Monoclonal antibody FW6 defines an epitope on alpha 3/4-monofucosylated polylactosaminoglycans expressed by fetal and colon carcinoma-associated mucins.

A mouse IgM monoclonal antibody FW6 was established after immunization of mice with mucins from human amniotic fluid and was characterized with regard to its binding epitope. According to a series of biochemical criteria the epitope is located on O-linked neutral carbohydrates of M(r) 700,000 and M(r) 570,000 mucins in human amniotic fluid. The epitope is presumed to contain alpha 3/4-linked fucose and terminal beta 3/4-linked galactose that are labile to the fucosidase I from almond emulsion or to the galactosidase from bovine testes, respectively. Immunoreactive fractions of glycan alditols from human amniotic fluid mucins were partially characterized by fast atom bombardment-mass spectrometry and methylation analysis to be composed of monofucosylated polylactosamine-type deca- or nonasaccharides. According to antibody competition studies, inhibition assays with defined carbohydrates and binding assays on neoglycolipids monoclonal antibody FW6 are presumed to recognize a novel epitope that is distinct from known carbohydrate markers of the Lex/Ley family associated with colonic carcinomas. The selective reactivity of this monoclonal antibody to the majority of human colonic carcinomas and its nonreactivity to normal colonic mucosa may render this antibody as a valuable tool in cancer diagnosis or cancer treatment.

Occurrence and distribution of glycoconjugates in human tissues as detected by the Erythrina cristagalli lectin.

We applied a horseradish peroxidase-Erythrina cristagalli agglutinin (HRP-ECA) conjugate for histochemical staining of tissue sections from various formalin-fixed, paraffin-embedded human tissue specimens. The HRP-ECA conjugate showed broad reactivity, but there was a distinct distribution of native (not masked by sialic acid) and sialic acid-masked ECA binding sites in the various organs. Free ECA binding sites could be detected on red blood cells, lymphocytes of thymus, tonsil, lymph node, and in mucous substances of different organs. Independent of blood group type, the vascular endothelium exhibited strong ECA reactivity. Free ECA binding sites occurred in the cytoplasm of Kupffer's cells in liver, in histiocytic cells of thymus, lymph node, tonsil, and in bone marrow. Podocytes of kidney glomerulus, syncytiotrophoblasts of placenta, megakaryocytes in bone marrow, myelin sheath of nerve, medullary thymocytes, and hepatocytes, as well as islet cells of pancreas, contained only sialic acid-capped ECA binding sites. Inhibiting studies with galactose, lactose, and N-acetyl-lactosamine, as well as other sugars, revealed that this lectin is specific for galactosyl residues. In comparison to galactose and lactose, N-acetyl-lactosamine exhibited the highest inhibitory activity on lectin binding, supporting the concept that this lectin is most reactive with N-acetyl-lactosamine-type (type 2 chain) glycoconjugates.

Lectin-binding sites in human parathyroid tissue.

The aim of this study was to demonstrate several lectin-binding sites in human parathyroid tissue and to correlate these results with functional activity. The following lectins were tested for binding sites with certain carbohydrates (in parentheses): Arachis hypogea (PNA) (galactose), Ulex europaeus I (UEA) (fucose) and concanavalin A (ConA) (mannose). In addition to normal parathyroids used as controls (13 cases), we examined adenomas associated with a clinical picture of primary hyperparathyroidism of differing severity (31 cases), atrophic glands contralateral to a hyperfunctioning adenoma (7 cases), and secondary (renal) hyperplasia (12 cases). Use of PNA (with and without neuraminidase treatment) and UEA yielded negative staining in normal glands, a wide variety of reactions in adenomas, and frequent dense precipitates in atrophic parathyroids, whereas ConA yielded positive staining in all kinds of parathyroid tissue. Assessment of functional activity of adenomas by clinical parameters (pre-operative serum levels of calcium and parathormone) displayed a significant correlation with the semiquantitative grading of the histochemical reactions after PNA and UEA. Lectin-binding sites in parathyroid chief cells of adenomas are believed to indicate some of the cell structures or products directly involved in the secretory process, including degradation. Although ConA may recognize constituent parathyroid glycoproteins, the binding sites for PNA and UEA are thought to be partially associated with secretory glycoprotein (SP-I), as is known from animal experiments. The positive reaction of the atrophic gland may result from degradation enforced by exposure of primarily non-terminal carbohydrate components.

Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. II. Lectin binding to the isolated glycoproteins of normal and malignant gastric mucosa.

Using affinity chromatography on HPA-, PNA-, Con A, and WGA-agarose columns only a part (10-30%) of the high molecular weight mucous glycoproteins could be isolated from the Triton X-100 solubilized components of normal as well as carcinomatous gastric mucosa. The main part of the mucus was not bound by the lectins, which corresponds to our earlier lectin histochemical observations on paraffin-embedded tissue sections. The lectin-bound mucous glycoproteins had a relatively lower molecular weight, ranging from about 250-1,000 kilodaltons, as indicated by polyacrylamide gradient gel electrophoresis and by gel filtration on Biogel A 1.5 m column. In gas chromatographic analysis the molar ratio of aminohexoses to galactose was found to be much higher (3:1) in the lectin-bound mucous substances than in the whole high molecular weight mucus (1:1). This finding indicates that lectins have a higher affinity to the hexosamine rich components of mucus, which may be special forms of mucous glycoprotein molecules or the incompletely glycosylated core and backbone regions of the oligosaccharide chains of mucus. Extremely high hexosamine values (10:1) were found in the PNA isolated mucus of gastric adenocarcinoma. Since it is known that PNA binds to the terminal disaccharide, beta-galactose-(1-3)-N-acetylgalactosamine, which is localized at the reducing end of the oligosaccharide chains of mucus, it is highly probable that the elongation of the oligosaccharide side chains is disturbed in gastric cancer cells.

Histochemical reactivity of the Geodia cydonium agglutinin (GCA) in human tissues.

We applied a peroxidase-antiperoxidase technique to study the distribution pattern and binding characteristics of the lectin from the marine sponge Geodia cydonium (Geodia cydonium agglutinin; GCA) in various human tissues. This lectin has been shown to possess a broad reactivity, but there was a distinct distribution of binding sites within the different organs. In the histochemical system GCA displayed no blood group specificity and labeled red blood cells, the vascular endothelium, and epithelial cells showing blood group antigen expression independent of the ABH blood group status. However, inhibition of GCA reactivity by simple sugars and complex carbohydrates demonstrated tissue-specific differences of lectin binding related to the ABH blood group status of the tissue and revealed information on the structural requirements of the histological lectin binding site. Tissues that totally lacked blood group antigens or that expressed only the H-antigen disclosed a GCA reactivity which was completely inhibited by lactose. In contrast, tissues that expressed blood group A- or blood group B-antigen exhibited a lactose-resistant lectin binding which was inhibited only by water-soluble blood group substance A from peptone A and by bovine glycophorin but not by other complex carbohydrates, including human glycophorin and human asialoglycophorin. Competitive inhibition studies in situ revealed that GCA binding was not inhibited by blood group type I/II carbohydrate sequence-specific lectins or by lectins with other sugar specificities. Inhibition by lactose of GCA binding to some histological sites indicates that the binding site consists of a beta-linked galactose-containing disaccharide. However, periodate oxidation of tissue sections had no effect on lectin binding, pointing to a subterminal location of the relevant sequence. The results obtained from inhibition studies with simple saccharides and complex carbohydrates in relation to the expression of ABH blood group antigens suggest a complex lectin combining site(s) in histological specimens. The lectin may possess either one binding site with a range of affinities for different carbohydrates (besides beta-linked disaccharides the GCA binding site accommodates to carbohydrate determinants carrying the blood group A or blood group B determinant), or may possess two different binding sites. Besides an acceptor site for beta-linked disaccharides, an additional binding site may exist accommodating to extended carbohydrate sequences related to A or B blood group structures. In conclusion, GCA represents a blood group-nonspecific lectin whose binding affinities are determined by the ABH blood group status of the tissue.

Characterization of glycoconjugates of human gastrointestinal mucosa by lectins. I. Histochemical distribution of lectin binding sites in normal alimentary tract as well as in benign and malignant gastric neoplasms.

Labeled lectins with binding specificity to the hexose components of mucus glycoproteins (HPA, RCA I, PNA, Con A, WGA, and UEA I) were used to demonstrate structural differences in the glycoprotein composition of various cell types of the normal, benign and malignant gastrointestinal mucosa. While in the RCA I, UEA I, and WGA binding of normal mucus secreting cell types only quantitative differences were observed, the mucus in the surface epithelial cells of gastric mucosa and in the colonic goblet cells was characterized by the absence of PNA, Con A, and PNA, HPA binding sites, respectively. These lectins, however, showed a strong binding to the supranuclear, Golgi-region in the undifferentiated or activated forms of these cells. Even the staining intensity of the luminal membrane surfaces of the non mucinous parietal and chief cells was often stronger by PNA, HPA, and RCA I lectins than that of the mucus secretions in the highly differentiated mucus cells. These results indicate the existence of either heterogeneous glycoprotein components or mucus molecules with variations in the degree of glycosylation of their oligosaccharide chains in the different cells. The latter seems more likely since in benign and malignant alterations lectin binding sites appear in great density, which were found to be characteristic of the undifferentiated mucus cells or for the non mucinous cells of the normal gastric mucosa. Similarly in some gastric cancers which do not stain with the periodic acid-Schiff reaction at all, large amount of free or neuraminic acid substituted PNA binding sites can be detected.

Immuno- and histochemical studies on galactan and human blood group-related receptors in the bovine lung.

Using a monoclonal anti-galactan antibody and streptococcus B type II antibody, the distribution of lung galactan could be demonstrated for the first time in a vertebrate organ. In addition to the immunochemical demonstration of the bovine lung galactan, a human blood group A-like glycoprotein is detectable by lectinological methods in the bovine lung tissue. Various other lectin-receptors, for instance those of the peanut lectin (PNA) or for lectins with L-fucose (UEA) and N-acetyl-lactosamine (ECA) specificity show a typical staining pattern in bovine lung.

Isolation, characterization and implications of anti-TF (Thomsen-Friedenreich) agglutinins from different sources.

Anti-TF agglutinins from peanut (Arachis hypogaea) and from vertebrate sera of different species have been successfully isolated by affinity chromatography on acid-activated Sepharose 4 B. The proteins were characterized by immunoelectrophoresis, polyacrylamide gel electrophoresis in the presence of SDS and with respect to their carbohydrate binding specificities. Anti-TF substances from sera showed one precipitin arc in immunoelectrophoresis, but quantitative immunoprecipitation revealed our human anti-TF to be a mixture of the three Ig-classes IgG, IgA and IgM. This finding was confirmed on SDS gel electrophoresis, where high molecular weight aggregates were found before reduction. Hemagglutination inhibition revealed that all isolated anti-TF compounds exhibit an exceptionally high affinity for the immunodominant group of the TF-antigen, namely the beta-D-galactosyl-(1 leads to 3)-N-acetyl-D-galactosamine disaccharide. On examination of formalin-fixed and neuraminidase treated tissue sections (kidney, mammary gland), fluorescein-labelled anti-TF from horse serum showed a virtually identical pattern when compared with fluorescein labelled peanut lectin. Likewise isolated IgA-class myeloma J 539, which shows specificity against beta-(1 leads to 6)-galactans, only bound to the appropriate Gal-beta-(1 leads to 6)-Gal structures, such as those found on bovine lung or the albumin gland of Helix pomatia. Rabbit anti-VCN (Vibrio cholerae neuraminidase) activity could be selectively abolished by beta-galactosyl-containing inhibitors, whereas papain F(ab) fragments from rabbit anti-VCN immunoglobulin did not compete with anti-TF for binding sites on VCN-treated human red cells. Anti-TF, on the other hand, did not compete with anti-VCN for active VCN.

Native and sialic acid masked Lewis(a) antigen reactivity in medullary thyroid carcinoma. Distinct tumour-associated and prognostic relevant antigens.

Forty-six medullary thyroid carcinomas (MTC) were subjected to a qualitative and quantitative characterization of native and sialic acid masked Lewis(a) (Le(a)) antigens. Immunohistochemical investigations included monoclonal antibodies (MABs) directed against alpha(2,3)-sialyl-Le(a), i.e. CA19-9 (MAB 19-9), native Le(a) (MAB anti Le(a)) and alpha(2,3)sialyl type 1 structure, i.e. CA 50 (MAB C50). To detect sialic acid masked Le(a) reactivity, MAB anti-Le(a) was also applied to native and enzymatically desialylated tissue sections with and without masking of sialic acid residues by sialic acid and sequence specific lectins. Only 7 MTC (15%) displayed a weak expression of CA19-9, while 16 (33%) showed moderate positive staining for native Le(a). Twenty-seven tumours exhibited a strong staining by the N'ase MAB anti Le(a) staining sequence. The latter could most effectively be inhibited by the simultaneous masking of alpha(2,3)-and alpha-(2,6)-linked sialic acid residues due to the competitive binding of sialic acid and sequence specific lectins: Maackia amurensis agglutinin (specific alpha(2,3)-linked sialic acid) and Sambucus nigra agglutinin (specific alpha(2,6)-linked sialic acid). Thus, in MTC the major portion of sialic acid masked Le(a) antigen reactivity is different from that detected by the MAB 19-9. The antigen reactivity is probably due to Le(a) structures containing both alpha(2,3) and alpha(2,6)-linked sialic acid residues. A highly significant correlation between the expression of CA50 and that detected by the N'ase MAB anti-Le(a) staining sequence indicates that the alpha(2,3)-sialyl type 1 chain represents a common intermediate structure within the pathway of the biosynthesis of sialylated Le(a) antigens, excluding the formation of CA19-9 via the formation of the disialyl type 1 structure. This is subsequently fucosylated to the corresponding sialic acid masked Le(a). Preliminary clinicopathological studies indicate that the sialic acid masked Le(a) antigens detected by the N'ase MAB anti-Le(a) staining sequence are related to biologically aggressive MTC.

Antigen CA 19-9: presence in mucosa of nondiseased müllerian duct derivatives and marker for differentiation in their carcinomas.

CA 19-9, a side branch of the Lewis blood group system, is a sialylated Lewis A antigen that is highly expressed by many adenocarcinomas of the digestive tract. The müllerian duct-derived mucosa of the uterus and fallopian tubes also synthesizes Lewis blood group antigens. To test whether the expression of CA 19-9 is enhanced in carcinomas of müllerian duct origin, we performed immunohistochemical staining for CA 19-9 in normal tissues from 33 women and in adenocarcinomas from 88 patients. In the normal uterine cervix, CA 19-9 was expressed in the cytoplasm of scattered glandular cells in 26 of 29 specimens. It was observed in the apical regions of mucosal cells in six of 26 normal endometrial samples and two of 13 normal fallopian tube specimens. These results are consistent with the presence of antigen CA 19-9 on a secretory product of the nondiseased mucosa of the müllerian duct. In adenocarcinomas of the endocervix, endometrium, and fallopian tubes, CA 19-9 was found in seven of 11, 57 of 71, and five of six samples, respectively. Progressive loss of differentiation was accompanied by disruption of subcellular localization of CA 19-9 and its secretion toward the glandular lumina. In well-differentiated regions of tumors, the antigen was detected mainly at the luminal surface of cancerous glands, whereas the staining was mostly cytoplasmic or vacuolar in less differentiated areas. The degree of CA 19-9 expression was inversely related to tumor differentiation (P less than .001).

The significance of lectin receptors in the kidney and in hypernephroma (renal adenocarcinoma).

Normal kidney tissue as well as hypernephromas were examined histochemically for the occurrence of lectin receptors. FITC- and rhodamine-labeled peanut agglutinin (PNA) and Ricinus communis agglutinin (RCA) were used for labeling different carbohydrate residues which may be of interest in the evaluation of the histogenesis of hypernephromas and possibly for concepts regarding the immunotherapy of these tumors. By using fluorescence microscopy, the receptor for PNA was found on the epithelial cells of the thin limb, distal convoluted tubules, and the collecting ducts. These binding sites occurred in a free as well as in a sialic-acid-substituted form and were mainly exposed on the luminal surface of the epithelial cells. They were absent, however, in the proximal convoluted tubules. This finding contrasts with the demonstration of RCA receptors in the brush border of these tubules. Moreover, RCA reacted with the epithelial cells of all tubules. The hypernephromas showed a wide range in the distribution and the quality of the lectin receptors, which were localized within the cytoplasm as well as in the cell membrane of the tumor cells. As demonstrated by our histochemical investigations, these kidney neoplasms may originate from any part of the tubules, and not only from the epithelial cells of the proximal convoluted tubules, as was suggested by earlier findings that employed antibodies against the brush-border antigens. In addition, the demonstration of sialic-acid-substituted and, in particular, of free PNA-receptors, which represent the immunodominant group of the Thomsen-Friedenreich antigen, may supply useful information on an immunotherapy concept.

Enhancement of carcinoembryonic antigen (CEA) expression in colorectal tumors by neuraminidase.

Twelve colorectal carcinomas with transitional mucosa and 10 colorectal adenomas which previously displayed weak or no carcinoembryonic antigen (CEA) expression were selected to verify whether neuraminidase unmasks CEA carbohydrate epitopes and, consequently, enhances the CEA expression. Peroxidase-antiperoxidase (PAP) method was performed on routinely processed tissues, without and with neuraminidase pretreatment of the sections. Lysine, without and with neuraminidase pretreatment of the sections. Lysine, as a modifier of electrostatic charge at cell surface, instead of neuraminidase was used to clarify whether the enzyme yields a specific or non-specific influence on CEA expression. All colorectal tumors exhibited more CEA after neuraminidase pretreatment, while previous negative specimens developed CEA expression. The same effect was observed in some transitional mucosa sections. This has not occurred in normal mucosa, probably owing to a resistant sialylation. The enhancement effect of lysine, although more weakly and not entirely superimposed to that or neuraminidase, suggests non-specific mechanisms of enzyme action. The removal of the negative charge at cell surface, especially due to sialic acid, allows more anti-CEA antibodies to react. The neuraminidase pretreatment of the sections is a useful method to demonstrate the real incidence of CEA in the colorectal tumors.

[Expression of the tumormarker p16INK4a in cytology specimens of the urinary bladder. A new means for early recognition and surveillance of urothelial cancer]. / Vorkommen einer p16INK4a-Expression in Zytologien der Harnblase. Ein neuer Weg zur Früherkennung und Verlaufskontrolle beim Urothelkarzinom.

BACKGROUND: This study was carried out to learn whether cytological specimens from urinary bladder lavages express the tumor suppressor gene p16INK4a, whether an abnormally increased expression indicates a cancerous state and whether cytological measurements are comparable regarding sensitivity and specificity with measurements made in histological sections of biopsies. PATIENTS AND METHODS: A total of 82 urine specimens of patients suspected of having a bladder tumor were examined for the presence of p16INK4a. RESULTS: Out of 46 patients with urothelial carcinoma 29 expressed p16INK4a in the cells in the urine specimens. Out of 36 patients free of cancer 30 expressed no p16INK4a in cytological specimens. The sensitivity of the expression proved to be 63% and the specificity 83%. Well-differentiated carcinomas seldomly expressed an increased p16INK4a (sensitivity 27%), whereas moderately differentiated carcinomas showed a sensitivity of 69% and poorly differentiated carcinomas a sensitivity of 77%. CONCLUSION: Compared to other minimally invasive tumor markers, such as NMP22, the expression of p16INK4a in cytology specimens of urine appears to be a sensitive marker for urothelial carcinoma, especially for the detection of poorly differentiated carcinomas. Its high specificity makes it ideal for use in tumor screening.

[Lectin histochemistry in the kidney and renal tumors]. / Lektinhistochemie an Niere und Nierentumoren.

In recent years histochemical methods using lectins with diverse carbohydrate binding specificities have proved useful tools for studying the distribution pattern of intracellular and pericellular glycoconjugates at the tissue level. Several studies of human kidneys have shown that the binding sites for certain lectins are strictly confined to various parts of the nephron. This allows the use of lectins as markers for the particular segments. The glomeruli exhibit abundant sialoglycoproteins at the free surface coat of the podocytes as detected by PNA after sialidase representing a major component of the filtration barrier; neoplastic podocytes of glomeruloid bodies in nephroblastomas as well revealed an intense lectin binding despite failing any vascularization. The lectin binding pattern of renal carcinomas varies within a wide range depending on their histological growth pattern. Whereas most renal carcinomas of clear and granular cell type rarely displayed a slight reactivity with lectins at the cell surface and at the luminal aspect of tubulopapillary tumors, a sub-group of carcinomas named chromophobe type exhibited a strong cytoplasmic staining with DBA and PNA after sialidase. Comparative evaluation of normal kidneys revealed an identical binding pattern exclusively in the intercalated cells of the collecting ducts probably indicating a histogenetic origin of these tumors from the collecting duct epithelium. This assumption derives further support from the detection of precursor lesions rarely detected in normal and tumor-bearing kidneys exhibiting the same lectin binding pattern. In accordance with recent observations in the literature the disclosure of a similar lectin binding pattern in renal oncocytomas and their precursor lesions exhibiting oncocytic transformation clearly favors the assumption of an identical histogenetic origin. On the other hand, few cases of a carcinoma mimicking collecting duct epithelium exhibited a broader lectin binding pattern revealing evidence of secretory activity; the histochemical similarities between this unusual carcinoma and transitional cell carcinoma confirmed the suggested origin from the ducts of Bellini. In conclusion, lectin histochemistry may be a useful tool for estimating the characteristics of renal tumors and elucidating their histogenesis.

[Opisthorchiasis simulating a malignancy]. / Die Opisthorchiasis in der Maske eines Malignoms.

We report on 2 patients from Siberia suffering from an infection with the parasite Opisthorchis felineus. The unusual course of their disease pretended in case 1 an eosinophilic leukemia and in case 2 a malignoma of the gallbladder. The Opisthorchiasis is endemic in large areas of Asia and Russia. Humans acquire the infection by eating raw fresh-water fish. Symptoms are nonspecific, but detection of eosinophilia in travellers or residents of endemic areas should induce analysis for specific antibodies against Opisthorchis species. Opisthorchiasis is known to be a precursor of cholangiocarcinoma. Malignoma which was initially suspected could be excluded in both cases and the patients were cured by oral administration of Praziquantel, 40-75 mg/kg body weight for 1 day in 3 divided doses.

Studies on the hydrolysis of 1-alkyl-sn-glycero-3-phosphoethanolamine in subcellular fractions of rat brain.

The formation of 1-alkylglycerol from 1-alkyl-sn-glycero-3-phosphoethanolamine in different cell fractions of rat brain is reported. The substrates used were labelled either with 14C or 3H in the alkyl residue or with 14C in the alkyl and 3H in the ethanolamine residue. The examination of the lipid- and water-soluble cleavage products showed that both ethanolamine and phosphoethanolamine are liberated from the substrate in the microsomal fraction of 14-day-old rat brain. The latter product is rapidly hydrolyzed. In comparison with other cell fractions, the microsomes contained the highest enzyme activities, which exhibited a pH optimum of 7.1--7.5. SH-group reagents are inhibitors, whereas diisopropylfluorophosphate has no effect. As the animals age, these enzyme activities decrease in brain homogenates.

[Responders and non-responders of surfactant therapy of very small premature infants. A clinical, histopathologic and immunohistochemical analysis]. / Responder und Nonresponder in der Surfactanttherapie sehr kleiner Frühgeborener. Eine klinische, histopathologische und immunhistochemische Analyse.

The administration of surfactants is highly effective and yields an immediate clinical response in cases of a lack of surfactant in the premature lung of preterm infants. The administration of surfactants might diminish the need for intensive care and artificial ventilation thereby also reducing or preventing the severe side effects associated with these therapeutic measures. However, the lack of clear clinical features precludes a reliable predictive forecast of the therapeutic outcome. Only the therapeutic follow-up period can disclose possible damage due to the administration of surfactants. Histological preparations of lung specimens from non-responding infants reveal cellular inflammatory infiltrations, destruction of the alveolo-capillary basement membrane and monocytic phagocytosis.