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Endo-β-galactosidase from Escherichia freundii cleaves polylactosaminyl structures as follows: R-GlcNAcβ1-3Galβ1-4GlcNAcβ1-R′ + H2O → R-GlcNAcβ1–3Gal + GlcNAcβ1-R′. By staining with Griffonia simplicifolia agglutinin-II following the enzyme digestion, the distribution of R-GlcNAcβ1–3Galβ1–4GlcNAc can be demonstrated in tissue sections. This carbohydrate chain is one of the backbone structures carrying the blood-group-related antigens and, thus, localization of this structure may provide detailed information about the distribution of variants with different backbone structures. Various formalin-fixed, paraffin-embedded tissue sections were stained by Griffonia simplicifolia agglutinin-II with or without prior enzyme digestion and the reactivity of the agglutinin imparted by enzyme digestion was studied in the following tissues and cells: pancreatic acinar cells, gastric surface mucosae, duct cells and mucous cells of salivary glands and tracheal glands, surface epithelium of trachea, goblet cells of large intestine, columnar epithelium of uterine cervical glands, distal and collecting tubules of kidney, certain cells of anterior lobe and colloid of middle lobe of pituitary glands, epithelial reticular cells and Hassall's corpuscles of thymus and Kupffer cells of liver. In gastric surface mucosae, the reactivity of the agglutinin appeared in non-secretor individuals but not in the secretor individuals, and in mucous cells of salivary and tracheal glands the reactivity appeared in Le(a - b -) non-secretor individuals but not in Le(a + b -) non-secretor or secretor individuals. In pancreatic acinar cells and duct cells of salivary glands from fetuses and newborn infants, prior fucosidase digestion markedly enhanced the Griffonia simplicifolia agglutinin-II reactivity elicited by endo-β-galactosidase digestion. Prior fucosidase digestion was also a prerequisite for revealing the reactivity of this agglutinin by endo-β-galactosidase digestion in gastric surface mucosae from secretor individuals. β-Galactosidase digestion disclosed reactivity of this agglutinin in pancreatic acinar cells and duct cells of salivary glands even after the removal of endo-β-galactosidase-labile lactosamine structures by sequential digestion with endo-β-galactosidase and β-N-acetylhexosaminidase. These results demonstrate that the procedures developed in this study provide a useful means for detecting different types of lactosamine structures which carry blood-group antigens in humans tissues.

Blood group ABH and related antigens are not expressed in normal thyroid glands. However, cells of thyroid papillary carcinoma, especially their luminal surfaces expressed ABH antigens in a manner compatible with the ABO blood group of patients. Sialyl Lewis a (CA 19-9) and sialyl precursor type 1 chain were likewise intensely and frequently expressed in these cells from most of the individuals examined. On the other hand, Lewis x, sialyl Lewis x and sialyl precursor type 2 chain were minor components of the carbohydrate antigens expressed in these cells. In contrast, type 2H, type 2A and presumably type 2B antigens were preferentially expressed in these cancer cells. Prior digestion with endo-β-galactosidase from Escherichia freundii eliminated or reduced the reactivity of monoclonal antibodies and lectins specific for these ABH and related antigens with carcinoma cells. Along with the elimination or reduction of staining with these reagents, binding sites with Griffonia simplicifolia agglutinin-II appeared in the corresponding tissue sites indicating the presence of poly-N-acetyllactosamine structures. These results suggested that poly-N-acetyllactosamine structure containing linear domain susceptible to endo-β-galactosidase digestion is a common and ubiquitous precursor of the blood group-related antigens and its synthesis is prerequisite for the oncofetal expression of these antigens in papillary carcinoma of the thyroids.

A Japanese girl with blood phenotype O who is complicated with a rare deficiency of selective anti-A agglutinin is described. (A) Tests for patient red blood cell (RBC): Commercial anti-A or anti-B serum did not agglutinate the patient RBCs. Neither anti-A nor anti-B agglutinin activity was recovered from patient RBCs by absorption-elution experiment according to the method of Landsteiner & Miller. Flowcytometric analysis revealed that patient RBC is totally lacked of immunoreactive material against both anti-A and anti-B antibodies. (B) Patient serum: Patient serum preferentially agglutinated RBCs from type B individuals, but not from type A individuals. Both the activities of A-and B-transferase in the patient serum were undetectable. In patient saliva, excretion of H-substance was indicated by inhibition test using anti-H lectin (Ulex-lectin). Furthermore, neither the presence of irregular antibodies nor quantitative decrease of immunoglobulins G, A, and M was observed in patient serum. Studies of her family members confirmed that the propositus was an only patient having blood phenotype O which lacked anti-A agglutinin activity despite of normal expression of anti-B agglutinin.

Blood group ABH and related antigens are expressed not only in red blood cells but also abundantly in many epithelial cells of human and other vertebrate species. Recent electron microscope observations have revealed the precise distribution of these antigens in various cellular constituents such as the Golgi complex, cell membrane, mucous granules and nuclear heterochromatin. These findings may contribute to elucidate the biosynthetic mechanism of these substances as well as their physiological and pathological function.

Endo-β-galactosidase from Escherichia freundii cleaves linear polylactosamine structure as follows: R-GlcNAc-β1-3Gal-β1-4GlcNAc-β1-R′ + H2O → R-GlcNAc-β1-3Gal + GlcNAc-β1-R′. Staining with Griffonia simplicifolia agglutinin II (GSA-II) following enzyme digestion reveals the distribution of R-GlcNac-β1-3Gal-1-4GlcNAc-β1-R′ structures in tissue sections. In this study, the procedure was applied to formalin-fixed, paraffin-embedded tissue sections from 26 cases of papillary carcinomas including 2 follicular variants, 8 follicular carcinomas, 7 adenomas, 1 anaplastic carcinoma and 1 medullary carcinoma in order to investigate whether different types of polyactosamine-containing structure are produced in these thyroid neoplasms. Simultaneously, the susceptibility of the ABH antigens expressed in these neoplastic cells to endo-β-galactosidase digestion was examined. Most of the papillary carcinoma cells from all the individuals examined were strongly stained by GSA-II following enzyme digestion. Without enzyme digestion, little or no reactivity with GSA-II was observed. Among other types of neoplasms, only one case of follicular carcinoma exhibited reactivity with GSA-II following enzyme digestion. ABH antigens were expressed in 22 cases of papillary carcinomas, 2 adenomas, 5 follicular carcinomas and 1 anaplastic carcinoma, and their expression was dependent on the ABO blood group of the patients. Endo-β-galactosidase digestion resulted in the elimination of these antigens not only in papillary carcinomas but also in other neoplasms. These results suggested that the polylactosamine-containing structures produced in papillary carcinomas are quite different from those in other neoplasms, and demonstrated that the procedure is a useful diagnostic means for distinguishing papillary carcinoma and other types of thyroid neoplasms.

The oligosaccharide structures of blood group antigens are not the primary gene products; they are constructed in a stepwise manner by adding particular sugar to precursor oligosaccharides via several glycosyltransferases coded for by different blood group genes (Watkins 1966, 1978, 1980). Consequently, final profiles of antigens expressed in each cell type are influenced by many different factors such as the intrinsic composition of glycosyltransferase species which are defined by the genotype of the individuals, relative activity or amount of these enzymes (repression, derepression or induction of the enzymes), competition between enzymes with overlapping substrate specificity, the organization of the enzymes in membranes, utilizability of precursors and specific substrate sugars, and the activity level of degradating enzymes. Changes in the antigen profiles during maturation, differentiation and malignant transformation are thought to be intimately related to the variability of these factors. Although great importance attaches to histo- and cytochemical information on the distribution and levels of glycosyltransferases and messenger RNA corresponding to the relevant enzyme, detailed and precise localization of the blood group antigens and their variants is the base line for analyzing these complex factors. On the basis of individual genotype and histochemical findings about the antigen distribution and the interrelationship between cells and cellular components producing different antigenic structures (cellular and subcellular mosaicism), we can deduce precursor oligosaccharide levels as well as the status of gene activation and its primary product, glycosyltransferases. Thus, these findings are a prerequisite for further analysis at the molecular genetic level. As emphasized in this article, lectin staining or immunostaining methods with MAbs combined with glycosidase digestion procedures are powerful tools for in situ analysis of carbohydrate structures in histochemical systems. Although in some cases valuable results have been obtained by applying the technique, our knowledge concerning the distribution of complex carbohydrate structures is still far from satisfactory. Along with well defined MAbs and lectins, the key to developing our methods further is successful introduction of glycosidases, in particular, endoglycosidases since these reagents are indispensable for analyzing the inner core structures and glycoconjugate species of the blood group antigens. Application of these techniques at the ultrastructural level is an alluring possibility, even though many difficulties must be overcome. Although their functional roles have not yet been determined, a diverse array of macromolecules is known to be decorated with blood group-related antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

Twelve different kinds of blood group-specific lectins have been used along with monoclonal anti-A, -B and -H antibodies for detecting the corresponding antigens in selected human tissues. Although most of the lectins recognized the antigens in the tissue sections examined, they displayed marked differences in their recognition patterns in certain tissues. Helix asparsa agglutinin (HAA), Helix pomatia agglutinin (HPA) and monoclonal anti-A antibody recognized A antigens in the mucous cells of salivary glands from blood group A or AB nonsecretor as well as secretor individuals, whereas Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin-I (GSA-I), Sophora japonica agglutinin (SJA) and Vicia villosa agglutinin (VVA) did not bind to them from nonsecretors. A antigens in endothelial cells, lateral membrane of pancreatic acinar cells and small mucouslike cells of submandibular glands from some individuals were likewise recognized by HAA and HPA but not by other blood group A-specific lections. In contrast, both HAA and HPA did not recognize the A antigens in mucous cells of Brunner's glands while other A-specific lectins and monoclonal anti-A antibody reacted specifically with the antigens. Such a difference was not observed with lectins specific for blood group B. However, the B antigens in Brunner's glands were recognized by these lectins but not with monoclonal anti-B antibody. The difference in labelling ability was also noted among the blood group H-specific lectins and monoclonal anti-H antibody in endothelial cells of blood vessels. Ulex europaeus agglutinin-I reacted with these cells irrespective of ABO and the secretor status of the individuals, while Anguilla anguilla agglutinin and monoclonal anti-H antibody reacted only with those cells from blood group O individuals. No reaction was observed with Lotus tetragonolobus agglutinin in these tissue sites. These results suggest a great diversity of blood group antigens in different human tissues.

We examined the distribution of blood group-related antigens using an indirect immunoperoxidase method with monoclonal antibodies (MAb) directed to A, B, H, Lewis a (Lea), Lewis b (Leb), Lewis x (Lex), and Lewis y (Ley) antigens and Type 1 precursor chain in human pancreas. Effects of prior digestion with exoglycosidases on MAb stainings were simultaneously investigated. A, B, H, Leb, and Ley antigens were detected in acinar cells and interlobular duct cells but not in centroacinar cells, intercalated duct cells, and islet of Langerhans cells. The expression of these antigens in acinar cells was not dependent on Lewis type and secretor status of the tissue donors, whereas that in interlobular duct cells was strictly dependent on secretor status. The distribution pattern of these antigens in acinar cells was not homogeneous, i.e., cells producing H antigens expressed both Leb and Ley antigens but not A or B antigens, whereas those producing A or B antigens did not secrete Leb and Ley as well as H antigens. Digestion with alpha-N-acetylgalactosaminidase or alpha-galactosidase resulted in the appearance of Leb and Ley antigens as well as H antigen in acinar cells producing A and/or B antigens. Type 1 precursor chain was not detected in pancreatic tissues from secretors but appeared in acinar cells producing H antigen after alpha-L-fucosidase digestion, which also disclosed Lex but not Lea antigen in acinar cells expressing both Leb and Ley. In some non-secretors, MAb against Type 1 precursor chain reacted with acinar cells without enzyme digestion. Although Lea antigen was not detected in acinar cells, it was found in centroacinar cells, intercalated duct cells, and interlobular duct cells from all individuals examined except two Le(a-b-) secretors. After sialidase digestion, Lex antigen appeared in centroacinar and intercalated duct cells from some individuals. Sialidase digestion also elicited reactivity with MAb against Type 1 precursor chain in islet of Langerhans cells from some individuals. These results demonstrate the complexity in the pattern of expression and regulation of blood group-related antigens in different cell types of human pancreas. Such complexity may largely be ascribed to differences in individual genotypes and in gene expression patterns of different cell types.

In a previous study(Ito et al. 1988), we compared the staining properties of several blood group A specific lectins in different human tissues and found that in submandibular glands from blood group A non-secretors, Dolichos biflorus agglutinin(DBA), Bandeiraea simplicifolia agglutinin I(GSA-I), and Sophora japonica agglutinin(SJA) did not react with mucous cells, whereas Helix pomatia agglutinin(HPA) and Helix aspersa agglutinin(HAA) reacted with them. In a preliminary study, we found that these two snail lectins cannot react with mucous cells of salivary glands from German Blood group A nonsecretors, suggesting the existence of racial difference in the expression of blood group antigens. As to the racial difference in antigenicity of erythrocytes, Gibbs et al.(1961) reported that the reactivity of blood group B erythrocytes with anti B serum was strongest among Negroes, weakest among Mongoloids and intermediate among Whites. Wiener et al.(1972) also showed that the racial difference in the reactivity of the red blood cells with anti H lectin. These observation led us to suggest that it might be possible to exist the racial difference in the expression of ABH antigens in human secretory organs.

In a series of previous studies, we have developed the lectin staining method in combination with exo- and endoglycosidase digestion procedures to localize and analyze the definite chemical structures of blood-group-related carbohydrate chains in human pancreas and submandibular glands (Ito et al. 1987, 1988a, b, 1989a, b). The results of these previous studies suggested that type 1, type 2 and 0- glycosidically linked, type 3 based ABH antigens are secreted in pancreatic acinar cells. In the present study, we developed immuno-staining method using several monoclonal antibodies(MoAbs) against blood-group-related antigens combined with exoglycosidase digestion procedures to provide more definite information as to tissue distribution and regulation of blood group related antigens in human pancreas.

Using lectin staining methods in combination with exo- and endo-glycosidase digestion procedures, we analyzed the chemical structure of different types of blood group-related substances in serous cells of formalin-fixed, paraffin-embedded human submandibular glands. Serous cells produced only H antigen; A and B antigens were not present, and the expression of H antigen is dependent on the secretor status of the tissue donor. Although reactivity with Ulex europaeus agglutinin I (UEA-I) was not markedly reduced by alpha-L-fucosidase digestion, an affinity for peanut agglutinin (PNA) was seen after fucosidase digestion in the cells from secretors. In those from nonsecretors, no PNA reactivity appeared after enzyme digestion. On the other hand, sialidase digestion elicited PNA reactivity in serous cells irrespective of the donor's secretor status. PNA reactivity observed after fucosidase or sialidase digestion was susceptible to endo-alpha-N-acetylgalactosaminidase (endo-GalNAc-dase) digestion. SBA reactivity in UEA-I-negative cells from secretors, or in cells from fetuses and newborn infants, was markedly reduced by beta-galactosidase digestion. After galactosidase digestion, reactivity with Griffonia simplicifolia agglutinin II (GSA-II) appeared in the corresponding cells. This GSA-II reactivity was almost completely eliminated by subsequent beta-N-acetylhexosaminidase digestion. Whereas PNA reactivity in these cells was not reduced by beta-galactosidase treatment, it was significantly diminished by endo-GalNAc-dase digestion. These results suggest that at least two kinds of precursor disaccharides are produced in submandibular serous cells, i.e., SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and PNA-reactive D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine (O-glycosidically linked Type 3 chain or T antigen). Final fucosylation and synthesis of these two types of precursor chain appear to be under the control of the secretor gene.

Using blood-group-specific lectins conjugated to horseradish peroxidase, distribution of A, B and H antigen was examined in formalin-fixed, paraffin-embedded pancreases from 27 human autopsy cases. Dolichos biflorus agglutinin, Griffonia simplicifolia agglutinin I-B4, and Ulex europaeus agglutinin I or Lotus tetragonolobus agglutinin were found to be satisfactory to demonstrate A, B and H antigen, respectively in acinar cells. No exception was encountered to the correspondence between blood type and lectin staining. In blood group O individuals, acinar cells producing H antigen were evenly distributed throughout the gland. However, in other blood groups, the distribution of antigens was not homogeneous. In blood group A individuals, some acini contained A antigen but not H antigen, others contained only H antigen and a few contained both A and H antigen. Such a mosaic distribution of antigens was also observed in B and AB individuals. Furthermore, in AB individuals, independent production of either A or B antigen in some acini was observed. The blood group antigens in acinar cells were demonstrated irrespective of secretor status of the tissue donor. Although Ulex europaeus agglutinin I reacted ubiquitously with vascular endothelia independently of the blood group, other blood-group-specific lectins could not react with the endothelia of any given blood group.The present study further demonstrated that the staining patterns of some blood-group-nonspecific lectins exhibit certain dependence on the blood group of the tissue donor. That is, soybean agglutinin reacted with the cells secreting A and/or H antigen but not those secreting B antigen; peanut agglutinin reacted with many acinar cells in all the nonsecretors examined, but in secretors, existence of such cells was very rare.

We examined the effects of alpha-L-fucosidase digestion on lectin staining in formalin-fixed, paraffin-embedded human pancreatic tissue from individuals of different blood groups. Digestion with the enzyme resulted in apparent diminished intensity of Ulex europaeus agglutinin-I (UEA-I) staining in the acinar cells. In addition to the decreased intensity of UEA-I staining, reactivity with soybean agglutinin (SBA) was increased in the enzyme-susceptible, UEA-I-reactive cells. The intensity of Griffonia simplicifolia agglutinin-II (GSA-II) staining performed after beta-galactosidase digestion in UEA-I-reactive acinar cells was markedly increased by prior treatment with fucosidase. GSA-II staining following sequential digestion with fucosidase and galactosidase was completely abolished by subsequent digestion with beta-N-acetylhexosaminidase. These results therefore substantiate the previous assumption that SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and GSA-II reactive beta-N-acetyl-D-glucosamine imparted following galactosidase digestion represent precursors of H antigen. The present study further demonstrated that intense peanut agglutinin (PNA) staining was imparted after digestion with fucosidase in UEA-I-reactive sites in secretors. In contrast, nonsecretors showed vivid PNA staining that was usually detected throughout the pancreas without prior enzyme digestion. Here, fucosidase digestion had if any little effect on PNA staining. These results suggest that in secretors a terminal trisaccharide, fucosylated D-galactose-(beta 1-3)-N-acetyl-D-galactosamine exhibiting positive PNA reaction after fucosidase digestion, exists in UEA-I-reactive acinar cells. It is assumed that the secretor gene could control the step of final fucosylation of D-galactose-(beta 1-3)-N-acetyl-D-galactosamine in human pancreas.

It is well known that HLA typing of lymphocyte is extremely useful for personal identification and is often used for paternal testing in the field of legal medicine. Direct application of this method to personal identification of cadavers, however, is sometimes difficult because of relatively low viability of lymphocytes in a cadaver. Other method for HLA typing of a cadaver such as elution method reported by Kashiwade (1986) or absorption method (lymphocyte cytotoxicity inhibition test) reported by Yoshimura (1982) also have some difficulty that, in the case of a cadaver with completely unknown HLA type, these methods require a large amount of highly expensive HLA antibodies, consumes tremendous time and, thus, is not applicable for practical purposes. In an effort to find easy and general application of HLA typing of cadaver tissues, we investigated the possibility of carrying out the test by using conventional typing tray. In this report, we will show that HLA typing of a cadaver can be successfully made by using cultured fibroblast and slightly modifying the conventional NIH method for HLA typing of lymphocytes (Bodmer, 1978).

We investigated localization of blood group antigens and their related substances in human labial salivary and submandibular glands by application of a post-embedding cytochemical staining procedure using lectin- or glycoprotein-gold complexes. Surgical tissue was obtained from 10 patients. Blood group-specific lectins, such as Dolichos biflorus agglutinin or Helix pomatia agglutinin (group A-specific), Griffonia simplicifolia agglutinin-I B4 (group B-specific), and Ulex europaeus agglutinin I (group H-specific) could recognize A, B, and H antigens, respectively, only in mature secretory granules (mature SG), which were found preferentially in cells in the late phase of the maturation cycle. In immature secretory granules (immature SG), which were found in cells in the early or middle phase of the maturation cycle, no binding with these lectins was observed. The Golgi complexes and endoplasmic reticula also were not labeled with these lectins. In blood group O and B secretors, blood group antigens were uniformly distributed throughout all the mature SG examined. However, in blood group A secretors, the distribution was heterogeneous, i.e., in some granules only H antigen was demonstrated, whereas in others both A antigens and a small amount of H antigens were detected. Among the blood group-nonspecific lectins, wheat germ agglutinin (WGA) was found to bind more preferentially to immature SG than to mature SG. This was demonstrated irrespective of the blood group and secretor status of the tissue donor, except that in blood group A secretors WGA bound strongly to some mature SG which possessed A antigen. We discuss the significance of cellular and subcellular mosaic distribution of blood group antigens in connection with morphological differences of secretory granules and the maturation cycle of mucous cells.

Renal biopsy tissue from 33 children with various glomerular diseases has been investigated by electron microscopy using a postembedding immunostaining technique with a protein A-gold complex in order to establish more precise correlations between immunopathologic and morphologic findings in glomeruli. This technique could detect immunoglobulins (IgG, IgA, and IgM), complement factor (C3c), and fibrinogen-related antigen. The immunoreactivity of these antigens was essentially confined to the mesangial, paramesangial, subendothelial, and subepithelial 'electron-dense deposits' in the glomeruli. Except for IgM and C3c in the case of glomerular sclerosis, the distribution of the mentioned factors was even in the electron-dense deposits, as could be shown by 'double-immunolabeling'. From the above-mentioned findings one can conclude that several of the localized factors are associated with the formation of electron-dense deposits, the ultrastructural hallmarks of glomerular disease.

In human pancreas, soybean agglutinin (SBA) conjugated to horseradish peroxidase reacted with the acinar cells secreting blood group A and/or H antigen, but not with those secreting only B antigen. For detailed histochemical characterization of SBA staining, the effects of treatment with unlabeled lectins and of digestion of certain enzymes on SBA staining were investigated in formalin-fixed, paraffin-embedded pancreatic tissue from individuals of different blood groups. Pre-incubation of sections with unlabeled Dolichos biflorus agglutinin to block A antigen eliminated subsequent SBA staining in the cells secreting A antigen, although failing to induce any effects in those secreting H antigen. In contrast, pre-incubation with unlabeled Ulex europaeus agglutinin-I (UEA-I) to block H antigen abolished SBA staining in cells secreting H antigen but not in those secreting A antigen. Treatment with galactose oxidase yielded the same results as those with unlabeled UEA-I, i.e., SBA reactivity was significantly diminished in cells secreting H antigen but not in those secreting A antigen. Digestion with beta-galactosidase resulted in a slight decrease of SBA staining in the cells secreting H antigen. Accompanying the decrease of SBA staining, reactivity with Griffonia simplicifolia agglutinin-II (GSA-II) appeared for the first time in the enzyme-susceptible, SBA-reactive cells secreting H antigen. Pre-treatment with galactose oxidase abolished this effect of beta-galactosidase. The GSA-II reactivity disclosed by treatment with galactosidase was completely eliminated by digestion with beta-N-acetylhexosaminidase, indicating that GSA-II staining after digestion with galactosidase is due to exposed penultimate beta-N-acetyl-D-glucosamine residues. These results demonstrate that at least two substances react with SBA in acinar cells of human pancreas, one being terminal beta-N-acetyl-D-galactosamine residues of A antigen, and the other being terminal beta-D-galactose-(1----3 or 1----4)-beta-N-acetyl-D-glucosamine dimers in the precursor of blood group H antigen. Such dimers may exist in close proximity to L-fucose residues of H antigen, since unlabeled UEA-I blocked SBA staining.

Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo- and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with alpha-N-acetylgalactosaminidase and alpha-L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with alpha-galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B4 reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo-alpha-N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from non-secretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.

We investigated the localization of blood-group antigens A, B, and H in human labial salivary and submandibular glands by applying a postembedding immunogold method using monoclonal antibodies in combination with the streptavidin-biotin bridge technique. The H, A, and B antigens were only detected in mature secretory granules (SGs), which were mainly found in cells in the late phase of the maturation cycle. In immature SGs, which were present in cells in the early or middle phases of the maturation cycle, these antigens were not detected. All other cytoplasmic organelles were not labeled by the monoclonal antibodies used. In blood-group-O secretors, H antigen was present in almost all of the mature SGs. In blood-group-A secretors, the labeling for H antigen exhibited a mosaic-like pattern, i.e. only some of the mature SGs contained H antigen. With respect to the A and B antigens, a similar mosaic-like pattern of staining was observed in blood-group-A and -B secretors, respectively. To the best of our knowledge, this is the first time that the distribution of blood-group antigens A, B, and H in human tissues has been demonstrated at the electron-microscope-level using monoclonal antibodies.

Effects of alpha-galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B4 positive acinar cells. beta-Galactosidase digestion following alpha-galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B4 positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple beta-galactosidase digestion as well as sequential digestion with alpha- and beta-galactosidase. However, when alpha-L-fucosidase digestion procedure was inserted between alpha- and beta-galactosidase digestion, UEA-I staining imparted by alpha-galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B4 positive acinar cells. Furthermore, after sequential digestion with alpha-galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B4 positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion.(ABSTRACT TRUNCATED AT 250 WORDS)