Masayoshi Hayama

Helicobacter pylori infection produces reversible glycosylation changes to gastric mucins

Abstract The protective ability of gastric mucins may depend largely on their oligosaccharide chains. We evaluated the effects of H. pylori infection on the glycosylation of gastric mucins. Gastric biopsy specimens from 20 H. pylori-infected patients before and after cure of the H. pylori infection and 8 normal uninfected volunteers were examined by immunostaining for simple mucin-type glycoproteins and blood-group-related antigens bearing type 1 chain backbone. The immunoreactivity in different gastric compartments was evaluated. Simple mucin-type glycoproteins and blood-group-related antigens were expressed in surface mucous cells. Simple mucin-type glycoproteins showed antrum-predominant expression in normal volunteers and were found in significantly fewer surface mucous cells in infected patients than in normal volunteers; their expression was restored after eradication of H. pylori. Sialyl Lewisa and Lewisb were expressed in fewer surface mucous cells after than before eradication. The patterns of glycosylation of gastric mucins vary in different gastric compartments and are reversibly altered by H. pylori infection. These alterations may affect the protective functions of gastric mucins.

Helicobacter pylori and two ultrastructurally distinct layers of gastric mucous cell mucins in the surface mucous gel layer

BACKGROUND AND AIMS Helicobacter pylori locate not only on the apical surface of surface mucous cells but also in the mucous gel layer covering the gastric mucosa. The present study was undertaken to observe the mucous gel layer itself and any H pylori in this layer at the electron microscopic level, and to determine whether H pylori proliferate in this layer. METHODS We examined resected human stomachs (five cases, fixed in Carnoys solution, paraffin embedded) under the light microscope, and gastric biopsy specimens (10 cases, fixed in glutaraldehyde with or without osmium, epoxy embedded) under the electron microscope. We performed histochemical staining for gastric mucins and immunostaining forH pylori, gastric gland mucous type mucins, and intestinal mucins. RESULTS Under the electron microscope, surface mucous cell type mucins and gland mucous cell type mucins in the mucous gel layer covering gastric mucosa without intestinal metaplasia showed reticular and band like structures, respectively. H pylori were frequently found as small aggregates within the mucous gel layer of surface mucous cell type mucins, and H pyloriwithin these aggregates were seen dividing.H pylori were frequently found in the mucous gel layer of the surface mucous cell type mucins along the border with the layer of gland mucous cell mucins. Occasionally, H pylori were trapped by frayed thin threads of the gland mucous cell type mucins. CONCLUSIONS The two types of gastric mucins in the mucous gel layer differ in ultrastructure. H pylori preferentially colonise and form microcolonies within the mucous gel layer of surface mucous cell type mucins. Mucins from gland mucous cells may disturb the movement of H pylori within the mucous gel layer.

Improved detection of medically important fungi by immunoperoxidase staining with polyclonal antibodies

This study was performed to identify pathological fungi of eight species [Aspergillus fumigatus, Candida albicans, Torulopsis (Candida) glabrata, Cryptococcus neoformans, Fusarium anthophilum, Rhizopus oryzae, Sporothrix schenckii and Trichosporon beigelii] in formalin-fixed, paraffin-embedded tissue sections by indirect immunoperoxidase staining. Mature albino rabbits were immunized with formalin-killed organisms. Antibodies were prepared by precipitation. Immunoperoxidase staining was applied to the paraffin-embedded tissue sections of experimentally infected mice and human autopsy and surgical specimens. Although the cell walls of each fungus stained clearly, many cross-reactivities appeared. However, it was possible to obtain specificity for the eight species by absorption and dilution of the antisera.

Proliferative activity of hepatocytes in chronic viral hepatitis as revealed by immunohistochemistry for proliferating cell nuclear antigen

Liver biopsy specimens of 65 cases of chronic viral hepatitis, including 29 cases of type B, 34 cases of type C, and two cases of non-A, non-B, non-C type, were immunohistochemically stained for proliferating cell nuclear antigen (PCNA) to evaluate the proliferative activity of hepatocytes. According to a histopathologic evaluation using the histology activity index (HAI) scoring system, chronic persistent hepatitis and chronic active hepatitis were clearly differentiated with no overlapping of the score. The labeling indices of PCNA of hepatocytes in chronic persistent hepatitis had a significant relationship with HAI scores (r = .54), suggestive of a contribution of lobular hepatocyte necrosis and/or portal inflammation to the regenerative rate of hepatocytes, but did not exceed 3.0%. On the other hand, 11 of 47 cases of chronic active hepatitis showed PCNA labeling indices higher than 3.5% without any significant relationship with the HAI scores. There was no significant difference, however, of distribution of HAI scores or PCNA labeling indices between hepatitis types B and C. Based on current concepts of the role of hepatocyte proliferation in the development of liver cirrhosis and hepatocellular carcinoma, the present results suggest that the high proliferative rate of hepatocytes subject to the persistent liver cell injury in chronic active hepatitis may be related to a reconstruction pattern of the liver in cases of progression to cirrhosis and development of hepatocellular carcinoma.

Helicobacter pylori Infection Up-regulates Gland Mucous Cell-Type Mucins in Gastric Pyloric Mucosa

Background.  Two types of mucous cell are present in gastric mucosa: surface mucous cells (SMCs) and gland mucous cells (GMCs), which consist of cardiac gland cells, mucous neck cells, and pyloric gland cells. We have previously reported that the patterns of glycosylation of SMC mucins are reversibly altered by Helicobacter pylori infection. In this study, we evaluated the effects of H. pylori infection on the expression of GMC mucins in pyloric gland cells.

Cell Lineage Specificity of Newly Raised Monoclonal Antibodies Against Gastric Mucins in Normal, Metaplastic, and Neoplastic Human Tissues and Their Application to Pathology Diagnosis

The specificity of monoclonal antibodies against gastric mucins (designated as HIK1083, PGM 36, and PGM 37) was studied immunohistochemically in normal, metaplastic, and neoplastic human tissues. These antibodies labeled class III mucin-producing cells identified by paradoxical concanavalin A staining in normal stomach, duodenum (Brunner gland), biliary tract, and main pancreatic duct; in mucinous metaplasia of pancreas and gallbladder; and in adenocarcinomas of stomach (90%), bile duct (80%), gallbladder (100%), pancreas (80%), lung (100% of goblet cell type adenocarcinomas), ovary (67% of mucinous carcinomas), and uterine cervix (100% of adenoma malignum tumors). Normal and neoplastic cells of esophagus, colon, salivary gland, kidney, endometrium, breast, prostate, and liver, as well as normal small intestine, lung, and uterine cervix, were all negative. The antibodies used should be valuable for the detection of class III mucin and class III mucin-producing cells in normal, metaplastic, and neoplastic tissues.

The application of immunoperoxidase staining for the detection of causative fungi in tissue specimens of mycosis I

This study was performed in order to identify the fungi of four species (Aspergillus fumigatus, Fusarium anthophilum, Candida albicans, Cryptococcus neoformans) in formalin-fixed, paraffin-embedded tissue sections by the indirect method of immunoperoxidase staining. Mature albino rabbits were immunized by formalin-killed organisms. The antibodies were prepared by precipitation at a 50% saturation of ammonium sulfate and were checked for cross-reactivities by Ouchterlonys double immunodiffusion and precipitin test. The immunoperoxidase staining was applied to the paraffin-embedded tissue sections of infected mice, human autopsy and biopsy specimens. Although each fungus was stained clearly the cell wall, cross-reactivities appeared among them, however it was possible to identify four fungi by absorption and dilution of the antisera.

Co-localization of TFF2 with gland mucous cell mucin in gastric mucous cells and in extracellular mucous gel adherent to normal and damaged gastric mucosa

Trefoil factor 2 (TFF2) is mucin associated peptide that has a mucosal barrier function in addition to participating in repair and healing. We examined the localization of TFF2 and gastric mucins in gastric mucous cells, the surface mucous gel layer (SMGL) adherent to normal gastric mucosa, and in the mucoid cap covering gastric erosions. Carnoy’s solution, or formalin/picric acid-fixed paraffin embedded materials from resected stomachs and formalin-fixed paraffin embedded gastric biopsy materials were used. Sections were immunostained for the TFF2 and histochemically stained for gastric mucins. In addition, thick sectioned gastric mucosa fixed in Carnoy’s solution were stained with FITC-labeled GSA-II lectin specific for gland mucous cell mucin and examined for three-dimensional images of the SMGL using a confocal laser scanning microscope. The TFF2 and gland mucous cell mucin were found intermixed together in the gastric gland mucous cells, in the SMGL in laminated layers, and in the mucoid cap. A laminated arrangement of continuous sheets of gland mucous cell mucin in the SMGL was demonstrated in the three-dimensional images. Co-localization of the TFF2 with gland mucous cell mucin suggests a physical interaction between the TFF2 and gland mucous cell mucin. The TFF2 trapped in the adherent mucins may be responsible for mucosal defense, healing, and repair.

Re-evaluation of melanin bleaching using warm diluted hydrogen peroxide for histopathological analysis

Excessive amounts of melanin pigments may hamper histopathological assessments of melanocytic lesions by obscuring cellular morphology and hindering antibody–antigen interactions. To determine the optimal melanin‐bleaching conditions for histopathological examination, heavily pigmented melanomas were treated with warm hydrogen peroxide (H2O2) diluted with various diluents (1% disodium hydrogen phosphate 12H2O ( (Na2 HPO4); phosphate buffer 0.05 M, pH 7.4 (PB); and PBS 0.05 M, pH 7.4) at varying temperatures (50°C, 55°C, and 60°C) and for varying incubation times (0.5, 1, 2, and 3 h). The effect of the sequential order of antigen retrieval and bleaching on preserving tissue morphology was then evaluated. Additionally, the effect of melanin bleaching using warm diluted H2O2 on the antigenicity of melanoma‐related markers (HMB‐45, MART‐1, and S‐100) and other markers used for histopathology was examined in amelanotic melanomas and tonsil tissue. Optimal and complete bleaching was achieved using warm 3% H2O2 in PB treatment at 55°C for 2 h following antigen retrieval with microwaving or digestion with trypsin. Under these conditions, the tissue morphology and antigenicity of various immunohistochemical markers were also well preserved. Bleaching with warm 3% H2O2 PB is a fast and efficient method of bleaching melanin pigments and performing immunohistochemical examination in heavily melanin‐pigmented lesions.

Anatomical and histological studies of so-called Müllerian duct cyst

Abstract  Background: We examined so‐called Müllerian duct cysts both histologically and immunohistochemically with anatomical observation to investigate the etiology of the ‘Müllerian duct cyst’.