Journal of Histochemistry and Cytochemistry, Vol. 46, 793-802, July 1998, Copyright © 1998, The Histochemical Society, Inc.


ARTICLE

Histochemical Reactivity of Normal, Metaplastic, and Neoplastic Tissues to {alpha}-Linked N-Acetylglucosamine Residue-specific Monoclonal Antibody HIK1083

Naoshi Nakamuraa,b, Hiroyoshi Otab, Tsutomu Katsuyamab, Taiji Akamatsuc, Kazuhiko Ishiharad, Makoto Kuriharae, and Kyoko Hottaf
a Second Department of Internal Medicine, Shinshu University School of Medicine, Matsumoto, Japan
b Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan
c Department of Endoscopy, Shinshu University School of Medicine, Matsumoto, Japan
d Department of Chemistry, Kitasato University School of Medicine, Sagamihara, Japan
e Isehara Research Laboratory, Kanto Chemical Co., Inc., Isehara, Japan
f Department of Biochemistry, Kitasato University School of Medicine, Sagamihara, Japan

Correspondence to: Tsutomu Katsuyama, Dept. of Laboratory Medicine, Shinshu Univ. School of Medicine, Asahi 3-1-1, Matsumoto 390, Japan.


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Monoclonal antibody (MAb) HIK1083, which is obtained by immunizing mice with a preparation of rat gastric mucins, has been shown to bind specifically to {alpha}-linked N-acetylglucosamine ({alpha}-GlcNAc). We investigated the specificity of MAb HIK1083 by immunostaining normal human organs, mucinous metaplasia of human pancreas, adenocarcinomas of human stomach, pancreas, and colon, and normal rat organs. The specificity was investigated by making comparisons with (a) a stain that labels Class III concanavalin A (ConA)-reactive mucin (Class III mucin), i.e., paradoxical ConA (PCS), and (b) staining with horseradish peroxidase (HRP)-conjugated Griffonia simplicifolia agglutinin II (GSA-II). In normal human and rat organs and in mucinous metaplasia of human pancreas, immunostaining with MAb HIK1083 and PCS showed similar specificities for mucins in glandular mucous cells. In adenocarcinoma of stomach and pancreas, GSA-II showed the most widespread positivity, PCS showed the least, and MAb HIK1083 showed a reactivity between those two extremes. Colon adenocarcinomas were labeled only with GSA-II. These results demonstrate that MAb HIK1083 could be a useful screening tool for Class III mucin in normal, metaplastic, and carcinoma tissues, and that the {alpha}-GlcNAc residue is one of the specific sugar residues found in Class III mucin. (J Histochem Cytochem 46:793–801, 1998)

Key Words: N-acetylglucosamine, gastric mucin, concanavalin A, immunohistochemistry, monoclonal antibody, human, rat, neoplasm


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Concanavalin A (ConA) is a lectin that binds selectively to mannose, glucose, and GlcNAc (Goldstein 1974 ). Combined pretreatment with periodate and sodium borohydride diminishes the ConA reactivity of most mucosubstances (such as those in the plasma membrane of various cells) but paradoxically imparts it to the mucin of some epithelial cells (Katsuyama and Spicer 1978 ). The latter type of mucin, Class III mucin, has been found only in gastric glandular mucous cells including cardiac gland cells, mucous neck cells, pyloric gland cells, Brunner's gland cells, and mucous gland cells along the biliary tract and pancreas.

Class III mucin is an essential component of the gastric surface mucous gel layer and plays an important role in protecting the gastric mucosa (Ota and Katsuyama 1992 ). Furthermore, the occurrence of Class III mucin in carcinoma tissues is consistently associated with the expression of gastric phenotypes. This is true not only in gastric carcinoma but also in carcinoma of the pancreas, biliary tract, lung, and other sites (Matsuzawa et al. 1992 ; Ota et al. 1995 ). Histochemical staining of Class III mucin is therefore indispensable to a proper understanding of the physiological role of the surface mucous gel layer and the phenotypic expression of various carcinomas.

The terminal sugar residue responsible for Class III ConA reactivity was suggested to be GlcNAc after biochemical and histochemical analysis (Hotta et al. 1982 ). Recently, Ishihara et al. 1996 introduced MAb HIK1083, which was obtained by immunizing mice with mucins prepared from rat gastric mucosa. This MAb reacts with oligosaccharides that have a nonreducing terminal of {alpha}-GlcNAc residues.

The present study was undertaken (a) to explore the localization of MAb HIK1083-reactive sites in normal human organs, metaplastic tissues of the pancreas, carcinoma tissues of the stomach, pancreas, and colon, and normal rat organs, and (b) to compare those results with the results of paradoxical ConA staining (PCS) for Class III mucin and the results of staining with GSA-II (which is also known to bind to Class III mucin). The results demonstrated the highly selective nature of the reactivity of HIK1083 with Class III mucin and reinforced the idea that it has potential diagnostic value.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Preparation of Tissues
Human tissues (20% formalin-fixed, routinely processed, and paraffin-embedded) were selected from the surgical pathology files of the Department of Laboratory Medicine Shinshu University School of Medicine. The following histologically normal tissues were selected: esophagus (n = 7), gastric cardiac mucosa (n = 7), fundic mucosa (n = 8), pyloric mucosa (n = 6), duodenum (n = 10), liver (n = 2), gallbladder (n = 9), common bile duct (n = 6), pancreas (n = 10), and colon (n = 10). In addition, gastric adenocarcinoma (10 cases of intestinal type, 10 cases of diffuse type), pancreatic duct carcinoma (five cases of papillary type, six cases of tubular type), and colon adenocarcinoma (six cases of well-differentiated type, four cases of moderately differentiated type) were also studied. The criteria used for the histological classification of gastric adenocarcinomas, pancreatic duct carcinomas, and colon adenocarcinomas were based on (a) Lauren's system, (b) the General Rules for the Study of Pancreatic Cancer issued by the Japan Pancreas Society, and (c) WHO guidelines, respectively.

In addition, tissues from male Sprague–Dawley rats (n = 5) weighing approximately 200 g were also examined. Tissues were extracted from these animals under sodium pentobarbital anesthesia (50 mg/kg IP). Small pieces of esophagus, stomach, colon, pancreas, salivary gland, lung, and kidney were fixed in one of the following: 20% buffered formalin (pH 7.0) at 4C for 24 hr; 70% ethanol at 4C for 2 hr; or Carnoy's solution at 4C for 2 hr. These materials were routinely processed and embedded in paraffin.

Monoclonal Antibody
MAb HIK1083 was generated by immunizing BALB/c mice with purified rat gastric mucins according to a previously reported method (Ishihara et al. 1993 ). The binding activity of MAb HIK1083 with the oligosaccharides of purified rat gastric mucins was confirmed by ELISA. The epitope sugar structure of such mucins is reported to be characterized by the presence of oligosaccharides containing peripheral {alpha}-GlcNAc residues (Ishihara et al. 1996 ).

Histochemistry
Serial 3-µm-thick sections were stained by the following methods. To produce five degrees of trypsin digestion, formalin-fixed sections of human gastric mucosa were treated with 0.2% trypsin (DIFCO; Detroit, MI) in 0.05 M Tris buffer, pH 7.6, together with 0.1% CaCl2 for 5, 15, 30, 45, or 60 min at 37C. These sections were then stained with MAb HIK1083 (dilution 20%), PCS (ConA was purchased from Sigma, St Louis, MO), or horseradish peroxidase (HRP)-conjugated GSA-II (GSA-II-HRP; EY Laboratories, San Mateo, CA) (dilution 1%). Immunostaining with MAb HIK1083 was performed by the indirect immunoperoxidase method. PCS and staining with GSA-II-HRP followed methods described previously (Katsuyama and Spicer 1978 ; Katsuyama et al. 1985 ). Diaminobenzidine was used as the chromogen for these stains. After the same tissues had been subjected to pretreatment (see below), all sections were stained with lectins and MAb HIK1083. A dual-staining method, galactose oxidase–cold thionine–Schiff (GOTS) followed by PCS (Ota et al. 1991 ), was used to differentiate gastric surface mucous cell-type mucins from gastric gland mucous cell-type mucins in mucinous metaplasia of the pancreas. All lectin stains were preceded by amylase digestion because both ConA and GSA-II react with glycogen.

Interference between GSA-II and MAb HIK1083 was investigated in sections of human fundic, pyloric, and duodenal mucosa. The procedures were as follows: (a) pretreatment with GSA-II and subsequent indirect immunoperoxidase staining with MAb HIK1083, and (b) pretreatment with MAb HIK1083 and subsequent GSA-II–HRP staining.

The effects of various pretreatments on the reactivity of MAb HIK1083, ConA, and GSA-II with human stomach and liver was investigated using the following procedures: (a) no pretreatment; (b) oxidation with 1% aqueous periodic acid (PA) for 10 or 60 min at room temperature (RT); or (c) interposed reduction with 0.2% NaBH4 in 1% Na2HPO4 for 2 min at RT after PA oxidation. The effect of amylase digestion was also evaluated for each protocol.

The reactivity for each stain was graded on the basis of the ratio of positive cells.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Comparative Studies of Fixation Protocols and Trypsinization Protocols
The binding of MAb HIK1083, ConA, and GSA-II was preserved when these tissues were fixed in 20% buffered formalin, 70% ethanol, or Carnoy's solution. The binding of MAb HIK1083 and lectins was best preserved in tissue sections fixed with either 70% ethanol or Carnoy's solution, to judge from the staining intensity, although immunostaining with MAb HIK1083 produced some background staining when these fixation methods were used. Formalin-fixed tissue sections gave satisfactory results whether immunostaining with MAb HIK1083 or lectin staining was performed.

Trypsin digestion of formalin-fixed, paraffin-embedded tissues enhanced the intensity of the reactivity to MAb HIK1083, a 10-min period of digestion providing the most favorable results. The studies detailed below were carried out using materials fixed with 20% buffered formalin and with 10-min trypsin digestion.

HIK1083 and Lectin Binding in Non-neoplastic Tissues
The results obtained in normal human and rat organs are summarized in Table 1 and Table 2, respectively. MAb HIK1083-reactive sites were identical to those visualized with PCS. In human and rat organs, MAb HIK1083 and PCS both labeled mucins in gastric gland mucous cells (including cardiac gland cells, mucous neck cells, and pyloric gland cells) and in Brunner's gland cells (Figure 1). In human organs, periductal glands of the common bile duct and the main pancreatic duct contained mucous cells that were both immunostained with MAb HIK1083 and positive for PCS. Mucinous metaplasia of the pancreas and gastric metaplasia of the gallbladder consisted of PCS-positive and GOTS-positive cells. The former (the gastric gland mucous cell type of metaplastic cells) also showed reactivity for MAb HIK1083 and GSA-II (Figure 2) and the latter (the gastric surface mucous cell type of metaplastic cells) also stained with GSA-II (Figure 2).



View larger version (111K):
[in this window]
[in a new window]
 
Figure 1. (a–d) Fundic mucosa of the human stomach prepared from serial sections. Sections are stained with H&E (a), paradoxical ConA staining (PCS) (b), with monoclonal antibody HIK1083 (c), or Griffonia simplicifolia agglutinin II–horseradish peroxidase staining (GSA-II–HRP) (d). Mucous neck cells, which have an irregular shape with somewhat angular nuclei located at their base, are distributed around the neck region of the fundic gland (a). PCS specifically labeled mucous neck cells (b). The distribution of the MAb HIK1083-reactive cells is identical with that of the PCS-positive cells (c). GSA-II labeled the supranuclear regions of the gastric surface mucous cells as well as the mucous neck cells (d, inset). Bar = 100 µm; inset = 25 µm.

Figure 2. (a–d) Mucinous metaplasia of the human pancreatic duct prepared from serial sections. (a, c, d) Staining as in Figure 1a, Figure 1c, and Figure 1d, respectively. (b) Staining with galactose oxidase–cold thionine–Schiff (GOTS) followed by PCS. The epithelium of the pancreatic duct has been replaced by mucous cells and shows a papillary structure (a). Among metaplastic cells, cells containing gastric glandular mucous cell mucins, which stain brown with PCS, predominate over cells containing gastric surface mucous cell mucins (arrows), which stain blue with GOTS (b). Glandular mucous cell type metaplastic cells (cf. b) are also labeled with MAb HIK1083 (c) or GSA-II (d). Bar = 25 µm.

Figure 3. (a–d) Normal human colon, prepared from serial sections stained as in Figure 1a–d, respectively. PCS- (b) and HIK1083-positive sites (c) are never seen in the human colon mucosa. Only GSA-II labels the Golgi area of goblet cells and absorptive cells of the colon (d). Bar = 50 µm.


 
View this table:
[in this window]
[in a new window]
 
Table 1. Staining reactions with HIK1083, PCS, and GSA-II in normal and metaplastic human tissuesab


 
View this table:
[in this window]
[in a new window]
 
Table 2. Staining reactions with HIK1083, PCS and GSA-II in normal rat tissuesa

GSA-II, but not HIK 1083 or PCS, also labeled the supranuclear Golgi regions in a variety of epithelial cells, including gastric surface mucous cells (Figure 1), intestinal absorptive cells, and goblet cells (Figure 3).

In rat colon, basal secretory cells were positive with MAb HIK1083, PCS, and GSA-II (Figure 4).



View larger version (120K):
[in this window]
[in a new window]
 
Figure 4. (a–d) Normal rat colon, prepared from serial sections and stained as in Figure 1a–d, respectively. Basal secretory cells of rat colon are positive with PCS (b), HIK1083 (c), and GSA-II (d). Bar = 25 µm.

Figure 5. (a–d) Adenocarcinoma of the human stomach, prepared from serial sections stained as in Figure 1a–d, respectively. The carcinoma tissue has the appearance of moderately differentiated adenocarcinoma (a). Carcinoma cells show reactivity with PCS (b), MAb HIK1083 (c), and GSA-II (d), although fewer cells are positive with PCS (b) than with MAb HIK1083 (c) or GSA-II (d). Bar = 100 µm.

Figure 6. (a–d) Adenocarcinoma of the pancreas, prepared from serial sections stained as in Figure 1a–d, respectively. The carcinoma tissue had the appearance of well-differentiated adenocarcinoma (a). Carcinoma cells show reactivity with PCS (b), MAb HIK1083 (c), and GSA-II (d). PCS-reactive mucins are the most restricted, being found in only a small number of carcinoma cells (b). Bar = 100 µm.

The following tissues were negative with PCS, MAb HIK1083, and GSA-II: human and rat esophagus, rat salivary gland, and rat kidney.

HIK1083 and Lectin Binding in Carcinoma Tissues
All 20 cases of gastric carcinoma were positive for MAb HIK1083 and GSA-II, and 19 cases (95%) were positive for PCS. Of the 11 cases of pancreatic ductal carcinoma, nine cases (82%), eight cases (73%), and all 11 cases were positive for MAb HIK1083, PCS, and GSA-II, respectively. The two cases of pancreatic ductal carcinoma that were negative for MAb HIK1083 were also negative for PCS.

In HIK1083-positive and PCS-positive carcinoma tissues, the apical cytoplasm of the tumor cells and the extracellular mucins were both stained. The intracytoplasmic reaction took the form of coarse or fine droplets. HIK1083-reactive mucins appeared to be somewhat more abundant than those stained by PCS (Figure 5 and Figure 6). GSA-II also labeled these regions and, in addition, the paranuclear region of the tumor cells. Staining with HIK1083, PCS, and GSA-II differed quantitatively from case to case. GSA-II provided the most widespread staining, and the GSA-II-positive regions included the HIK1083- and PCS-reactive regions.

All 20 cases of colon adenocarcinoma consistently exhibited reactivity for GSA-II but lacked reactivity for MAb HIK1083 and PCS (Figure 7).



View larger version (167K):
[in this window]
[in a new window]
 
Figure 7. (a–d) Adenocarcinoma of the colon, prepared from serial sections stained as in Figure 1a–d, respectively. The carcinoma tissues have the appearance of well-differentiated adenocarcinoma (a). PCS- (b) and HIK1083-positive (c) sites are never seen in carcinoma tissue. Carcinoma cells reveal reactivity only with GSA-II (d). Bar = 100 µm.

Figure 8. (a–d) Normal human pyloric mucosa, prepared from serial sections. Pyloric gland cells are positive with MAb HIK1083 (a). Pretreatment with GSA-II prevents the binding of MAb HIK1083 to pyloric gland cells (b). Pyloric gland cells are positive with GSA-II (c). Pretreatment with MAb HIK1083 does not prevent the subsequent binding of GSA-II to pyloric gland cells (d). Bar = 100 µm.

Interference Between GSA-II and MAb HIK1083
Pretreatment with GSA-II prevented the binding of MAb HIK1083 to human mucous neck cells and to pyloric and Brunner's glands (Figure 8a and Figure 8b), but pretreatment with MAb HIK 1083 did not prevent the subsequent binding of GSA-II to gastric and duodenal epithelial cells (Figure 8c and Figure 8d).

Effect of Pretreatment on Staining
Oxidation and interposed reduction had no significant influence on the affinity for MAb HIK1083 and GSA-II shown by the gastric fundic mucosa. Without pretreatment, ConA labeled most mucous neck cells and some surface mucous cells weakly. Pretreatment involving PA oxidation for 10 min or 60 min, followed by NaBH4 reduction, increased the staining intensity of gastric glandular mucous cells with ConA (Class III ConA reactivity) but abolished ConA reactivity at other sites in the gastric mucosa, as reported previously (Katsuyama and Spicer 1978 ).

Pretreatment involving PA oxidation for 10 min or 60 min induced ConA reactivity in hepatocytes. This induced reactivity persisted after interposed NaBH4 reduction, as reported previously (Katsuyama and Spicer 1978 ). GSA-II also showed an affinity for hepatocytes. The reactivity of hepatocytes for GSA-II persisted after pretreatment involving PA oxidation for 10 min or PA oxidation for 10 min followed by NaBH4 reduction, but this reactivity was abolished by PA oxidation for 60 min followed by NaBH4 reduction. Pretreatment by amylase digestion completely abolished the PA oxidation-induced cytoplasmic reaction shown by hepatocytes on PCS or GSA-II-HRP staining, but did not influence HIK1083 reactivity. Hepatocytes were negative for MAb HIK1083 irrespective of the type of pretreatment used.


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

In this study we confirmed that MAb HIK1083 showed an affinity for Class III ConA-reactive mucin not only in normal tissues but also in metaplastic and carcinoma tissues.

Although the biochemical and physiological processes affecting gastric mucins under various conditions have been extensively studied (Kawano et al. 1990 ; Komuro et al. 1992 ), little attention has been paid to the fact that there are two types of mucous cell (i.e., surface mucous cells and glandular mucous cells) in the gastric mucosa. In fact, the two types of mucins secreted by these two types of mucous cells exhibit considerably different histochemical properties, suggesting fundamental differences in their sugar moieties (Katsuyama and Spicer 1978 ; Suganuma et al. 1981 ; Katsuyama et al. 1985 ; Ota et al. 1991 ). As demonstrated earlier (Ota and Katsuyama 1992 ), these cells collaborate to form the surface mucous gel layer of the stomach, which is one of the most important agents protecting the gastric mucosa. Use of MAb HIK1083 would facilitate the study of these two types of mucin.

PCS requires pretreatment, consisting of PA oxidation and borohydride reduction. A slight change in the conditions used during such pretreatment might have a considerable influence on the results obtained. Immunostaining with HIK1083, on the other hand, is a simple method that allows both detection of Class III ConA-reactive mucin and characterization of the gastric phenotype expressed in a given metaplastic or carcinomatous lesion. Furthermore, this MAb could be easily applied to ultrastructural histochemistry.

Biochemical analysis suggests that the most likely candidate for the sugar residue responsible for Class III ConA reactivity is GlcNAc (Hotta et al. 1982 ). Those authors extracted mucin-type glycoproteins from human gastric mucins using a ConA–Sepharose column after oxidation and reduction of the type used in the PCS sequence. They suggested that GlcNAc residues at the nonreducing terminal of the oligosaccharides were responsible for the Class III ConA reactivity. This idea was based on the fact that, of the three specific sugar residues of ConA, mannose and glucose are absent from epithelial mucins. In line with this idea, Ishihara et al. 1996 had already established that the epitope recognized by MAb HIK1083 consisted of oligosaccharides containing peripheral {alpha}-GlcNAc residues. The present study provides further confirmation that the reactivities of mucins to MAb HIK1083 and GSA-II are stable despite the use of PA oxidation followed by NaBH4 reduction, as was Class III ConA-reactive mucin.

Two MAbs with specificities similar to that of MAb HIK1083 have been reported. Hanisch et al. 1993 introduced MAb 2B5, which reacts with terminal ß-GlcNAc residues. MAb 2B5 was found to label glandular mucous cells of the human stomach, Brunner's gland cells, metaplastic cells of pancreas and gallbladder, and gastric differentiated carcinomas of the stomach, pancreas, and gallbladder. However, histochemical comparison between the specificity of MAb 2B5 and that of PCS has not been made. In addition, Hughes et al. 1994 and Hughes and Bhathal 1997 introduced MAb 5HL-5D11-D10 (D10). This showed the same reactivity as PCS in human normal and carcinoma tissues, although the reactivity of this MAb in rat tissues has not been investigated nor has its epitope structure been elucidated. The similar specificities shown by these two antibodies may indicate that Class III mucin contains more than one specific oligosaccharide.

In the present study, GSA-II prevented the subsequent binding of HIK1083 to mucous neck cells and to pyloric and Brunner's glands, but MAb HIK1083 did not prevent GSA-II binding. This result is consistent with biochemical reports showing that GSA-II binds to both {alpha}- and ß-GlcNAc residues (Ishihara et al. 1996 ) and that the epitope recognized by MAb HIK1083 includes oligosaccharides containing peripheral {alpha}-GlcNAc residues (Ishihara et al. 1996 ).

Colon cancers and some gastric and pancreatic cancers showed GSA-II reactivity but not PCS or MAb HIK 1083 reactivity. In fact, GSA-II binding glycoproteins in human colon carcinoma have been reported to carry GlcNAc at their nonreducing terminals (Nakayama et al. 1985 ) and, because MAb HIK 1083 recognizes only {alpha}-GlcNAc residues and ConA preferentially binds to {alpha}-GlcNAc, our results suggest that colon carcinomas may carry ß-GlcNAc residues.

GSA-II has been employed to label glandular mucous cell-type mucins of the stomach (Ihida et al. 1988 ). However, in the present study GSA-II also showed affinities for mucins in gastric surface mucous cells, mucins in the gastric surface mucous cell type of metaplastic cell of the pancreatic duct, colon adenocarcinoma cells, and the supranuclear region of various epithelial cells. To judge from these observations, GSA-II is not suitable for histochemical analysis of Class III ConA-reactive mucin and might be replaced by MAb HIK1083 for identification of glandular mucous cell-type mucins.

MAb HIK1083 may have several potentially useful applications. First, histological detection of Class III mucin by this antibody should be useful both for pathological diagnosis and for investigating the histogenesis of adenocarcinomas expressing gastric phenotypes. Second, its application to the biochemical analysis and detection of Class III mucin may be useful for the analysis of gastric mucins. Finally, estimation of Class III mucin in serum or pancreatic juice could serve for the detection of precancerous or carcinomatous lesions.


  Acknowledgments

We are indebted to the staff of Kanto Chemical Company for providing MAb HIK1083 and to Masanobu Momose (Central Clinical Laboratory, Shinshu University Hospital) for excellent technical assistance. We also gratefully acknowledge the encouragement given to us by Prof Kiyosawa (The Second Department of Internal Medicine, Shinshu University School of Medicine).

Received for publication September 3, 1997; accepted March 3, 1998.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Goldstein IJ (1974) Studies on the combining sites of concanavalin A. Adv Exp Med Biol 53:35-42

Hanisch FG, Koldovsky U, Borchard F (1993) Monoclonal antibody 2B5 defines a truncated O-glycan, GlcNAc ß1-3Gal ß1-4GlcNAc ß1-6 (GalNAc), on mucins from deep gastric and duodenal glands as well as metaplasia and neoplasia of gastric differentiation. Cancer Res 53:4791-4796[Abstract]

Hotta K, Goso K, Kato Y (1982) Human gastric glycoproteins corresponding to paradoxical concanavalin A staining. Histochemistry 76:107-112[Medline]

Hughes NR, Bhathal PS (1997) Gastric mucous neck cell and intestinal goblet cell phenotypes in gastric adenocarcinoma. J Clin Pathol 50:741-748[Abstract]

Hughes NR, Bhathal PS, Francis DMA (1994) Phenotypic identity of gastric mucous neck cells and mucous cells of cardiac, pyloric, and Brunner's glands. J Clin Pathol 47:53-57[Abstract]

Ihida K, Suganuma T, Tsuyama S, Murata F (1988) Glycoconjugate histochemistry of the rat fundic gland using Griffonia simplicifolia agglutinin-II during the development. Am J Anat 182:250-256[Medline]

Ishihara K, Kurihara M, Eto H, Kasai K, Shimauchi S, Hotta K (1993) A monoclonal antibody against carbohydrate moiety of rat gastric surface epithelial cell-derived mucin. Hybridoma 12:609-620[Medline]

Ishihara K, Kurihara M, Goso Y, Urata T, Ota H, Katsuyama T, Hotta K (1996) Peripheral {alpha}-linked N-acetylglucosamine on the carbohydrate moiety of mucin derived from mammalian gastric gland mucous cells: epitope recognized by a newly characterized monoclonal antibody. Biochem J 318:409-416[Medline]

Katsuyama T, Ono K, Nakayama J, Nanai M (1985) Recent advances in mucosubstance histochemistry. In Kawai K, ed. Gastric Mucus and Mucus Secreting Cells. Amsterdam, Excerpta Medica;, 3-18

Katsuyama T, Spicer SS (1978) Histochemical differentiation of complex carbohydrates with variants of the concanavalin A–horseradish peroxidase method. J Histochem Cytochem 26:233-250[Abstract]

Kawano S, Tsujii M, Nagano K, Ogihara T, Tanimura H, Hayashi N, Ito T, Sato N, Kamada T, Tamura T (1990) Different effect of Helicobacter pylori on the human gastric antral and body mucosal intracellular mucin. Scand J Gastroenterol 25:997-1003[Medline]

Komuro Y, Ishihara K, Ohara S, Saigenji K, Hotta K (1992) Effects of tetragastrin on mucus glycoprotein in rat gastric mucosal protection. Gastroenterol Jpn 27:597-603[Medline]

Matsuzawa K, Akamatsu T, Katsuyama T (1992) Mucin histochemistry of pancreatic duct cell carcinoma, with special reference to organoid differentiation simulating gastric pyloric mucosa. Hum Pathol 23:925-933[Medline]

Nakayama J, Katsuyama T, Ono K, Honda T, Akamatsu T, Ota H (1985) Large bowel carcinoma-specific antigens detected by the lectin, Griffonia simplicifolia agglutinin-II. Jpn J Cancer Res 76:1078-1084[Medline]

Ota H, Katsuyama T (1992) Alternating laminated array of two types of mucin in the human gastric surface mucous layer. Histochem J 24:86-92[Medline]

Ota H, Katsuyama T, Akamatsu T, Fujimori Y, Matsuzawa K, Ishii K, Honda T, Nakayama J, Furihata K, Ono K, Maeda K (1995) Application of mucin histochemistry for pathological diagnosis—expression of gastric phenotypes in metaplastic and neoplastic lesions and its relation to the organoid differentiation. Acta Histochem Cytochem 28:43-53

Ota H, Katsuyama T, Ishii K, Nakayama J, Shiozawa T, Tsukahara Y (1991) A dual staining method for identifying mucins of different gastric epithelial mucous cells. Histochem J 23:22-28[Medline]

Suganuma T, Katsuyama T, Tsukahara M, Tatematsu M, Sakakura Y, Murata F (1981) Comparative histochemical study of alimentary tracts with special reference to the mucous neck cells of the stomach. Am J Anat 161:219-238[Medline]