The ultrastructural basis for the identification of cell types in the pancreatic islets

Summary The pancreatic islets of rabbit, dog and opossum have been studied by light and electron microscopy. Silver-positive cells in the rabbit are predominantly sandwiched between the peripheral A and central B cells, and by electron microscopy are identified as D cells. Pancreatic islets in the tail of the dog pancreas have A, B, and D (silver-positive) cells, but the islets in the uncinate process of the dog pancreas lack phosphotungstic acid hematoxylin-positive A cells. By electron microscopy the characteristic D cells are found in both tail and uncinate process, but A cells are confined to the tail islets, confirming the identification of cell types. A unique cell type termed the F cell is found in the dog uncinate islets and it is characterized by secretory granules of angular profiles. In the opossum, the A cells contain considerable amounts of glycogen demonstrable by both light and electron microscopy. A unique cell type is also present in the opossum islets termed an E cell (Thomas, 1937), which has large secretory granules (400–500 m). The physiological implications of a multiplicity of cell types in pancreatic islets is discussed.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases. The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donahue.     

An immunocytochemical and electron-microscopical study of endocrine cells in the gut and pancreas of a stomachless teleost fish,Barbus conchonius (Cyprinidae)

Summary Four immunoreactive endocrine cell types can be distinguished in the pancreatic islets of B. conchonius: insulin-producing B cells, somatostatin-producing A₁ (= D) cells, glucagon-producing A₂ cells and pancreatic poly-peptide-producing PP cells. The principal islet of this species contains only a few PP cells, while many PP cells are present in the smaller islets. Except for the B cell all pancreatic endocrine cell types are also present in the pancreatic duct.At least six enteroendocrine cell types are present in the gut of B. conchonius: 1. a cell type (I) with small secretory granules, present throughout the intestine, and possibly involved in the regulation of gut motility; 2. a C-terminal gastrin immunoreactive cell, probably producing a caerulein-like peptide; these cells are located at the upper parts of the folds, especially in the proximal part of the intestinal bulb; 3. a met-enkephalin-immunoreactive cell, present throughout the first segment; 4. a glucagon-immunoreactive cell, which is rare in the first segment; 5. a PP-immunoreactive cell, mainly present in the first half of the first segment; 6. an immunoreactive cell, which cannot at present be specified, located in the intestinal bulb. The latter four cell types are mostly located in the basal parts of the folds, although some PP-immunoreactive cells can also be found in the upper parts.Most if not all enteroendocrine cells are of the open type. The possible functions of all enteroendocrine cell types are discussed.Abbreviations BPP bovine pancreatic polypeptide - CCK cholecystokinin - GEP gastro-entero-pancreatic - GIP gastric inhibitory peptide or glucose-dependent insulin releasing peptide - PPP pig pancreatic polypeptide - VIP vasoactive intestinal polypeptide     

Eosinophils and mast cell homogeneity of the guinea pig eyelid skin, conjunctiva, and ileum

Mast cell heterogeneity has been described on the basis of differential staining reactions, light microscopic morphology, anatomic location, degranulation after polyamines, biochemical contents, growth requirements, and reactions to lymphokines. We have demonstrated typical "connective-tissue mast cells" by using anatomic criteria, histological staining reactions, electron microscopy, and reaction to compound 48/80 in the guinea pig conjunctiva, eyelid skin, and ileum. A second, much larger population of cells in the ileal mucosa and the conjunctiva, and rarely in the eyelid skin stained reddish-blue with acid toluidine blue in tissue fixed in ethanol-acetate-lead subacetate (BLA) and with alkaline Giemsa in formaldehyde-fixed tissue, did not stain with ethanolic or acid toluidine blue in formaldehyde-fixed tissue or with alkaline Giemsa in BLA-fixed tissue, and did not degranulate after 48/80 treatment. These are features of the rat intestinal "mucosal mast cells"; however, ultrastructural and light microscopic studies with the orcein Giemsa stain demonstrated these cells in the guinea pig to be eosinophils. Tissue culture, biochemical, and immunological studies indicate the existence of a second type of mast cell (bone-marrow-derived mast cell), ultrastructurally almost indistinguishable from the connective tissue mast cell. Our studies demonstrate only one mast cell type in the guinea pig and support the contention that other forms of mast cells are immature forms or variants of the connective-tissue mast cell.     

Innervation of the pancreas by vasoactive intestinal polypeptide (VIP) immunoreactive nerves

The vasoactive intestinal polypeptide (VIP) has been shown to exert effects on endocrine and exocrine pancreatic secretion. Immunocytochemistry reveals that VIP immunoreactive nerves occur in the porcine, canine, feline and avian pancreas. In the pancreas of pig and cat VIP nerves are abundant around non-immunoreactive nerve cell bodies of the intrapancreatic ganglia but scarce in the islets and in the exocrine parenchyma. In the dog pancreas, however, the intrapancreatic ganglia contain strongly immunoreactive VIP nerve cell bodies which give off axons that seem to heavily innervate vessels as well as endocrine and exocrine cells. We suggest that in the pig and cat the pancreatic VIP nerves mainly affect the activity of a second type of intrapancreatic neuron, whose transmitter is unknown, whereas in the dog pancreas VIP nerves directly contact their putative effector structures.     

哺乳动物胰腺体部胰多肽（PP）免疫反应细胞的比较研究

采用SPA-GDN免疫组化染色技术,对人、大鼠、小鼠、豚鼠、猪、狗和猫等七种哺乳动物胰腺体部胰多肽(PP)免疫反应细胞的分布和形态进行比较研究,结果表明,上述七种动物PP细胞的分布和形态有明显的种间差异。人、大鼠和小鼠PP细胞主要位于胰岛周边部,形成环形结构,少量PP细胞散布在外分泌部的腺泡和导管;而豚鼠、猪和猫的PP细胞则主要分布在外分泌部腺泡和导管上皮间;狗的PP细胞在内、外分泌部均有分布。PP细胞的形态在上述动物间也有明显的差异,这可能与该细胞在不同动物的作用途径及功能不同有关。     

A combined stain for identifying epithelial cells of the gastric mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: paradoxical concanavalin A staining (PCS) to identify mucous neck cells, periodic acid Schiff-concanavalin A staining to distinguish mucous neck cells from surface mucous cells, and a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: Feulgen hydrolysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; Feulgen hydrolysis-PAS-concanavalin A-Bowie staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

A Combined Stain for Identifying Epithelial Cells of the Gastric Mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: A) paradoxical concanavalin A staining (PCS) to identify mucous neck cells, B) periodic acid Schiff-concana-valin A staining to distinguish mucous neck cells from surface mucous cells, and C) a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: 1) Feulgen hydroIysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; 2) Feulgen hydrolysis-PAS-concanavalin A-Bowic staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

Differentiation of Cells in the Hypophysis and in Pancreatic Islets

Deparaffinized, 3-5μ, sections are brought to water, oxidized 3.5 min in an equal-parts mixture of 0.3% H₂SO₄ and 0.3% KMnO₄, and decolorized with 4% K₂S₂O₅. Nuclei are stained with Gomori's (1939) chromium-hematoxylin, and cell granules with Cason's (1950) mixture. The eosinophilic cells of the hypophysis and the alpha cells of pancreatic islets (of Langerhans) stain carmine red; basophilic and beta cells stain dark blue. Heidenhain's _susa is the most suitable fixative for hypophysis, Bouin's fluid for pancreas; but a satisfactory result is obtainable after formalin-sublimate or plain formalin. Besides studying the ratio of the cell types in the hypophysis or in pancreatic islets, it is possible to estimate the granule content of the cells. The method works on human autopsy material provided fixation of hypophysis occurs within 24 hr, and. pancreas, 12 hr post mortem, and it is suitable also for quite fresh organs.     

常用实验动物不同组织部位肥大细胞异质性的组织学特点比较

目的探讨并比较六种常用实验动物不同部位肥大细胞异质性的形态学特点。方法应用麻醉后处死的方法,取小鼠、大鼠、豚鼠、兔、犬和猴的皮肤、肺脏、乙状结肠和脾脏组织,经4%中性缓冲甲醛溶液或Bouin’s液固定后,制作常规组织切片,分别作HE、甲苯胺蓝、阿尔新蓝-藏红和P物质免疫组织化学染色,镜下观察并应用彩色病理图像分析软件进行形态学分析;另取皮肤组织固定于磷酸缓冲戊二醛溶液,制作常规超薄切片,透射电镜下观察肥大细胞超微结构。结果六种动物不同组织中肥大细胞各有其分布特点,且在形态大小、异染性及染色特性等方面表现各异,肥大细胞密度和形态学参数差异有显著性（P〈0.05）;豚鼠、犬和猴皮肤肥大细胞颗粒具有特殊亚微结构;小鼠皮肤组织内可见P物质免疫阳性肥大细胞和神经纤维,犬脾脏内可见P物质免疫阳性肥大细胞。结论在六种实验动物的同一组织中以及同一种动物不同组织中,肥大细胞具有明显的异质性,此异质性对采用实验动物开展与肥大细胞功能相关的动物实验研究具有重要参考价值。     

Species differences in the immunoreactive patterns of calcitonin gene-related peptide in the pancreas

Summary In the pancreas, calcitonin gene-related peptide (CGRP) immunoreactivity has been described in nerve fibers and in distinct types of islet cells. This unique, apparently species-specific cell-type expression prompted the present investigation to clarify further the pattern of CGRP immunoreactivity in different mammalian species (i.e., different strains of rats, mice, guinea pigs, rabbits, cats, dogs, pigs, and humans) commonly used for functional and anatomical studies of the pancreas by means of immunohistochemistry using three different CGRP antibodies. In each species, CGRP-immunoreactive neurites innervate the exocrine and endocrine compartments, the vasculature, and the intrapancreatic ganglia, where they form dense networks encircling unstained cell bodies. The only exception is the pig pancreas, where the islets appear to be devoid of immunoreactive fibers. The overall density of immunoreactive pancreatic axons in different species is as follows: rat, mouse, and rabbit>guinea pigpig and cat> >dog and human. CGRP-immunoreactive endocrine cells appear to be restricted to the rat pancreas, where they form a subpopulation of somatostatin-containing D cells. In contrast, in mouse, guinea pig, cat, dog, and human pancreas, a homogeneous staining of the core of the islets, where insulin-producing B cells are located, was visualized in sections incubated with the rabbit CGRP antiserum at 4°C, but not at 37°C (an incubation temperature that does not affect the islet cell staining in the rat nor the fiber labeling in any species). Furthermore, the staining of islet B cells was not reproductible with all the CGRP antibodies used, all of which comparably stain nerve fibers in each species, and islet D cells in the rat. Immunoreactive islet cells were not visualized in pig and rabbit pancreas. These results are consistent with the hypothesis that the expression of CGRP in nerve fibers is a common feature of mammalian pancreas, whereas its expression in endocrine cells appears to be restricted to the D cells of the rat pancreas.     

Stains for A, B, and D cells in fetal rat islets.

Ten techniques often used for identification of A, B, and D cells in adult islets of Langerhans were applied to fetal rat pancreas. Modifications were tried with many of these techniques. Two indole methods (xanthydrol and postocoupled benxylidene reactions) and a cryostat technique using o-phthaladehyde failed to stain fetal islets. Phosphotungstic acid hematoxylin and lead hematoxylin lightly stained fetal A cell granules in Helly's fixed tissue. The Grimelius silver nitrate technique stains adult rat A cells but failed to stain fetal cells. A modification of this technique stained fetal A cells and a possible 4th cell type. The specificity of this method was confirmed by restaining stained cells with a fluorescent antibody technique and with pseudoisocyanin. B cells, as previously reported, were readily stained by the aldehyde fuchsin technique. Fetal D cells were not stained by the Hellerstrom-Hellman alcoholic silver nitrate method, nor did they display pseudoisocyanin metachromasia after acid hydrolysis; they did fluoresce brightly with this technique when viewed with UV light. It was thus possible to distinguish the three usual cell types, plus a possible fourth type, in the fetal rat pancreas.     

Ontogeny of the endocrine pancreas in sea bass (Dicentrarchus labrax): an ultrastructural study. II. The big and secondary islets

The big and secondary islets of sea bass larvae were characterized ultrastructurally from, 25 to 60 days after hatching. From the 25th day, big islets consisted of inner type II and III, external type I and peripheral type IV cells. From the 55th day, type V cells appeared in limited peripheral areas. Secondary islets, first found in 32-day-old larvae, were made up of inner type II and III, external type I, and peripheral either type IV and V cells (type I islets), or only type V cells (type II islets). Type I cells contained secretory granules with a fine granular, low-medium electron-dense material, whereas the secretory granules of type II cells were smaller and had a high electron-dense core with diffused limits; needle and rod-like crystalloid contents were occasionally found. Type III secretory granules posessed a homogeneous, high or medium electron-dense material with or without a clear halo. Type IV cells had secretory granules with a polygonal dense core embedded in a granular matrix and granules containing a high or medium electron-dense material. Type V cells had secretory granules with a fine granular, high or medium electron-dense content. These cell-types correlated with cells previously identified immuno-cytochemically, as regards to their distribution in the islets, and related to those characterized ultrastructurally in adult specimens. Thus, types I, II, III, IV and V correspond to D₁, B, D₂, A and PP cells, respectively. From the 32nd day onwards, endocrine cells of all the different types were found grouped, type V cells also being observed in isolation close to pancreatic ducts and/or blood vessels. Small groups consisting of type I and II cells were found in 40-day-old larvae. A mitotic centroacinar ductular cell containing some secretory granules similar to those of type I cells, was seen adjacent to a type I cell. As the larvae grew older, the endoplasmic reticulum developed, the number of free ribosomes decreased, and the number and size of the secretory granules increased. Dark type I, II, III, IV and V cells were found in the islets and cell clusters from the 55th day onwards.     

Comparative studies on the gastric glycopeptide in eleven animal species.

1. Glycopeptides in the stomachs of eleven mammalian species, including human, rabbit, horse, cow, pig, goat, sheep, dog, cat, guinea pig and rat were assayed by determining the carbohydrate content of materials which remained after proteolysis. 2. The glycopeptide content was higher in the mucosa than in the muscular layer including serosa, especially in the porcine stomach and the fourth stomachs of the ruminants than in the stomachs of any other animals. 3. The glycopeptide, which was stained with both alcian blue and PAS, was absent or sparingly present in the mucosae of the human, rabbit, horse stomachs and in the mucosae of the first to third stomachs of the cow, goat and sheep, whereas in the mucosae of the pig, dog, cat, guinea pig and rat stomachs and in the mucosae of the fourth stomachs of the cow, goat and sheep, it was found in noticeable extents.     

Staining of different endocrine cells with hydrochloric acid-toluidine blue in Epon embedded rat tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 hr in 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

Staining of Different Endocrine Cells with Hydrochloric Acid-Toluidine Blue in Epon Embedded Rat Tissue

The usual HCl-toluidine blue staining of different endocrine cells is applicable to paraffin embedded material. A modification for Epon embedded tissue suitable for consecutive light and electron microscopic studies is described which makes it possible to find the same stained cell, both in a semithin section and in subsequent ultrathin sections. This method facilitates the search for scattered specific endocrine cells. Without removing the resin, sections of Epon embedded tissues were hydrolyzed for 17 hr in 1% HCl at 65 C and stained for 2 turn 0.1% toluidine blue in McIlvaine buffer, pH 5.8. The following cells were stained: C cells in thyroid glands; A and D cells in pancreatic islets; B cells in anterior pituitary; G, D and Ec cells in the gastrointestinal tract; Ad cells of the adrenal medulla.     

间接免疫过氧化物酶技术鉴定猪和牛的肥大细胞

用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体ＡＡ１,ＡＡ３及ＡＡ５的间接免疫过氧化物酶技术对经Ｃａｒｎｏｙ液或中性缓冲福尔马林固定的猪和犊牛空肠,舌及胸腺的石蜡切片进行了免疫染色。对猪和牛的肥大细胞特异性免疫染色与常规的组织化学染色的结果进行了比较。     

Islet alpha cell number is maintained in microencapsulated islet transplantation

Islet transplantation can reverse hyperglycaemia in Type 1 diabetes patients. One problem in islet transplantation is a loss of beta cell mass as well as blunted glucagon responses from the grafted islets. It has been suggested that alpha cell loss is associated with close contact of the alpha cells with the implantation organ. In the present study we made use of microencapsulation, where transplanted islets are not in direct contact with the host implantation site. After transplantation, the number of glucagon cells stained per microencapsulated islet section was increased whereas the number of insulin cells stained was decreased. DNA content of the islets was reduced, as was insulin content, whereas glucagon content was unchanged. This indicates that cell number in transplanted microencapsulated islets diminishes, which can be accounted for by loss of beta cells. However, in contrast to previous studies using non-encapsulated islets, alpha cell number seems to be maintained.     

Organogenesis and differentiation of the pancreas in the toad Bufo bufo L.

Summary In Bufo bufo at stage III₆ the first endocrine islets appear in the part of the pancreas corresponding to the dorsal anlage. At stage IV₂, 5 days later, the pancreatic duct develops and new islets arise by budding off from the ductal epithelium. The ultrastructural study of the secretory granules morphology of endocrine cells has distinguished four different cell types: B-cells (stage III₉), A-cells (stage IV₃), D-cells (stage IV₃) and a fourth type not yet identified (stage IV₃). By immunocytology insulin and corticotropin-releasing factor (CRF) cells have been demonstrated at stage III₉, and glucagon and somatostatin cells at stage IV₁. Lastly, endocrine islets can be homogeneous (predominantly containing insulin cells, rarely glucagon cells) or heterogeneous (insulin cells at the centre and glucagon or somatostatin cells at the periphery). Hypotheses are put forward for the origin and the constitution of the different generations of endocrine islets and isolated cells.     

Light- and electron-microscopic immunocytochemical investigation of the subcommissural organ using a set of monoclonal antibodies against the bovine Reissner's fiber

Ten monoclonal antibodies (Mabs) against glycoproteins of the bovine Reissner's fiber (RF) have been used in a structural and ultrastructural immunocyto-chemical investigation of the bovine subcommissural organ (SCO) and RF. The SCO of other vertebrate species has also been studied. For comparison, polyclonal antibodies against bovine RF (AFRU) were used. The SCO and RF of ox, pig and dogfish and the SCO of dog, rabbit, rat and frog were submitted to light-microscopic immunocytochemistry using AFRU and Mabs. Postembedding ultrastructural immunocytochemistry was applied to sections of bovine SCO using AFRU and Mabs. Bovine SCO consists of ependymal and hypendymal cell layers, the latter being arranged as cell strands across the posterior commissure, or as hypendymal rosette-like structures. All cytoplasmic regions of the ependymal and hypendymal cells were strongly stained with AFRU. Six Mabs showed the same staining pattern as AFRU, one Mab stained RF strongly and SCO weakly, two Mabs stained RF but not SCO, and, finally, one Mab (3B1) exclusively stained the apices of the ependymal and hypendymal cells. All Mabs recognized the SCO and RF of the pig. Two Mabs bound to the SCO of the dog. One Mab stained the SCO of the rabbit and another the SCO of the rat. The SCO of frog and dogfish were totally negative. Bovine SCO stained with AFRU, showed label in the rough endoplasmic reticulum (RER) and the secretory granules (SG) of the ependymal and hypendymal cells. The former, in the form of parallel cisternae, reticulum or concentric rings, was seen throughout all cytoplasmic regions. SG were abundant in the apical pole of the ependymal and hypendymal cells. Only one Mab showed a staining pattern similar to AFRU. Five Mabs showed strong reactions in the SG but weak labeling of the RER. Mab 3B1 showed the label confined to the SG only. Our results suggest that: (i) in the bovine tissue, some epitopes are present in both precursor and processed materials, whereas others are characteristic of mature glycoproteins present in SG and the RF; (ii) the bovine SCO secretes at least two different compounds present in ependymal and hypendymal cells: (iii) both compounds coexist in the same secretory granule; (iv) there are conserved, class-specific, and species-specific epitopes in the glycoproteins secreted by the SCO of vertebrates.     

Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.