Mathias Vierbuchen

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.

Renal oncocytoma. II. Lectin and immunohistochemical features indicating an origin from the collecting duct.

SummaryThe present study is aimed to gain more insight into the histochemical properties of renal oncocytomas. Ten oncocytomas and normal kidneys were investigated using several lectins (peanut agglutinin - PNA, Dolichos biflorus agglutinin - DBA and Ulex europaeus agglutinin - UEA) and antibodies against epithelial membrane antigen (EMA), Tamm-Horsfall glycoprotein (THG) and lysozyme. Lectin histochemistry revealed a characteristic binding pattern in renal oncocytomas, with strong DBA-binding and, in some cases, a weaker staining with UEA apparent in the cytoplasm of the oncocytes. PNA binding sites were evident only after enzymatic cleavage of sialic acid by neuraminidase. Comparative evaluation of normal kidneys exhibiting a strict compartmentalization of saccharide moieties in the various nephron segments revealed a similar binding pattern exclusively in interspersed collecting duct epithelium. This striking resemblance suggests that renal oncocytomas may originate from the collecting duct system. Further support for this assumption has been provided by the demonstration of strong cytoplasmic EMA reactivity in the oncocytes. In normal kidneys prominent labeling for EMA was apparent in the very same interspersed cells of the collecting ducts. THG and lysozyme failed to react in renal oncocytomas. In accordance with observations recently reported in the literature, these results clearly favor a histogenetic origin of renal oncocytomas from the collecting duct epithelium.

Renal oncocytoma. I. Cytochrome c oxidase in normal and neoplastic renal tissue as detected by immunohistochemistry--a valuable aid to distinguish oncocytomas from renal cell carcinomas.

SummaryUsing a polyclonal antibody raised against bovine heart cytochrome c oxidase, the occurrence of this mitochondrial marker enzyme has been investigated in 63 kidney tumors (ten renal oncocytomas, 43 renal cell carcinomas and ten tubulopapillary adenomas) as well as in normal renal tissue by an immunoperoxidase method (PAP-technique). The differentiation between renal oncocytomas and mitochondria-rich carcinomas represents a problem of histopathology since these tumors have a different prognosis and require different patient managements. The strong immunoreactivity in renal oncocytomas contrasted with the much weaker reactivity in renal carcinomas and adenomas. Even mitochondria-rich (granular cell type) carcinomas exhibited only moderate staining intensity. Furthermore, single strongly stained oncocytes or small complexes were sometimes detected in normal renal tissue. The demonstration of marked differences in enzyme content between renal oncocytomas and granular cell carcinomas renders this method suitable for unequivocal distinction between these renal neoplasms. The antibody proved to be a valuable marker for detecting “true” oncocytic transformation in renal tumors and was useful in defining even single oncocytes or small oncocytic lesions.

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 Kupffers 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.

Sialylated glycoconjugates in chromophobe cell renal carcinoma compared with other renal cell tumors : indication of its development from the collecting duct epithelium

SummaryThe present study was designed to shed light on the extraordinary histochemical properties of the chromophobe cell renal carcinoma detected by Hale’s colloidal iron reaction. Special emphasis was laid on the lectin histochemical analysis of cytoplasmic glycoconju-gates. Binding of peanut agglutinin (PNA) and Erythrina cristagalli agglutinin (ECA) after enzymatic release of sialic acid and direct binding of Dolichos biflorus agglutinin (DBA) correlates well with the expression of binding sites for Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA) revealing abundant sialylated carbohydrate moieties within the cytoplasm. This characteristic binding pattern differs considerably from the faint staining observed in the majority of other renal carcinomas, thus confirming that the chromophobe cell renal carcinoma is a distinct entity. However, the lectin binding pattern of renal oncocytoma obviously resembles that of chromophobe carcinoma indicating a close relationship between these renal tumors. Detailed analysis of adjacent renal parenchyma revealed a lectin binding pattern quite similar to that described in the chromophobe carcinomas exclusively in the intercalated cells lining the collecting duct. This finding suggests that the chromophobe cell renal carcinoma originates from the collecting duct epithelium. The detection of small complexes consisting of altered epithelia which display the morphological characteristics of chromophobe carcinoma and the histochemical properties of intercalated cells probably indicates the emergence of preneoplastic lesions preceding the development of chromophobe carcinoma. Even though further studies are clearly needed to elucidate the physiological role of the cellular glycoconjugates detected, the present results already provide valuable insight into the histogenesis and pathogenesis of the chromophobe cell renal carcinoma.

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.

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.

Blood group antigen expression in medullary carcinoma of the thyroid : an immunohistochemical study on the occurrence of type 1 chain-derived antigens

SummaryUsing monoclonal antibodies (MoABs) against blood group determinants and related carbohydrate sequences, it is now possible to clarify their carcinoma-associated modulation at a molecular level. In the present study a panel of MoABs against different type 1 chain derived blood group antigens, comprising A, B, H type 1, Lea, sialyl-Lea (CA 19-9), sialyl type 1 structure (CA 50), and Leb was used to investigate their immunoreactivity in 38 medullary carcinomas of the thyroid (MTC) and in normal thyroid tissue. The antigens were not expressed in normal follicular or C-cells but were expressed to a various extent in MTC.The studies revealed some characteristic anomalies in the frequency and patterns of tumor-associated antigen expression. The MoAB C 50 stained 32 of the 38 tumors, H type 1 (Led) was demonstrated in 21 and the Leb antigen in 27. The Lea- and the A antigen were detected in 10 and 12 tumors and the B antigen in one. From the results some rules about the pathways for tumor-associated re-expression of these antigens can be deduced. Lea antigen expression was significantly correlated with the CA 50 and Leb antigens. The significant relation observed between A-, H1-, and Leb antigen formation in MTC suggests the existence of a carcinomaassociated fucosyltransferase committing the type 1 precursor chain along the H1-antigen pathway, and by further glycosylation to an A-, B-, or a Leb antigen. Comparative studies of tumor-associated H type 1 and H type 2 antigen expression revealed that H type 2 antigen synthesis was significantly related to a blood type 0 in the host. On the other hand, H1 antigen reactivity was independent of the ABO blood type of the hosts and was also detected in H type 2 antigen-negative tumors. These findings support the proposal that even in tumor tissue, H antigen expression is still determined by the interaction of at least two different genes. Despite the occurrence of the precursor substance (CA 50) and the formation of the Lea- and Leb antigens, indicating the presence of a α1,4-fucosyl-transferase (Lewis-enzyme), only two tumors showed the formation of CA 19-9.In conclusion, the investigations demonstrated the dominant re-expression of three type 1 chain-derived structures in MTC, namely H type 1, Leb, and CA 50. These findings support the general concept demonstrated in other carcinomas, that fucosyl- and sialytransferases are preferentially activated in MTC. Complex carbohydrates affect many cellular properties and the aberrant expression of sialylated and fucosylated antigens may influence the biological potential of tumor cells.

Blood group antigen expression in malignant tumors of the thryoid: a parallel between medullary and nonmedullary carcinomas

Blood group antigen (BGA) expression has been described in many fetal, adult, and tumorous tissues. Synthesis of BGA in the thyroid gland is regarded as oncofetal due to blood group structures that are detected in fetal and carcinomatous tissues but not in the normal adult organ. This study examined the prevalence of type 1 (CA-50, CA-19-9, Lea, Leb, A, B, H type 1) and type 2 (Lex, Leyy, A, B, H type 2) antigens in normal thyroid (n=25), papillary (n=104), follicular (n=52), anaplastic (n=33), and medullary (n=48) carcinomas of the thyroid. While normal thyroid tissue expressed no BGA, there was a significant increase of BGA expression in carcinomas of the thyroid gland. There are two theories about the possible origin of C cells, from wich the medullary carcinomas arise. Some authors postulate that C cells belong to the amine precursor uptake and decarboxylation system and therefore derive from the neural crest, while others believe that C cells originate from the fifth pharyngeal pouch, as do the follicular cells. The results obtained in this study show that medullary and nonmedullary carcinomas correspond to one another in their BGA expression profile. Therefore it is concluded that medullary carcinomas may have the same origin as nonmedullary tumors of the thyroid.ZusammenfassungDie Expression von Blutgruppenantigenen (BGA) wurde in vielen fetalen, adulten und tumorösen Geweben beschrieben. Die Synthese von BGA in der Schilddrüse wird als onkofetal angesehen, da Blutgruppensubstanzen in fetalen und karzinomatös veränderten Geweben, nicht aber im normalen, adulten Organ gefunden werden. In der vorliegenden Studie wurde das Vorkommen von Typ-1-Antigenen (CA-50, CA-19-9, Lea, Leb, A, B, H type 1) and Typ-2-Antigenen (Lex, Leyy, A, B, H type 2) in der normalen Schilddruse (n=25) und in papillären (n=104), follikulären (n=52), anaplastischen (n=33) und medullären (n=48) Karzinomen untersucht. Während normales Schilddrüsengewebe keine BGA exprimierte, waren die Karzinome der Schilddruse durch eine progressive Expression dieser Strukturen gekennzeichnet. Über den möglichen Ursprung der C-Zellen, aus denen die medullären Karzinome etitstehen, werden 2 Theorien diskutiert. Einige Autoren postulieren eine Zugehörigkeit der C-Zellen zum APUD-(Amine-precursor-uptake-and-decarboxylation-) System and somit eine Abstammung von der Neuralleiste. Andere gehen davon aus, daß die C-Zellen aus der 5. Schlundtasche stammen, wie auch die follikulären Zellen. In der vorliegenden Untersuchung wurde festgestellt, daß medulläre and nichtmedulläre Malignome Übereinstimmungen in der Expression von BGA zeigen. Es wird der Schluß gezogen, daß medulläre Karzinome den gleichen Ursprung wie nichtmedulläre Tumoren der Schilddrüse besitzen könnten.

Expression of alpha-3/4-monofucosylated polylactosaminoglycan epitope, as defined by monoclonal antibody FW6, is a marker of the colorectal adenoma–carcinoma sequence

Background. The expression of a distinct alpha‐3/4‐monofucosylated polylactosaminoglycan epitope, which is detected by monoclonal antibody FW6, was investigated by comparative immunohistochemical analysis of colorectal tissue specimens exhibiting different grades of premalignant and malignant transformation. The presence of this peculiar epitope was compared with different Lewis type 2 blood group antigens.