rhythm curves were plotted from the combined data for each species for S-phase and M-phase. Each group of studies showed a general consensus on timing, and the composite curve showed a regular sinusoidal pattern. The rhythms in mice and rats were the same, whereas those in humans were in the opposite phase and had reduced amplitudes. In the rodents, S-phase peaked at about 3:30 A.M. and M-phase peaked at about 8:30 A.M. In humans S-phase peaked at about 3:30 P.M. and M-phase peaked at about 11:30 P.M. If the timing of the circadian rhythms in cell proliferation can be firmly established, it may be possible to schedule drug treatments to take advantage of the differences in cell proliferation rates at different times of day.
We investigated the effectiveness of very low doses of the contact sensitizer dinitrofluorobenzene in sensitizing BALB/cJ mice. Surprisingly, the ear swelling reactions were greater with lower dinitrofluorobenzene doses, down to one-twentieth of doses commonly used. Although it is common practice to use much lower doses at challenge than at sensitization, we found greater reactions with lower doses at sensitization than at challenge. We also studied the timing of the development and waning of reactivity to dinitrofluorobenzene, dinitrochlorobenzene and oxazolone. Reactivity peaked at day 5 for dinitrofluorobenzene and dinitrochlorobenzene, and at day 3 for oxazolone. Reactivity waned by 3 weeks with dinitrofluorobenzene and oxazolone, and by day 7 with dinitrochlorobenzene. Pretreatment with cyclophosphamide caused a delay in the development and waning of reactivity.
Previously, we describe a unique binding site for the Fc region of IgG in human intestinal goblet cells, but regulation of the intestinal IgG Fc binding site (Fc gamma IBS) has not been clarified. In this work, we examined the effects of tumour necrosis-alpha (TNF-alpha) and interferon gamma (IFN-gamma) on expression of the Fc gamma IBS in HT29-N2 colonic cancer cells, which differentiate readily into goblet cells containing the binding site when grown in galactose-containing medium. Expression of the site was monitored immunocytochemically and by ELISA on homogenates of the cells. TNF-alpha in doses of 0.1-100 ng/ml caused a reduction in expression of the Fc gamma IBS and the proportion of cells positive for mucin (as demonstrated by Alcian blue stain), without affecting the viability of the cells. The effects of TNF-alpha on the FC gamma IBS and mucin production could not be attributed to a decreased proliferative rate of the cells, as the cells' incorporation of 5-bromo-2'-deoxyuridine was unaffected. By contrast with TNF-alpha, IFN-gamma (i) did not affect the proportion of cells expressing the Fc gamma IBS, (ii) decreased the viability of the cells, and (iii) increased cell proliferation. Additional evidence of specificity of the TNF-alpha effect on the Fc gamma IBS was that TNF-alpha did not affect expression of the polymeric immunoglobulin receptor (secretory component), whereas IFN-gamma increased it. We conclude that TNF-alpha may suppress expression of the Fc gamma IBS by colonocytes and oppose differentiation of the cells towards mucin-producing cells.
Various pathophysiological relationships between IgA and the asialoglycoprotein receptor on hepatocytes have been proposed. As a step toward defining these relationships, we purified the receptor from human livers by means of p-aminophenyl beta-D-thiogalactopyranoside-agarose affinity chromatography. Unexpectedly, we found the asialoglycoprotein receptor to be associated specifically with IgG. Evidence for the specific association included the following: (a) IgG, but little IgA or IgM, was associated with asialoglycoprotein receptor that had been bound to asialoorosomucoid in an enzyme-linked immunosorbent assay; (b) the asialoglycoprotein receptor-associated IgG was predominantly IgG2 and IgG4 subclasses, whereas serum IgG is predominantly IgG1; and (c) some asialoglycoprotein receptor was specifically recovered together with IgG from an anti-IgG affinity column. The association of IgG with asialoglycoprotein receptor was not mediated by receptor recognition of carbohydrate moieties because IgG neither bound directly to asialoglycoprotein receptor nor inhibited binding of asialoglycoprotein receptor to asialoorosomucoid in vitro. The association could not be attributed to antigen-antibody interaction either. We conclude that IgG is associated in vitro with the asialoglycoprotein receptor by a mechanism not yet fully defined and speculate that the IgG serves an immunological function.