An in vitro model of pancreatic carcinoma. Morphology and in vivo growth.

Pancreas rudiments from 13-day rat embryos were cultured in the presence of dimethylnitrosamine (DMN) for up to 10 weeks. Pancreas morphogenesis and differentiation occurred during the first week of culture. Acinar cell degeneration and necrosis began on the fifth day of culture and resulted in almost complete loss of acinar cells, islet cells, and fibroblasts by the end of the third week. This was associated with proliferation of cells without zymogen granules (centroacinar, ductal, or undifferentiated?). Theses cells formed glandular structures which extended to the surface of the explant. By the end of the fourth week, explants resembled ductal hyperplasia with foci of carcinoma in situ. The distribution pattern of neoplasia in 343 explants examined after 10 weeks of DMN treatment was as follows: 79% resembled ductal cell carcinoma; 9%, ductal hyperplasia; and 3%, acinar cell carcinoma. Nude mice injected with cell suspensions prepared from 10-week-old culture developed subcutaneous nodules. These nodules resembled duct cell carcinoma with desmoplastic reaction.     

Long-term organ culture of embryonic rat pancreas in a chemically defined medium.

Embryonic rat pancreas anlagen have been grown in a chemically defined medium, subdivided biweekly and recultured for a total time of 10 weeks. The total mass increment during this time was in excess of 1000-fold. Samples were removed for light and electron microscope examination and for periodic measurement of enzymatic activity. Morphogenesis and cytodifferentiation occurred, peripheral outgrowth of epithelial buds was followed by the formation of interconnecting tubular structures and, eventually, by the appearance of distinctive acinar cells with zymogen granules. Mitotic figures became conspicuous at the periphery of explants within a day after each subdivision resulting in the formation of new tubular structures and acini. In general, the central area of the explants presented more mature acini with zymogen granules than was manifested at the periphery. The enzymatic activities of amylase, lipase, and chymotrypsin developed maximally during the first week of culture, reached a plateau level by the second week, and remained at a relatively constant level throughout the 10-week culture period.     

Regeneration of submandibular salivary gland autografted in the rat tongue

Autologous SMG fragments were implanted in tongues of male rats which were sacrificed 15–20 min, 24 hr, 72 hr, 1 week, or 8 weeks after implantation. The tongues were excised, fixed, and processed for light and electron microscopy. In addition, some rats were injected with [³H]-thymidine 1 hr before sacrifice and the labeling indices (L.I.) of the salivary epithelial and interstitial cells were calculated. Twenty-four hours after implantation, SMG autografts showed massive central necrosis with some acini and ducts surviving at the periphery of the lobules. There was marked infiltration of the autografts with neutrophils and macrophages. Also the basal laminae surrounding the necrotic acini and ducts remained intact. The morphology of the autografts after 72 hr was similar to that after 24 hr except that there was additional necrosis and acini and ducts could no longer be identified in the autografts. By 1 week after implantation, the autografts showed lobular morphogenesis, ductal branching, and revascularization. At this time, the regenerating salivary epithelium appeared undifferentiated with no evidence of secretory granules. The L.I. of interstitial and ductlike structures showed significant increases over control values at 1 week after implantation, and then declined toward control levels by 3 weeks after implantation. By 8 weeks after implantation, there was evidence of acinar and striated ductal cytodifferentiation in two autografts. The results emphasize the potential of SMG autografts to regenerate subsequent to severe tissue necrosis.     

What's new in in vitro studies of exocrine pancreatic cell injury?

Exocrine pancreas in vitro models are useful for the study of pancreatic differentiation, secretion mechanisms, cell injury, and lysosomal processing of secretory product. Syrian hamster pancreas in explant organ culture undergoes a series of morphologic changes which parallel in vitro acinar cell injury, differentiation, and phenotypic alteration. Within 48 hours, the cultured acinar cells show morphologic evidence of sublethal cell injury. Autophagy and crinophagy are particularly striking. The autophagic processes can be inhibited by the addition of the protein synthesis inhibitor cycloheximide or by culture at lowered temperatures (20 degrees C). Acinar cells lethally damaged show pyknotic nuclei, high amplitude swelling, and necrosis. Approximately 25% of each explant is viable after 72 hr in culture and the viability remains constant at 25-35% for up to 60 days of culture. The morphological changes of the explants are consistent with many of the features of pancreatitis and carcinoma of the exocrine pancreas. There is an increase in the ductal elements and a decrease in acini over time in culture. This may be due to: (a) an increased replication of ductal epithelial cells concomitant with necrosis of acinar epithelial cells and/or (b) phenotypic alteration of acinar cells to ductal cells. Acinar cell necrosis and phenotypic alterations may in part be due to the activation of lysosomal degradation pathways. Processes which inhibit lysosomal activation proved protective against these alterations, while processes which promote zymogen activation were deleterious.     

Regeneration of rat submandibular gland following partial extirpation. A light and electron microscopic study

A wedge of parenchymal tissue was excised from the left submandibular gland of six week old male Sprague-Dawley rats. The animals were randomly grouped by body weight and killed at intervals of one day to five weeks following the operation. Tissue from and adjacent to the site of injury was removed and prepared for routine light and electron microscopy. Light microscopic findings consisted of degeneration and necrosis of the parenchymal tissue during the first 24 hours, followed by hyperemia and endothelial as well as epithelial proliferation from one to three days. Extensive epithelial proliferation occurred during the next two weeks, followed by regeneration of new lobules, beginning at the periphery of the injured lobes. Ultrastructurally, the new parenchymal tissue appeared to have regenerated from residual duct cells. Dedifferentiated epithelial cells gave rise to two different cell lines: one line which transformed into terminal tubules and acinar cells, and another which became striated ducts. These differentiating cells were organizing into lobules as early as three weeks after the operation. Because of their proximity to cells of regenerating striated ducts as well as intercalated ducts and acini, the myoepithelial cells appeared to be of epithelial origin.     

Early lesions of pancreatic ductal carcinoma in the hamster model.

Syrian golden hamsters were treated weekly with 10 mg/kg body weight N-nitrosobis (2-oxopropyl) amine for life (Group 1) or 6 weeks and were sacrificed at biweekly intervals from 2 weeks (Group 1) and 8 weeks (Group 2) after initiation of the experiment. The pancreas was examined in step sections, and the sequential alterations noted for each interval were recorded. Lesions were found in intrapancreatic and extrapancreatic ducts. Equivalent alterations consisting of hyperplasia, metaplasia, atypia, and lesions characteristic of carcinoma in situ developed ubiquitously and simultaneously in pancreatic ducts of different sizes and in ductules, but not in acinar cells. Among the most significant findings were intrainsular ductular formations, their proliferation, and sequential malignant alteration comparable to the involved preexisting ductules. Differences between the two experimental groups were of a quantitative rather than qualitative nature. The incidence and multiplicity of neoplastic lesions at each interval according to group, sex, and anatomic locations of adenocarcinomas are outlined. Predilected areas for some lesions were found. Results indicate a common origin of all induced tumors from a pluripotent cell populating the pancreatic ductal system.     

Proliferation of normal breast epithelial cells as shown by in vivo labeling with bromodeoxyuridine.

The proliferative activity of normal acinar and ductal breast epithelial cells was studied by in vivo labeling with 5-bromodeoxyuridine (BrdUrd) in 26 cases with concurrent breast carcinoma. The BrdUrd-labeled cells were recognized in histologic sections of paraffin-embedded tissue, using an anti-BrdUrd antibody and an immunoperoxidase reaction. The percentage of BrdUrd-labeled cells showed great variability for both acinar (0% to 2.66%; mean, 0.70%; standard deviation [SD], 0.80%) and ductal cells (0% to 1.99%; mean, 0.51%; SD, 0.57%). The fraction of proliferating epithelial cells declined with the age of the patients and was significantly higher in premenopausal women (1.16% +/- 0.85% for acinar and 0.94% +/- 0.60% for ductal cells) as compared with the postmenopausal women (0.27% +/- 0.46% for acinar and 0.17% +/- 0.22% for ductal cells), P less than 0.01 for acinar and P less than 0.001 for ductal cells, respectively. In some patients, great variability in distribution of proliferating acinar and ductal cells among different lobules and ducts was observed. No difference was found in the number of proliferating acinar and ductal cells situated near or far from their corresponding tumors. No correlation was seen between cell proliferation of normal acinar or ductal cells and cell proliferation of the respective tumors.     

Changing myoepithelial cell distribution during regeneration of rat parotid glands

The distribution of the myoepithelial cells during regeneration of the rat parotid gland after atrophy induced by one week of parotid duct ligation was investigated by immunohistochemistry for actin and transmission electron microscopy (TEM). Immunohistochemically, residual ducts were surrounded by actin-positive cells when clips were removed from the duct. Three days later, most of the newly formed acini originating from the residual ducts were also embraced by actin-positive cells. After 10 days, actin-positivity tended to be seen as dots around acini that decreased in number day by day. On day 21 actin-positive cells mainly surrounded intercalated ducts with only a few positive reactions identified at the acinar periphery. Electron microscopically, residual ducts and newly formed acini were peripherally embraced by myoepithelial cells before day 5. After day 7, shift of myoepithelial cells from the periphery of acini to the duct-acinar junctional region was identified. Then few myoepithelial cells were identified at the periphery of acini. These observations indicate that myoepithelial cells migrate from the acinar periphery to the duct-acinar junctional region during rat parotid regeneration, and that such behaviour is closely related to that seen during rat parotid development.     

Fine structure of the ventral and dorsal lobes of the prostate in the young adult syrian hamster,Mesocricetus auratus

The normal ventral and dorsal prostatic lobes of the young adult Syrian hamster were examined at the light and electron microscopic levels. Each lobe is composed of branched tubular secretory units separated from each other by loose interacinar connective tissue and draining into the urethra. The lumen of each acinus is lined by a simple epithelium composed of columnar secretory cells with occasional small basal cells. The epithelial layer, with the thin underlying lamina propria, forms a mucosa that is often highly folded. The whole acinus is bounded by a thick muscular stroma. In each of the ventral lobes, there are three main ducts, each one formed of tubular branched tributary secretory units. The walls of the secretory acini are moderately folded. Microvilli dominate the lumenal surface of the secretory epithelial cells. The Golgi complex is very extensive and shows dilated cisternae and secretory vesicles and vacuoles of various sizes. Membrane-bounded secretory granules populate the Golgi and apical areas and are released into the acinar lumen by exocytosis. The rough endoplasmic reticulum is dispersed throughout the cytoplasm, except in the region of the Golgi apparatus. In each of the dorsal lobes, there are several main tubular ducts that open into the urethra. Both proximal (ductal) and distal portions of the glandular tree are secretory in nature. Microvilli and cytoplasmic bulges and blebs dominate the lumenal surface of the secretory cells. The cells are also characterized by highly dilated cisternae of rough endoplasmic reticulum. The secretory cells show heterogeneity in the degree of dilation and distribution of rough endoplasmic reticulum, and this heterogeneity may reflect location in the glandular tree. Large dilated cisternae with irregular outlines are common in the basal portion of some cells, mainly those of the distended proximal (ductal) portions of the acini. The more highly folded distal portions of acini show smaller regular cisternae distributed throughout the cytoplasm. The Golgi complex is poorly to moderately developed, and membrane-bounded secretory granules are absent or sparse. Apocrine secretion predominates in the dorsal lobe.     

Morphometric and immunohistochemical characterization of human liver regeneration.

Regeneration in human liver is characterized in part by the formation of ductular structures, so-called ductular hepatocytes in massive hepatic necrosis and bile ductules in mechanical biliary obstruction. In an attempt to characterize the liver regenerative process, we performed image analysis and immunohistochemical staining of the ductular structures in these well defined human liver disorders, 13 cases of massive hepatic necrosis and 9 cases of mechanical biliary obstruction. The proliferation index was determined and the expression of several antigens was localized by immunohistochemical staining using antibodies to alpha-fetoprotein, alpha-1-antitrypsin, albumin, and cytokeratin 19. The ductular structures in adult human liver were compared with the developing ductal plates in 11 fetal livers, ranging in age from 9 to 36 weeks of gestation. Image analysis demonstrated that the mean total area, mean nuclear area, and mean cell size of ductular hepatocytes were significantly larger than those of bile ductules (p < 0.05). The proliferation index of ductular hepatocytes and bile ductules was significantly higher than that of hepatocytes of normal livers (p < 0.02). Bile ducts, bile ductules in mechanical biliary obstruction, ductular hepatocytes in massive hepatic necrosis, and the ductal plate cells in fetal liver showed strong staining for cytokeratin 19, which characterizes intermediate filaments associated with bile duct epithelial cells. Albumin, a liver-specific protein, and alpha-1-antitrypsin, a protease inhibitor, were strongly expressed in ductal plate cells of fetal liver, hepatocytes, and ductular hepatocytes, whereas bile duct cells and bile ductules were negative for albumin. In summary, ductular hepatocytes demonstrate morphometric and immunophenotypic features of both hepatocytes and biliary epithelial cells, whereas bile ductules share characteristics primarily with fetal ductal plates and mature bile ducts. These findings suggest that ductular hepatocytes in massive hepatic necrosis may serve as bipotential progenitor cells, and bile ductules in mechanical biliary obstruction are related to ductal plates of fetal liver.     

Ultrastructural aspects of calf submandibular glands

Submandibular gland biopsies from four calves were examined by electron microscopy. Most of the parenchyma consists of mucous acini capped by seromucous demilunes. Secretory product of the demilunes reaches the acinar lumen via intercellular canaliculi located between adjacent demilunar cells or by narrow apical extensions of demilunar cells bordering the lumen in common with acinar cells. Intercellular canaliculi are absent between mucous acinar cells, but intercellular space is present at junctions of demilunar cells, acinar cells, and intercalated duct cells. Intercalated ducts are short and connect mucous acini with striated ducts. Striated ducts show more basal infoldings and mitochondria than those of bovine parotid glands. Nuclear bodies are present in most epithelial cell types of the gland but are larger and more easily recognized in nuclei of striated duct cells. Attempts are made to correlate the structure of bovine submandibular glands with its secretion of small amounts of hypotonic saliva relative to the larger volume of isotonic saliva secreted by parotid glands of the same animal.     

Immunohistochemical studies of endocrine cells in heterotopic pancreas

Summary Twenty-one specimens of heterotopic pancreas were investigated using the indirect immunoperoxidase method for insulin, somatostatin, glucagon, pancreatic polypeptide (PP) and gastrin. Ten specimens showed ducts, acini and islets, seven showed ducts and acini, and four showed a ductal component alone. Pyloric gland-like mucous glands were occasionally identified in association with the ductal component. In eight of ten lesions containing islets, the islets were round and had a clearly defined outline with many glucagon cells and either none or a modest number of PP cells (dorsal type). In the remaining two lesions, the islets showed varying sizes and irregular outline with many PP cells and a few or no glucagon cells (ventral type). In either type of islets, insulin and somatostatin were detected, but gastrin cells were absent. Some isolated endocrine cells were also present among the acinar and ductal components. Their occurrence in ducts was more frequent in lesions or areas mainly composed of the ductal compoment than in those with less prominent ductal tissue. In eight lesions a few gastrin cells were found in the ductal component which showed goblet cell metaplasia and pyloric gland metaplasia. An intimate relationship between goblet cell metaplasia and appearance of G cells is noteworthy.     

Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction

Acinar cell regeneration in the rat parotid gland after atrophy induced by a one week period of duct obstruction was examined using histology, immunohistochemistry and transmission electron microscopy (TEM). For immunohistochemistry, antibodies to 5-bromo-2'-deoxyuridine (BrdU), injected one hour before tissue collection, and cytokeratin were employed. When clips were removed from the duct, only ductal epithelial cells remained; all acinar cells had been deleted. Some duct cells were BrdU positive. After three days, newly-formed acini comprising immature acinar cells had appeared; many of the cells were BrdU positive and mitotic figures were readily identified. Thereafter progressive acinar cell maturation and proliferation occurred, parotid gland weight returning to control levels by 7 days. Peak BrdU labelling indices for duct and acinar cells were on days 0 and 4, respectively. By TEM, cytoplasmic organelles in epithelial cells of transitional duct-acinar structures seen at 2 days were poorly developed. Immature acinar cells seen on day 3 contained zymogen granules and had increased endoplasmic reticulum and mitochondria. By day 5, maturing acinar cells had abundant endoplasmic reticulum and zymogen granules, resembling acinar cells in control glands. These observations indicated origin of acinar cell precursors from duct cells during regeneration of the acinar cell-free atrophic gland. Subsequent expansion of the acinar cell population was dependent on maturation and proliferation of these newly-formed cells.     

Ultrastructural study of acinar and intercalated duct organization of submandibular and parotid salivary gland

According to some current hypotheses, the morphology and organization of the intercalated duct/acinar interface of salivary gland have implications for the induction of tumors in this organ. However, this region has received limited detailed investigation. To study the organization of the terminal ductal segments of salivary gland, conventional transmission electron microscopy of human parotid and submandibular glands and canine submandibular gland was combined with 3-dimensional observations of polymer casts of the canine submandibular ductal system; the latter were prepared by retrograde injection of acrylic resin via the main excretory duct with subsequent digestion of the gland tissue. The division of intercalated ducts, into first- and second-order branches, and acinar arrangement is more complex than previously suggested. The entire surface of each elongated second-order intercalated duct is covered with acini projecting in all directions. In the human gland, some acini abut directly on the intercalated duct surface, whereas others are connected by a short stalk of intercalated duct cells; in comparison with canine submandibular gland, the latter may be a modification producing a third-order of the intercalated duct unit. All of these features combine to produce a highly efficient secretory apparatus with a large proportion of acinar cells to each intercalated duct.     

Redistribution of the sheep neonatal Fc receptor in the mammary gland around the time of parturition in ewes and its localization in the small intestine of neonatal lambs

Maternal immunity is mediated exclusively by colostral immunoglobulins in ruminants. As the neonatal Fc receptor (FcRn) is suggested to be involved in the transport of immunoglobulin G (IgG) in the mammary gland, we cloned this receptor from sheep and analysed its expression in the mammary gland around the time of parturition and also in the small intestine from the newborn lamb. FcRn heavy-chain mRNA was detected (by using in situ hybridization) exclusively in the acinar and ductal epithelial cells in mammary gland biopsies both before and after parturition. Immunohistochemistry revealed that the cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition. A remarkable difference was observed in the pattern after lambing, where the apical side of the cells was strongly stained. The presence of the FcRn in the acinar and ductal epithelial cells of the mammary gland, and the obvious change in distribution before and after parturition, indicate that the FcRn plays an important role in the transport of IgG during colostrum formation in ruminants. Immunohistochemical analysis detected a strong apical and a weak basal FcRn signal in the duodenal crypt cells of a neonatal lamb, which have been previously demonstrated to secrete IgG1 in newborn ruminants. The FcRn was not detected in the duodenal enterocytes, which absorb intact IgG from the colostrum in a non-specific manner. These data suggest that FcRn is involved in IgG1 secretion in ruminant epithelial cells.     

Expression of tenascin, type IV collagen and laminin during human intrahepatic bile duct development and in intrahepatic cholangiocarcinoma

Expression of tenascin, type IV collagen and laminin during human intrahepatic bile duct development and in cholangiocarcinoma was examined by immunohistochemistry. In the developing hilar bile ducts, tenascin was expressed in the mesenchyme around the epithelial cells migrating from the ductal plate into the mesenchyme at 10–14 weeks of gestation. Tenascin was also expressed in the mesenchyme around newly formed hilar bile ducts at 15-20 weeks of gestation, but its expression disappeared after 21 weeks of gestation. Type IV collagen and laminin were expressed around the ductal plate, around epithelial cells migrating from the ductal plate into the mesenchyme, and around newly formed hilar bile ducts, and their expression was present throughout fetal life. By contrast, in the development of peripheral bile ducts, tenascin expression was not found. Type IV collagen and laminin were identified around the ductal plate, migrating epithelial cells and peripheral bile ducts. In cholangiocarcinoma, tenascin and type IV collagen were expressed in the stroma, but laminin was not identified. These findings suggest that tenascin may play a role in hilar bile duct development and that type IV collagen and laminin may play a role in both hilar and peripheral bile duct development. Expression of tenascin and type IV collagen in the stroma of cholangiocarcinoma may be the result of malignant transformation of intrahepatic biliary epithelium; tenascin in peritumoral stroma may stimulate carcinoma cell proliferation and growth in cholangiocarcinoma.     

Differential distribution of sialic acid in exocrine pancreas and parotid gland

The sialic acid-specific lectin limulin (LPA, from Limulus polyphemus hemolymph) was used for the investigation of the distribution of accessible sialoglycoconjugates on the surface of cells from rat and rabbit parotid gland and exocrine pancreas. Fluorescence microscopy with rhodamine-conjugated LPA on fresh-frozen and fixed-frozen sections of the parotid gland revealed lectin binding sites on acinar and ductular epithelial cells. The staining was localized only at the periphery of the acini and ducts and was absent from the apical and lateral surface of epithelial cells. This staining pattern contrasted with that found in epithelial cells of acini and ducts in exocrine pancreas, where the luminal surface was intensely labeled by the fluorescent lectin. The luminal content of ductular tract and acini in parotid gland and pancreas was devoid of lectin-reactive sialoglycoconjugates. Connective tissue surrounding ducts and blood vessels bound the lectin heavily in both glands. These results outline that cells with similar structure and function, but constituents of different exocrine glands, exhibit differential distribution of sialoglycoconjugates on their corresponding plasmalemmal domains.     

Expression of vimentin in proliferating and damaged bile ductules and interlobular bile ducts in nonneoplastic hepatobiliary diseases.

Biliary epithelial cells express characteristically cytokeratin in their cytoplasm in normal and diseased livers. The present study disclosed that vimentin was frequently expressed in the cytoplasm of proliferating and damaged bile ductules and interlobular bile ducts, while their normal counterparts were negative for vimentin. Although this expression itself seemed nonspecific to any of the hepatobiliary diseases examined, bile ductules and interlobular bile ducts were frequently positive in chronic cholestatic and necroinflammatory liver diseases. In biliary epithelial cells, vimentin was localized around the nucleus or in the subnuclear regions, when present. Immunoelectron microscopically, reaction products for vimentin and for cytokeratin were found on bundles of intermediate filaments in the cytoplasm of biliary epithelial cells. The former was found mostly in the paranuclear and subnuclear regions, while the latter detected around the desmosomes, in addition to the paranuclear cytoplasm. Vimentin and cytokeratin were also seen together under immunoelectron microscopy on the same intermediate filaments. It seems likely that aberrant expression of vimentin in bile ductules and interlobular bile ducts and heterogeneous antigenic expression of intermediate filaments in the same biliary epithelial cells may be related to proliferation of, reorganization of, or damage to the ductular and ductal biliary cells in a variety of hepatobiliary diseases.     

Chronic sialadenitis

Summary The cellular changes in salivary gland parenchyma with chronic inflammation were studied immunocytochemically with a panel of antibodies. Myoepithelial cells were labelled with antimyosin, duct cells with a polyclonal anti-callus prekeratin, a monoclonal anti-keratin CAM 5.2 and a monoclonal anti-keratin 7 (RPN 1162), and a subpopulation of basal duct cells with a monoclonal anti-keratin 16 a. The wide range of changes observed were similar to those described following experimental duct ligation. One of the most striking features was the survival of myoepithelial cells surrounding persisting acini and ductal structures. Most of these ductal structures appeared to be either surviving intercalated ducts or were altered acinar cells. There was no evidence of myoepithelial or ductal hyperplasia. The 16a positive basally located duct cells which are conspicuous in normal glands, pleomorphic adenomas and in the epithelial islands in lymphoepithelial lesions (Palmer et al. 1985; 1986) were virtually absent, except in one specimen with mild inflammatory changes. If this cell type represents a reserve cell, then loss of it may preclude recovery of the remaining parenchyma following resolution of inflammation.