Cellular proliferation activity in duct-ligated submandibular glands (SMGs) of rats were evaluated by using index of proliferating cell nuclear antigen (PCNA) immunostaining and ³H-thymidine incorporation. The normal SMGs showed a very low index (<0.1%) of PCNA immunostaining and ³H-thymidine incorporation in the acinar and all the ductal segments. On day 2 and 3 of duct ligation, index of PCNA staining and ³H-thymidine incorporation increased and reached a peak in degranulated granular convoluted tubules (GCTs), duct like structures (DLSs) and dilated excretory ducts (EDs). On day 5 to 10, the glands showed fibrous proliferation around the DLS, and cells in DLSs and dilated EDs had decreasing index of PCNA staining and ³H-thymidine incorporation. On day 14 and 21, the PCNA staining and ³H-thymidine incorporation had decreased and was mainly confined to the DLSs and basal cells of keratinizing epithelial masses. In the unligated SMG of the opposite side of the duct-ligated animals, proliferating cells were found mainly in the acinar compartment from the 2nd to 5th day of ligation.
These results suggested that in case of acceleration of secretry function of SMG, secretory cells may proliferate as for the functional adaptation. However, in the case of obstructive lesions of SMG, GCT cells and ductal basal cells may have more potential for proliferation and these cells may be the major participant cells during glandular regeneration and possibly in the histogenesis of the majority of salivary gland tumors, if not all.

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Administration of acetylsalicylic acid (ASA) to rats caused an alteration in intraperoxisomal localization of enzymes in liver peroxisomes as investigated by means of immunoelectron microscopy. Rats were fed on a diet containing 0.5% ASA for 2 weeks and livers were fixed by perfusion. Vibratome sections of the liver tissue were processed to routine electron microscopy (EM), alkaline 3, 3′-diaminobenzidine (DAB) reaction for catalase, and post embedding immunocytochemistry for several peroxisomal enzymes. By light microscopy (LM) of the DAB stained materials, many large spindleform peroxisomes were noted in the cytoplasm of hepatocytes, among which normal size of peroxisomes was also observed. By EM, such large peroxisomes contained fibrillar inclusions in the matrix in addition to the crystalloid core. The fibrillar inclusions were more strongly stained than the matrix after the DAB reaction for catalase. By postembedding immunoelectron microscopy, gold particles showing catalase, acyl-CoA oxidase, L-α-hydroxy-acid oxidase A and serine: pyruvate/glyoxylate aminotransferase (SPT) were closely associated with the fibrillar structures. Urate oxidase was confined exclusively to the crystalloid core, on which a few gold particles showing L-α-hydroxy-acid oxidase A was also noted. D-amino acid oxidase was associated exclusively with a clear spot located adjacent to the crystalloid core. In this clear spot, no other enzymes examined were present. The results show that the fibrillar structure induced by ASA includes at least catalase, acyl-CoA oxidase, L-α-hydroxy-acid oxidase A and SPT, but not urate oxidase and D-amino acid oxidase. D-amino acid oxidase is one of elements that constitute the clear spot. Taken together, the present results demonstrate that some subcompartments exist in the experimentally altered liver peroxisomes.

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This study was designed to clarify the role of growth factors and vascular proliferation in the growth of colon tumor. On immunohistochemical observation, the ratios of positive cells for PCNA, bFGF and VEGF in carcinomas and adenomas were higher than in non-neoplastic mucosa. On reverse transcriptasepolymerase chain reaction (RT-PCR) study, the expression of bFGF mRNA, FGF-receptor (FGFR) mRNA and VEGF mRNA was confirmed in all cases including non-neoplastic tissues, adenomas and carcinomas. On in situ hybridization, bFGF mRNA was expressed in carcinoma cells, vascular cells and fibroblastic cells, and the expression ratio of bFGF mRNA in carcinomas was higher than in non-carcinomatous mucosa. VEGF mRNA was expressed in carcinoma cells, medial smooth muscle cells and fibroblastic cells. These results suggest that bFGF and VEGF may contribute to tumor growth in association with neovascularization.

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To elucidate the role of extracellular matrix and basement membrane associated proteins at the ectodermal-ectomesenchymal interface of developing salivary glands, human fetal submandibular glands (n=100), gestational age varying from 10 to 40 weeks, were evaluated for expression of tenascin, type IV collagen and laminin by using immunohistochemical methods and the results were compared with their expression in adult glands (n=20). Immunoreactive tenascin was present in the epithelial-mesenchymal interface of developing salivary ducts beginning from 10 weeks of gestational age during the early developing stages. In the late developmental stages, the intensity of immunoreaction was reduced around the proximal striated ducts and no immunoreaction was seen around the intercalated duct and acinar cells. Adult submandibular glands had immunoreactive tenascin confined to the periductal connective tissue of distal segment of striated and excretory ducts. Type IV collagen and laminin were observed in the basement membrane of salivary ducts and acini beginning from 16-18 weeks, and the intensity of reaction was more prominent during intermediate developing stages (19-32 weeks). The intensity of reaction was, however, less prominent in late developmental stages (33-40 weeks) which was similar to that in the adult glands. The present study suggests that tenascin forms an epithelial-ectomesenchymal interface before the organization of collagen IV and laminin in the basement membrane of salivary ducts and acini in developing human submandibular glands. The accumulation of the basement membrane proteins, however, are associated with stages of more active cytodifferentiation and morphodifferentiation of the glands.

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Using an array of histochemical and immunohistochemical techniques, we have characterized the various types of mucins in the normal, metaplastic and neoplastic stomach, pancreas and biliary ducts, in the normal and neoplastic esophagus, colon and lung, and in tumors of the ovary. Gastric phenotypes were variously expressed in metaplastic and neoplastic cells in these organs. Their expression was usually associated with an organoid differentiation simulating the gastric pyloric mucosa.
In gastric intramucosal carcinoma tissues, neoplastic cells often proliferated forming structures resembling non-neoplastic gastric mucosa. Thus, carcinoma cells in the more superficial layers expressed strong galactose oxidase cold thioneine Schiff reactivity (a surface mucous cell type property), whereas cells in the deeper layers were stained by paradoxical concanavalin A staining (a glandular mucous cell type property). Proliferative cells were perferentially located in the middle layers. This organized differentiation is lost in portions of the tumor that have invaded beyond the muscularis mucosae. Adenocarcinoma cells in both the bile duct and the pancreatic duct, in the bronchioloalveolar carcinoma of the lung and in mucinous cyst adenoma of the ovary showed gastric phenotypes and organoid differentiation. In papillary portions of these tumors, cells in protruded portions of the neoplastic papillae tended to express phenotypes of surface mucous cell, while in the indented portions they expressed phenotypes of glandular mucous cell. The goblet cell metaplasia in areas adjacent to pancreatic ductal carcinoma is identical to gastric pyloric metaplasia.
We conclude that the systematic use of mucin histochemistry, a relatively simple technique that can be implemented in most histopathology laboratories, may provide important information about structure and property of neoplastic tissues and could enhance our understanding of carcinogenesis in many organs.

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The techniques of immunocytochemistry and in situ hybridization can supplant the flawed ‘mitotic index’ in providing critical information regarding cell cycle status of human tumors. While methods such as bromodeoxyuridine (BrdU) incorporation can provide accurate measurements of S phase indices in tissue samples, this method cannot generally be used for routine pathology specimens. Monoclonal and polyclonal antibodies have been developed which can, under appropriate fixation conditions, detect cells in specific phases of the cell cycle, including: the Ki-67-defined nuclear cell proliferation related antigen, expression of which is characteristic of all cycling (non-G₀) cells; proliferating cell nuclear antigen (PCNA; a marker of S-phase); and statin, a marker of G₀-phase. Antibodies to PCNA however, cannot be employed with reliability in deparaffinized, formalin-fixed tissues owing to the variable retention of non-repliconassociated PCNA in the nucleus in non-S-phase cells; ‘PCNA indices’, however, in methacarn fixed tissue can provide information comparable to that of BrdU incorporation. Statin is a 57kd protein, expression of which is associated with cell senescence and cell quiescence; immunolocalization is possible only in frozen or methacarn-fixed, deparaffinized tissues. Antibodies to PCNA and statin have been applied to a series of 51 methacarn breast specimens, confirming an inverse correlation between statin and PCNA indices; furthermore, a direct relationship between statin index and long term survival was observed. The most recently described marker associated with cell proliferation, in situ hybridization using cDNA probes to histone H3 and/or H4 mRNA, offers the unique feature of identifying a cytoplasmic S-phase marker, permitting double labeling studies with antibodies to nuclear proteins. These methods, in aggregate, represent reasonable alternatives to flow cytometrically determined cell proliferation indices.

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In order to demonstrate the intracellular localization of the peroxisome proliferator in the hepatocytes, we have examined the localization of silver grains due to ¹⁴C-labeled bezafibrate in cultured rat hepatocytes by means of light and electron microscopic radioautography. On light microscopic radioautograms of the epoxy resin sections at each incorporation period, about 90% of all the silver grains were localized over the cytoplasmi Then, statistical significance was evaluated on grain density in the cytoplasm. On electron microscopic radioautograms of whole mount cultured cells observed by high voltage electron microscopy, silver grains were localized on the cytoplasmic matrix, especially over the endoplasmic reticulum. From these results, it is concluded that bezafibrate was localized over the endoplasmic reticulum. This fact suggests than the receptor of peroxisome proliferator should be associated with the endoplasmic reticulum or the receptor exists on the endoplasmic reticulum. Thus, it is demonstrated that the peroxisome proliferator acts on the endoplasmic reticulum of hepatocytes to proliferate peroxisomes.

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Background: The basic histopathologic patterns of Helicobacter pylori gastritis have been described, but the details of the interaction between bacteria, epithelial cells and inflammatory cells are poorly understood. One of the limiting factors is the lack of a staining technique allowing the simultaneous visualization of bacteria and the morphologic details of the infected mucosa.
Purpose: To use a new stain to analyze intensity and distribution of H. pylori and gastritis in patients with H. pylori gastritis without ulcer, with duodenal ulcer, and with gastric ulcer, in an attempt to elucidate the role of H. pylori in the pathogenesis of peptic ulcer disease.
Methods: patients underwent esophagogastro-duodenoscopy and gastric biopsies were obtained from 3 sites on the antral lesser curvature, 2 on the antral greater curvature, and 6 on the corpus. Formalin-fixed biopsy specimens were processed, oriented, embedded in paraffin, cut in 4-μm sections and stained with a combined stain developed in our laboratory (a combination of hematoxylin and eosin with a silver stain and Alcian Blue at pH 2.5). This stain allows the simultaneous visualization of H. pylori, the gastric mucosal morphology, and the inflammatory infiltrate. Density of H. pylori, neutrophils and mononuclear cells were scored on each specimen using a scale from 0 to 5.
Results: Density and distribution of H. pylori were essentially similar in the non-ulcer infected patients and in patients with gastric ulcer. Patients with duodenal ulcer had a higher density of bacteria in the antrum than in the corpus. All patients had significantly greater inflammatory responses in the antrum than in the corpus, but this gradient was much more pronounced in patients with duodenal ulcer than in those with gastric ulcer and those with no ulcer. Surprisingly, the strongest intensity of gastritis (both in the antrum and corpus) was found in this latter group.
Conclusions: The gastritis gradient is more important than the intensity of inflammatory responses per se in determining the outcome of H. pylori infection with respect to the development of peptic ulcer. Larger studies, using new methodologies that allow a better assessment of the numbers of bacteria in the gastric mucus, should be performed to better elucidate the relationship between magnitude of infection, intensity of the mucosal inflammatory responses, and outcome of H. pylori gastritis.

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