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Active specific immunotherapy of a murine mammary adenocarcinoma using a synthetic tumor-associated glycoconjugate.

A synthetic tumor-associated glycoconjugate (S-TAG) "vaccine" formulation was developed for active specific immunotherapy of a murine mammary adenocarcinoma (TA3-Ha). An S-TAG composed of the Thomsen Freidenreich hapten coupled to a conventional carrier protein (keyhole limpet hemocyanin) and emulsified in Ribi adjuvant, when administered s.c. (in four doses at 3 to 6 days apart) into hosts bearing TA3-Ha tumors, provided 25% long-term survival. When administration of this synthetic glycoconjugate was preceded by treatment with cyclophosphamide (100 mg/kg i.v.), 50% long-term survival was observed for hosts in which the tumor had been established for 5 days and up to 90% long-term survival for groups of mice with tumors established for 1 to 2 days. In contrast, a significantly (P less than 0.025) lower level of survival was observed when cyclophosphamide treatment was preceded by active immunizations with the S-TAG tumor vaccine. Surviving tumor-challenged mice that had been treated with cyclophosphamide and the S-TAG vaccine had relatively good IgG antibody and delayed-type hypersensitivity responsiveness to the synthetic Thomsen Friedenreich determinants. About 30% of these animals were also able to resist and sustain long-term survival when rechallenged with a high dose (1 x 10(4] of TA3-Ha tumor cells. Lymph node cells obtained from surviving animals were highly inhibitory to tumor growth in a Winn-type assay.

Adenocarcinoma↗

Effects of ozone on lamb tracheal mucosa. Quantitative glycoconjugate histochemistry.

Whether or not the previously reported O3-induced abnormality in the postnatal development of tracheal secretory function in lambs is accompanied by changes in epithelial cell populations and their glycoconjugate composition was determined. Six lambs were killed at birth and 12 lambs at age 2 weeks. Of the latter 12, six were exposed to O3 (1 ppm, 4 hours daily for 5 days during the 1st week of life) and five had air-sham exposures (controls). Tracheal glycoconjugates were localized in situ with lectins to detect N-acetyl-galactosamine (galNAc), alpha-D-galactose (alpha-gal), beta-D-gal(1----3)-galNAc (beta-gal), and fucose (fuc). Mean (+/- SD) epithelial cell density (cells/mm basal lamina) was 418 +/- 57 in the newborns, 385 +/- 63 in controls (P was not significant), and 342 +/- 47 in O3-exposed lambs (P less than 0.05). Mucous cell density was 87 +/- 12 in newborns, 63 +/- 10 in controls (P less than 0.05), and 76 +/- 10 in O3 exposed lambs (P was not significant). Ciliated cells remained unchanged from birth to 2 weeks (P was not significant), but decreased (P less than 0.05) in O3-exposed lambs. All counted mucous cells contained fuc and galNAc at birth and retained these residues after sham and O3 exposure. The alpha-gal-containing mucous cells declined from 97 +/- 13 to 7 +/- 1 (P less than 0.05) and beta-gal containing cells from 39 +/- 5 to 25 +/- 4 in controls. In contrast, cells containing alpha-gal 71 +/- 10 remained at newborn levels (97 +/- 13) and beta-gal-containing cells increased from 40 +/- 5 at birth to 58 +/- 8 in O3-exposed animals (P less than 0.05). It was concluded that early postnatal exposure of lambs to O3 causes a decrease in epithelial cell density, but retards the developmental decrease in the number of tracheal mucous cells and alters the lectin detectable carbohydrate composition of mucus in these cells. These developmental defects were interpreted to be the morphologic correlates of the previously shown effects of O3 on the maturation of secretory function and mucus transport.

Acetylgalactosamine↗

The identification of membrane glycoconjugates in Leishmania species.

The membrane glycoconjugates of 8 different species of Leishmania were compared by lectin blotting. Five different lectins with various sugar specificities were examined: concanavalin A, Lens culinaris, Ricinus communis, soybean agglutinin, and peanut agglutinin. Concanavalin A and Lens culinaris reacted with every Leishmania tested. The patterns observed for these 2 lectins, as well as the various species of parasites, were different. However, a common 41,000-52,000 and a 160,000-185,000 Mr component was present in almost all the parasite isolates examined. Ricinus communis only recognized a nondiscrete galactose-containing glycoconjugate similar to Leishmania-excreted factor. Soybean and peanut agglutinins reacted with a few low molecular weight parasite components. Soybean agglutinin reacted with all the Leishmania species tested, whereas peanut lectin only recognized 3 isolates. The latter lectin bound to discrete components migrating with the dye front and with Mr's of 35,000 and 52,000. Increased glycosylation was noted on avirulent L. major promastigotes and was associated with the appearance of several new peanut agglutinin-binding glycoproteins.

Agglutination↗

Lewis-like activities of and rabbit antisera against glycoconjugates present in edible organs of phanerogamia and cryptogamia.

13 species of phanerogamia and cryptogamia were found to contain substances cross-reacting with anti-Lewis agglutinins in ethanol precipitates from their edible organs such as leaves. Crude glycoconjugates were isolated from some species by Sephacryl S-200 gel filtration, immunization with some of which resulted in the production in rabbits of anti-Lea-like agglutinins with a high titer. Production of anti-Lea-like precipitin with high specificity was also confirmed in a rabbit antiserum against the glycoconjugate prepared from young leaves of Aralia elata.

Animals↗

Chronic systemic treatment with epidermal growth factor in pigs causes pronounced urothelial growth with accumulation of glycoconjugates.

Epidermal growth factor (EGF) is present in large amounts in the urine, but the effects of systemically administered EGF on the urinary tract have not been described previously. In the present paper, we describe a potent growth induction of EGF on the urinary tract. Goettingen minipigs were treated with solvent (n = 5), EGF 30 micrograms/kg/day (n = 6) for 4 weeks, or EGF 30 micrograms/kg/day for 5 weeks followed by 3 weeks of recovery (n = 5). The ureters and bladders were examined by routine histology and electron microscopy and were immunostained for proliferating cell nuclear antigen. Four weeks of EGF treatment increased the median cross sectional area of the ureter fourfold with growth of all wall layers. The urothelium was widened from 5 cell layers in the controls to 10 in the EGF-treated animals. Proliferating cell nuclear antigen immunostaining revealed an increased mitotic activity in the basal zone of the urothelium. In the luminal zone, glycoconjugates accumulated in goblet cells, in cells with intracytoplasmic lumina, and beneath the luminal cell membrane in the umbrella cells. Our studies present a new experimental approach to growth induction of the urinary tract. The findings implicate the EGF system in regulating urothelial growth and glycoconjugate biosynthesis.

Animals↗

Light microscopic histochemical detection of sugar residues of glycoconjugates in human nasal mucosa using HRP-lectins.

A battery of horseradish peroxidase-conjugated lectins (ConA, WGA, PNA, SBA, DBA, UEAI and LTA) was used to study the content and distribution of glycoconjugate sugar residues of the normal human nasal mucosa. Our findings indicate that differences exist between the types of sugar residues found in the secretory product of the mucous goblet-like cells and those found in the mucous glands secretion. The saccharide content of the epithelial basal cells, which has never been previously investigated, is reported. Differences in the content and distribution of glycoconjugate sugar residues were found between human nasal mucosa and that of the other human extrapulmonary tracts.

Adolescent↗

Monomeric, monovalent derivative of Maackia amurensis leukoagglutinin. Preparation and application to the study of cell surface glycoconjugates by flow cytometry.

A stable subunit of Maackia amurensis leukoagglutinin (MMAL) was prepared by the selective reduction of disulfide bridges between the subunits followed by alkylation with 4-vinylpyridine. MMAL failed to precipitate fetuin, whereas it retained its ability to bind to the same glycoprotein coated on a plastic plate, indicating the monovalency of this derivative. This binding to immobilized fetuin was inhibited by a haptenic sugar, Neu5Ac alpha 2-3lactose, with the same inhibitory potency as against the native M. amurensis leukoagglutinin. Microscopic observation as well as flow cytometric analyses showed that Chinese hamster ovary cells were clearly stained with fluorescein isothiocyanate-labeled MMAL without any detectable agglutination. This staining was inhibited by the addition of fetuin or by the sugar chains of fetuin. Differences in the types of sialylated glycoconjugates on the cell surface of several cell lines were detected by the combined use of fluorescein isothiocyanate-labeled MMAL and the monomeric derivative of elderberry bark lectin (specific for the Neu5Ac alpha 2-6Gal/GalNAc sequence) by flow cytometry. These results demonstrate the usefulness of these monovalent derivatives of sialylated oligosaccharide-specific lectins as probes for the analysis of cell surface glycoconjugates containing sialic acid by the technique of flow cytometry.

Amino Acids↗

GPI-anchored glycoconjugates from Trypanosoma cruzi trypomastigotes are recognized by lytic anti-alpha-galactosyl antibodies isolated from patients with chronic Chagas' disease.

The target molecules on the cell surface of Trypanosoma cruzi trypomastigotes reacting with lytic anti-alpha-galactosyl antibodies from chronic patients with Chagas' disease (Ch anti-Gal) have been purified by solvent extraction and identified as glycoconjugates migrating in the 74-96-kDa range (F2 antigen) and in the 120-200-kDa range (F3 antigen) on SDS-PAGE. The F3 antigen was tested for binding to Ch and normal human serum (NHS) anti-Gal and to MoAb 3C9. We observed that Ch anti-Gal and MoAb 3C9, but not NHS anti-Gal, bind strongly to the trypomastigote glycoconjugates. These antibodies, however, did not compete with each other for binding to F3 molecules, indicating that they are recognizing different epitopes. Binding of Ch anti-Gal to F3 antigen is abolished by treatment of these molecules with alpha- but not beta-galactosidase. Binding of 3C9 MoAb is abolished by treatment of F3 with sialidase. F2/F3 antigens absorbed Ch anti-Gal as well as lytic antibodies from total chagasic sera. These antigens also specifically discriminate between the serum reactivity of patients with active Chagas' disease and those of sera from cured patients, drug-treated patients with dissociated serology (positive conventional serology, negative trypanolytic activity), healthy individuals, and patients with several other infectious diseases. We also observed that F2/F3 antigens are anchored to the parasite membrane via glycosylphosphatidylinositol (GPI). The alpha-galactosyl epitopes recognized by Ch anti-Gal are present in a series of O-linked oligosaccharide chains in the mucin-like glycoprotein component of the complex.

Animals↗

Glycoconjugate mediated endothelial cell adhesion to Dacron polyester film.

PURPOSE: The purpose of this study was to explore new strategies for enhancing specific cell type attachment to biomaterials using immobilized lectins for cell surface glycoconjugates. The lectin Ulex europaeus I (UEA I) has a high affinity for human vascular endothelial cell surface glycoconjugates. METHODS: UEA I was covalently bound to polyethylene terephthalate (Dacron) with the cross-linking agent 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride to achieve oligosaccharide-mediated endothelial cell attachment to this otherwise nonadherent surface. RESULTS: Experiments with radiolabeled UEA I demonstrated covalent linkage of as much as 1.35 micrograms/cm2. The lectin binding site is available after the reaction, as demonstrated in experiments a neoglycoprotein. Adhesion studies reveal a 100-fold increase in endothelial cell attachment for the UEA I/polyethylene terephthalate surface (99.7 +/- 29.6 cells/high-power field) when compared with untreated (0.7 +/- 0.5), crosslinking agent (0.4 +/- 0.3), and denatured UEA I (1.2 +/- 1.1) control groups. Five vascular endothelial cell lines adhered to the UEA I/polyethylene terephthalate surface, whereas monocytes, smooth muscle cells, and fibroblasts did not. CONCLUSION: These results imply new strategies for endothelialization of prosthetic grafts and promotion of selective cell adherence to biomaterials, with emphasis on carbohydrate interactions. Moreover, this experimental system offers a model for exploring the biologic significance of the endothelial cell-UEA I ligand.

Cell Adhesion↗

Differential distribution of glycoconjugates in human reproductive tract.

OBJECTIVES: To study the distribution of glycoconjugates in the epithelia of various regions of the human female reproductive tract and to examine whether lectins can be used as specific probes to define cell populations in the human female reproductive tract. DESIGN: Nineteen fluorescein isothiocyanate (FITC)-labeled lectins, which recognize different oligosaccharide structures, were applied to frozen sections of human fallopian tubes (distal and proximal segments) and endometrium. The differential binding patterns of the lectins to the epithelia were evaluated under fluorescent microscopy. RESULTS: Four of 19 FITC-labeled lectins tested demonstrated distinct binding patterns along the reproductive tract. Arachis hypogaea (PNA) and Ricincus communis II selectively reacted with epithelial cells of endometrium and distal, but not proximal tube. In addition, PNA distinguished ciliated from secretory cells of the distal tube. Maclura pomifera binding followed a gradient of decreasing intensity from distal to proximal tube and was negative in the endometrium. Dolichos biflorus reacted exclusively with proximal tubal epithelium. CONCLUSIONS: Analysis of the FITC-lectin binding pattern and the sugar specificities indicate that the distribution of galactosyl residues varies among different regions of the reproductive tract, whereas glucosyl, mannosyl and fucosyl residues are more evenly distributed. The differential expression of glycoconjugates may be involved in the distinct biological function of each region in female reproductive tract.

Endometrium↗

Histochemical analysis of lizard testicular glycoconjugates during the annual spermatogenetic cycle.

The distribution of glycoconjugates in the testis of lizard Podarcis s. campestris De Betta was investigated by HRP-conjugated lectins during the annual spermatogenetic cycle. In addition, pretreatments of sections with neuraminidase and removal of alkali-labile O-linked sequences by beta-elimination allowed the structure of glycoconjugates to be further explored. Leydig cells displayed changes of lectin-binding sites during annual cycle, and during the abortive spermatogenesis period lacked N-linked sialylgalactosyl glycans. Sertoli cells stained by Con A, WGA, RCA120, BS I-B4, showed, except in July, O-linked sialylgalactosyl glycans. Spermatogonia bound Con A and WGA. Spermatocytes bound also BS I-B4, SBA, UEA I, and during spring spermatogenesis, revealed O-linked sialylgalactosyl glycans. The acrosomes of spermatids were also stained by RCA120 and PNA, whereas the heads of spermatozoa did not bind SBA and PNA. During the abortive spermatogenic period, the acrosomes showed O-linked sialylgalactosyl glycans and N-linked glycans terminating in beta-galactosyl residues. During the reproductive period, the acrosomes of spermatozoa expressed O- and N-linked sialylgalactosyl glycans and beta-galactosyl terminal residues on O- and N-linked glycans. This, in the testis of lizard, the two spermatogenesis periods show the emergence of different types of glycosylation.

Acrosome↗

Glycoconjugate vaccines: future combinations.

There is a large and growing base of knowledge about the protective immune response to encapsulated bacterial pathogens. This information is a clear advantage to the development of glycoconjugate vaccines. It is well understood for such pathogens as Haemophilus b, pneumococcus, meningococcus and group b streptococcus that the induction of a functional anti-saccharide antibody response is likely to be protective. The most important question for glycoconjugate vaccines is the magnitude of the response required for protection. Even after extensive field trials with Haemophilus b vaccines, there is not a precise antibody correlate of immunity. In fact, cited values still rely on older studies with polysaccharide vaccine [11]. For that reason, vaccine such as the multivalent pneumococcal formulations will require field trials to measure their efficacy and hopefully determine the appropriate correlates of immunity. However, the addition of new serotypes into a multivalent pneumococcal formulation after efficacy trials are completed will probably have to be based on antibody correlates since it will not be possible to do efficacy studies on individual serotypes especially since they will have low prevalence. A similar dilemma may be faced with a vaccine for the group C meningococcus where the low disease incidence will make efficacy studies difficult. As was the case with mixing DTP and Hib vaccines, in the future combinations of effective antigens will continue to rely on comparative immunogenicity studies to ensure efficacy. It is clear that the immune system is capable of responding to a diversity of antigens simultaneously. However, it is also clear that vaccine components can interfere with each other and this interference is not necessarily predictable [12]. The ability to measure the stability of each of the components of a combined product reliably will become even more critical as the complexity of the mixtures increases.

Animals↗

Modulation of the glycoconjugate expression in the tracheo-bronchial epithelium during sustained hypovitaminosis A.

The aim of this study was to determine the influence of sustained marginal vitamin A deficiency on the morphology of glycoconjugate expression in the tracheobronchial epithelium of guinea pigs. The distribution of oligosaccharide chains was investigated by applying a panel of 24 lectins. Glycosaminoglycans were detected by histochemical techniques. Number as well as morphology of ciliated cells showed no significant alterations in hypovitaminosis A. In contrast, the quantity of goblet cells was constantly decreased. A considerable reduction of secretory granules was also observed in these cells. Cytomembranes of ciliated cells (especially in the area of ciliar extensions) showed constant alterations in the patterns of lectin binding in vitamin A-depleted guinea pigs. Our results demonstrate a significant augmentation of accessibility of fucosyl molecules in proximal domains of glycoconjugates of ciliary membranes, whereas the presence of mannose structures seemed unchanged. In distal bronchioli, terminal N-acetylgalactosamine molecules were expressed. During marginal vitamin A deficiency, ciliary cells were specially labelled by GSA I(B), indicating presentation of terminal galactose molecules in alpha-position. Additionally, the cytoplasm of epithelial cells demonstrated enhanced concentrations of polyantennary oligosaccharide core structures. Staining of epithelial cells by VVA was restricted to control specimens. Abundance of N-acetylglucosamine residues on the non-reducing terminus of oligosaccharides was significantly enhanced in the connective tissue of depleted animals as demonstrated by the binding patterns of GSA II. We suggest that altered oligosaccharide patterns may contribute to enhanced predisposition to tracheobronchial infection in marginal vitamin A deficiency.

Animals↗

Lectin-histochemical study of O-linked glycoconjugates in dysplastic retina of Norrie disease.

The carbohydrate chains of O-linked glycoconjugates in dysplastic retina from a Japanese female infant with Norrie disease were examined by lectin histochemistry. The avidin-biotinylated peroxidase method was used. The retina was highly dysplastic and composed of undifferentiated embryonic tissues containing a number of rosettes of varying sizes. The lumina of the rosettes were stained by peanut agglutinin, which recognizes the Gal beta 1,3GalNAc sequence of O-linked glycans. However, the lumina were not labeled by wheat germ agglutinin, which reacts with sialic acid and/or N-acetylglucosamine. These observations suggest that the O-linked glycoconjugates in the lumina of rosettes were not sialylated in the present case. Their lack of terminal sialic acids may be related to the rosette formation.

Acetylglucosamine↗

[Anti-glycoconjugate antibodies and dysglobulinemic or dysimmune peripheral neuropathies].

Different anti-glycoconjugate auto-antibodies are described to be associated with peripheral neuropathies; the anti-MAG monoclonal IgM activity is determined by ELISA or immunoblotting essays, anti-glycolipid activity by ELISA or chromatography and monoclonal gammopathies by commercially available kits. These investigations are limited by possible cross-reactions due to the common glycosilate epitope. The peripheral neuropathies with anti-glycoconjugate auto-antibodies are different: Guillain-Barré syndrome and its variants (anti-GM1, GQ1b, LM1, GD1, GD1a gangliosides or sulfatides), motor neuropathy (anti-GM1 ganglioside), sensory neuropathies (anti-GD3, GD1b, GD1a, GQ1b gangliosides or sulfatides), and chronic myelinic neuropathy with IgM gammopathy (anti-MAG). We review the major clinical and electrophysiological features and discuss the underlying pathophysiological mechanisms.

Autoantibodies↗

Glycoconjugates during the annual sexual cycle in lizard epididymal ductuli efferentes: a histochemical study.

The epididymal ductuli efferentes of the lizard Podarcis sicula campestris De Betta are lined with simple, columnar, nonciliated and ciliated cells. The use of lectin histochemistry has provided information about changes of sugars associated with glycoconjugates of epithelial cells and intraluminal spermatozoa which are conveyed from the longitudinal canal to the cranial region of the ductus epididymis. Epithelial cells exhibited residues of alpha-D-mannose, N-acetylglucosamine, and beta-D-galactose-(1-4)-N-acetylglucosamine as revealed with lectins Con A, WGA and RCA120, respectively, throughout the sexual cycle. An increase of RCA120 staining was observed on microvilli of nonciliated cells and in the cytoplasm of ciliated cells during the reproductive period. However, during the following refractory period, when the organ was in regression, there was a decreased staining with Con A on microvilli and the absorbent surface of nonciliated cells, with WGA in nonciliated cells and the cytoplasm of ciliated cells, and with RCA120 on microvilli and the cytoplasm of both cell types. Terminal alpha-D-galactose residues were increasingly stained from autumn up to the reproductive period with BS I-B4 on microvilli, the absorbent surface and cilia, whereas they were entirely lacking during the refractory period. UEA I revealed alpha-L-fucose residues on the absorbent surface of nonciliated cells during the abortive and reproductive periods, increasing in the latter period when cilia also expressed this sugar. Terminal alpha/beta-D-N-acetylgalactosamine was evidenced with SBA on the absorbent surface of nonciliated cells during the reproductive period. The terminal beta-D-galactose-(1-3)-N-acetylgalactosamine dimer was never found with PNA, whereas O- and N-linked sialoglycoconjugates were present only during the reproductive period. The spermatozoa head exhibited N-linked glycans with high-mannose content, and beta-D-galactose-(1-4)-N-acetylglucosamine as well as O- and N-linked sialoglycoconjugates throughout the year. During the reproductive period, oligosaccharides with alpha-D-mannose residues increased, and oligosaccharides with terminal alpha-D-galactose, alpha-L-fucose and sialic acid-N-acetylgalactosamine dimer were also present. Unlike spermatozoa of seminiferous tubules, the spermatozoa head of the lizard epididymal ductuli efferentes exhibited seasonal variability in the lectin binding pattern which may be related to time-dependent changes in the glycoconjugate profiles of epithelial cells.

Animals↗

Glycoconjugate profile in plasma and erythrocytes of gastric cancer patients.

The present study has examined the glycoconjugate profile in plasma and erythrocyte membranes of 24 adult male gastric cancer patients and an equal number of age and sex-matched controls. Protein-bound hexose, hexosamine and sialic acid were significantly increased in plasma and erythrocytes of gastric cancer patients compared to controls. Elevation of glycoconjugates in circulation is suggested to be a result of increased shedding by the tumor cells or increased synthesis by liver, due to acute phase response.

Adenocarcinoma↗

Light and electron microscopic cytochemistry of glycoconjugates in the rectosigmoid colonic epithelium of the mouse and rat.

The several cell types in mouse and rat rectosigmoid colon have been examined with light and electron microscopic methods for localizing and characterizing complex carbohydrates. Mucous cells, also termed vacuolated cells, and goblet cells comprised most of the deep crypt epithelium in both species, and absorptive columnar cells and goblet cells mainly populated the more superficial epithelium of the upper crypts and main lumen. Occasional tuft cells and enteroendocrine cells were also encountered. Transitional cells structurally intermediate between mucous cells and absorptive cells contained granules characteristic of mucous cells and vesicles like those of columnar absorptive cells. These intermediate cells supported the concept of replacement of mucous by absorptive cells through transformation of mucous into absorptive cells. The intermediate cells also contained numerous lysosomes often in apparent fusion with mucous granules, indicating crinophagic disposal of mucous granules as a mechanism in the cell transformation. Glycoconjugate in absorptive cell vesicles resembled that coating the apical plasmalemma and appeared to represent the source of the glycocalyx of the brush border. Complex carbohydrate in these vesicles differed cytochemically from that of the mucous cell granules, which release their content into the crypt lumen. The absorptive cell vesicles, therefore, constitute an organelle distinct from the mucous cell granules rather than an atrophic form of the latter in a more mature cell. Goblet cells differed in failing to transform morphologically with age but changed in the cytochemical characteristic of their secretion during migration up the crypts. Terminal N-acetylglucosamine residues diminished, while terminal sialic acid-galactose dimers increased during the upward migration, indicating activation of glycosyl transferase synthesis in relation to goblet cell maturation. Glycoconjugate in secretion of mucous cell granules differed markedly from that in goblet cell granules, and content of both organelles differed from that of absorptive cell vesicles. However, secretion in mucous cell granules appeared generally similar for mice and rats with minor exceptions, and secretion in goblets of mice generally resembled that in goblets of rats. Cells interpreted tentatively as Kulchitsky cells stained for high content of fucose with the Ulex europeus I lectin. Globoid leukocytes infiltrating the epithelium of the rat but not the mouse rectosigmoid colon resembled globoid leukocytes in rat tracheal epithelium and, like the latter, appeared to derive from mast cells.

Animals↗