Electron Microscopic Definition of Intestinal Endocrine Cells: Immunogrold Localization and Review

Intestinal mucosal biopsy specimens processed during the past 25 years were used to example the ultrastructural characteristics of intestinal endocrine cells. The cells were defined on the basis of morphologic criteria and, when feasible, with specific antisera and immunogold staining. The hypothesis was that each endocrine cell, once well defined, should be identifiable on the basis of standard morphologic criteria not requiring specific immunostaining. This was not the case. D, G, EC₁, EC₂, ECn, D₁, and intestinal gastrin cells have characteristic secretory granules and, when sufficient granules are present, can be identified consistently on the basis of morphologic criteria. Absolute identification of D, G, IG, and TG cells requires staining with specific antisera, a condition easily obtainable only for D, G, and IG cells. D₁, EC₁, EC₂, and ECn cells must be identified morphologically until secretory products specific for each of these cells are identified. I, L, N, and K cells are remarkably similar in appearance and must be distinguished by specific staining. Mo, S, and P cells were not identified by either morphologic appearance or immunostaining. It is suggested that a cell similar to the D₁ cell but with exceptionally small granules may be the P cell. Absolute identification of intestinal enteroendocrine cells by electron microscopy requires specific staining. The characteristic appearance of the secretory granules of many of these cells (D, G, EC₁, EC₂, ECn, D₁, and IG) permits morphologic identification when numerous secretory granules are present.     

Ultrastructural Localization of Synaptophysin to the Secretory Granules of Normal Glucagon and Insulin Cells in Human Islets of Langerhans

Immunogold labeling of the pancreatic islets in humans by means of monoclonal antibodies to synaptophysin resulted in a distinct localization of gold particles to the secretory granules of glucagon-immunoreactive cells. The same type of immunoreactivity was noted with antiserum to chromogranin A. Glucagon immunoreactivity was concentrated in the dense central core of the secretory granules. Some immunoreactivity for synaptophysin was also found in the secretory granules of the insulin-producing cells, although it was weaker in this location.     

Ultrastructural Localization of Synaptophysin to the Secretory Granules of Normal Glucagon and Insulin Cells in Human Islets of Langerhans

Immunogold labeling of the pancreatic islets in humans by means of monoclonal antibodies to synaptophysin resulted in a distinct localization of gold particles to the secretory granules of glucagon-immunoreactive cells. The same type of immunoreactivity was noted with antiserum to chromogranin A. Glucagon immunoreactivity was concentrated in the dense central core of the secretory granules. Some immunoreactivity for synaptophysin was also found in the secretory granules of the insulin-producing cells, although it was weaker in this location.     

Pancreatic endocrine cells in Bufo bufo: immunocytochemistry and ultrastructure

The endocrine pancreas of the toad consists of rounded islets of various sizes embedded in the exocrine tissue. Isolated cells are also present. At least 4 types of endocrine cell are distinguishable by shape, size and electrondensity of the secretory granules as well as by their immunoreactivity with different antisera: insulin, somatostatin, pancreatic polypeptide, and glucagon cells. Insulin cells can be divided into 2 types according to their cytoplasmic electrondensity. Colocalisation of different hormones in the same cell is rarely observed. The close contact between endocrine and exocrine cells and the scarcity of nerve supply is indicative of a paracrine control of hormone secretion.     

An Electron Microscopic Demonstration of Induction of Chondrogenesis in Neonatal RAT Muscle Outgrowth Cells in Monolayer Cultures

Second passage fibroblast-like cells grown from explants of neonatal rat muscle continue to demonstrate fibroblast-like properties for many days when cultured on plastic surfaces. Such cells can be induced to change to a chondrocyte-like mode of expression by the addition of effector materials prepared from bovine cortical bone decalcified with 0.6 NHCl. Other studies show that similar demineralized bone particles and extracts from them have, in vivo, osteoinductive properties. Optimum conditions for this differentiation in monolayer culture were found in the use of 2% fetal calf serum with Dulbecco's modified Eagles medium. At 10% fetal calf serum the chondrogenic changes could not be detected. Light microscopy showed a sequence of morphological changes, after 36 h in culture, which resembled those seen at the beginning of osteogenesis in vivo Induced cultures showed abundant extracellular proteoglycan production. Isotope incorporation studies showed stimulation of glycosaminoglycan synthesis in response to effector materials in soluble form. Type II collagen could be detected after three days. Electron microscopic analysis of induced and control cultures showed unequivocal evidence for marked production of an extensive extracellular matrix in the region of effector particles. The cells themselves change shape and develop an abundant system of lysosome-like vesicles and a very active, highly engorged endoplasmic reticulum and Golgi apparatus. After nine days in culture, evidence for the formation of a ruthenium red stained structure on the surface of the cells in contact with inductive particles, was observed. The simple monolayer culture system described provides a direct means by which the presence of active chondrogenic fractions may be assessed, and in which the mechanism of action of the effectors can be studied.     

Granular Cell Myoblastoma: An Electron Microscopic and Cytochemical Study Illustrating the Genesis of Granules and Aging of Myoblastoma Cells

Seven typical granular cell myoblastomas, 4 from the skin (2 multicentric) and 1 each from the tongue, vulva and breast, were studied with the electron microscope and with cytochemical procedures for the visualization of lysosomes, endoplasmic reticulum, mitochondria, Golgi apparatus and membranes. With these parameters, all of the lesions were found to be virtually identical. To the authors' knowledge, the apparent formation of the specific small granule from the Golgi apparatus and the large granule (cytolysome) by segregation of portions of cell cytoplasm as well as the apparent aging process in myoblastoma cells is described for the first time. The small granules resemble lysosomes, but do not stain with the lysosomal markers employed. The large granules (cytolysomes) contain acid phosphatase but only a few contain thiolacetic acid esterase activity, suggesting that there are at least two varieties of cytolysomes in myoblastoma. It is concluded that myoblastoma is a tumor-like lesion of Schwann cell origin, which is either a reactive cellular response or, more likely, a true neoplasm.     

Atypical Endocrine Granules in Atypical Endocrine Tumor (AET) of the Lung: An Immunoelectron Microscopic Study

Highly dense granules are a hallmark for recognizing atypical endocrine tumor (AET) of the lung. We report a case of AET with many atypical neurosecretory type granules: moderately dense granules (mean size 373.7nm) and "target" granules with a central dense core (425.1 nm), both apparently larger than the highly dense granules (223.3nm). lmmunoelectron microscopical studies demonstrated that all three types of granule were positive for gastrin releasing peptide (GRP), human chorionic gonadotropin α-subunit (hCGα), calcitonin or serotonin. Although the size profiles of positive granules were similar for calcitonin and hCGα, they were different from those of GRP or serotonin granules. The presence of atypical granules and the different size profiles of hormonal products in AET indicate that caution is required in ultra-structural evaluation of granules in lung carcinomas.     

Serous Cells in the Parotid Glands of Two Species of Tamarins: Polarized Secretory Granules

The parotid glands of two species of tamarins were examined by electron microscopy. Endpiece cells are typical in appearance, with an extensive rough endoplasmic reticulum, prominent Golgi apparatuses, and numerous serous granules. In the saddleback tamarin, the secretory granules contain a dense spherule pressed against the inner aspect of the limiting membrane, leading to a surface bulge. During the course of merocrine secretion (a form of exocytosis), such morphologically polarized granules approach the luminal plasma membranes with the bulge in the vanguard. It is these protuberances that fuse with the plasmalemma. In contrast, although serous granules in the cotton top tamarin contain a spherule, they lack surface bulges and their docking on luminal membranes seems to be a random event with respect to their surface morphology. Moreover, certain other types of cells in a taxonomically wide spectrum of species have granules with a less obvious structural polarity, as well as cells whose granules lack morphological polarity but have a functional polarity that comes into play during exocytosis of such secretory granules. Anat Rec, 291:1254–1261, 2008. © 2008 Wiley‐Liss, Inc.     

Myofilament Localization and Immunoelectron Microscopic Detection of Muscle-Specific Actin in Neoplastic Myoepithelial Cells in Pleomorphic Adenomas and Myoepitheliomas

Elucidating the cellular characteristics of the nonluminal or myoepithelial cells of pleomorphic adenomas is one approach to establishing the diagnostic criteria for myoepitheliomas. Ultrastructural features of nonluminal tumor cells in 22 pleomorphic adenomas and of tumor cells in 9 myoepitheliomas were assessed from micrographs of routinely fixed and epoxy resin-embedded samples. Recognizable myofilaments were only moderately prominent in 1 myoepithelioma. In the rest of the cases, irrespective of whether nonluminal cells of pleomorphic adenomas or tumor cells of myoepitheliomas were spindle, angular, round, or plasmacytoid in form, myofilaments were noted only in one third of the cases and were present even in these in a small proportion of the tumor cells. Intermediate filament accumulations and basal lamina were more frequent findings associated with nonluminal tumor cells. Six pleomorphic adenomas and 2 myoepitheliomas had been fixed in half-strength glutaraldehyde and embedded in LR White resin for immunoelectron microscopic detection of muscle-specific actin. In 3 (2 pleomorphic adenomas and myoepitheliomas) of these 8 cases, readily visualized bands of filaments in many tumor cells were strongly labeled by the colloidal gold probe detecting muscle-specific actin even when myofilaments were minimal and infrequent in 2 cases and undetectable in the third by routine transmission electron microscopy. Lack of myofilament detection by immunocyto-chemistry or routine electron microscopy does not exclude a diagnosis of pleomorphic adenoma or myoepithelioma when growth patterns and cytology indicate such diagnoses. Immunoelectron microscopy, in fact, shows that muscle-specific actin can be detected even when myofilaments or muscle actin are apparently absent or minimal by routine electron microscopy or immunohistochemistry, respectively. Because examples of pleomorphic adenoma and myoepithelioma each with similar histologic and cytologic features of the myoepithelio-matous cells can have variable degrees or complete absence of expression of myofilaments or muscle-specific actin, the time-honored term myoepithelial for the nonluminal cells of pleomorphic adenomas and the term myoepithelioma are legitimate even in the absence of those markers that are specific for normal myoepithelial cells.     

Myofilament Localization and Immunoelectron Microscopic Detection of Muscle-Specific Actin in Neoplastic Myoepithelial Cells in Pleomorphic Adenomas and Myoepitheliomas

Elucidating the cellular characteristics of the nonluminal or myoepithelial cells of pleomorphic adenomas is one approach to establishing the diagnostic criteria for myoepitheliomas. Ultrastructural features of nonluminal tumor cells in 22 pleomorphic adenomas and of tumor cells in 9 myoepitheliomas were assessed from micrographs of routinely fixed and epoxy resin-embedded samples. Recognizable myofilaments were only moderately prominent in 1 myoepithelioma. In the rest of the cases, irrespective of whether nonluminal cells of pleomorphic adenomas or tumor cells of myoepitheliomas were spindle, angular, round, or plasmacytoid in form, myofilaments were noted only in one third of the cases and were present even in these in a small proportion of the tumor cells. Intermediate filament accumulations and basal lamina were more frequent findings associated with nonluminal tumor cells. Six pleomorphic adenomas and 2 myoepitheliomas had been fixed in half-strength glutaraldehyde and embedded in LR White resin for immunoelectron microscopic detection of muscle-specific actin. In 3 (2 pleomorphic adenomas and myoepitheliomas) of these 8 cases, readily visualized bands of filaments in many tumor cells were strongly labeled by the colloidal gold probe detecting muscle-specific actin even when myofilaments were minimal and infrequent in 2 cases and undetectable in the third by routine transmission electron microscopy. Lack of myofilament detection by immunocyto-chemistry or routine electron microscopy does not exclude a diagnosis of pleomorphic adenoma or myoepithelioma when growth patterns and cytology indicate such diagnoses. Immunoelectron microscopy, in fact, shows that muscle-specific actin can be detected even when myofilaments or muscle actin are apparently absent or minimal by routine electron microscopy or immunohistochemistry, respectively. Because examples of pleomorphic adenoma and myoepithelioma each with similar histologic and cytologic features of the myoepithelio-matous cells can have variable degrees or complete absence of expression of myofilaments or muscle-specific actin, the time-honored term myoepithelial for the nonluminal cells of pleomorphic adenomas and the term myoepithelioma are legitimate even in the absence of those markers that are specific for normal myoepithelial cells.     

Selective Localization of Histamine to Electron Densen Granules in Antigen-Challenged Sensitized Rat Mast Cells and to Similar Granules Isolated from Sonicated Mast Cells: An Electron Microscope Autoradiographic Study

The subcellular localization of histamine was studied in sensitized rat mast cells following antigen challenge and in granules obtained from sonicated cells, using an electron microscope autoradiographic technique. The mast cells were furnished with labelled histamine by incubation in ³H-histidine. The silver grain distribution (reflecting the localization of radioactive histamine) was highly non-random. The highest silver grain densities occurred over homogeneous, electron dense (normal) granules and moderately electron dense granules. Swollen, less electron dense (“changed”) granules with a reticular appearance and devoid of a limiting membrane had the lowest density of all subcellular structures studied and were therefore probably almost free of histamine. There was a good correlation between the percentage of electron dense granules, the histamine content and the silver grain density in saline-washed granule fractions isolated after sonication of mast cells for different times. These results support the hypothesis that histamine release occurs during the sequential exocytosis of storage granules, and during the sonication of mast cells, probably as a cation exchange between the amine, which is ionically bound to the heparin-protein complex of the granule matrix, and cations from the extracellular fluid. The exchange will occur as soon as the perigranular membrane becomes permeable to water and cations.     

Apoptosis in Cells of Rat Sympathetic Ganglia during Early Ontogeny: Electron Microscopic Study

Postnatal ontogeny of sympathetic ganglia includes both proliferative processes and programmed cell death. Electron microscopy helps to evaluate the intensity and the relationship between these processes.     

Electron Microscopic Analysis of Myeloma Cells in Relation to Drug Response and Prognosis

Myeloma cells obtained from 33 patients were analyzed by electron microscopy to determine whether there is any correlation among ultrastructural characteristics of myeloma cells, drug response, and prognosis. The median survival time after diagnosis of responders (18 cases) and nonresponders (15 cases) was more than 423 days and 139 days, respectively. Nuclear immaturity and three cyto-plasmic abnormalities —scattered pattern of mitochondria, single-sac looplike structures, and numerous intramitochondrial granules —were found to be associated with poor outcome. Although the cytoplasm of almost all the myeloma cells examined was judged to be mature or intermediate, the degree of nuclear immaturity was considered to correspond closely to the grade of nuclear-cytoplasmic asynchrony. Thus the electron microscopic examination of myeloma cells is of use to assess accurately their immaturity and abnormality and might provide clues for the prediction of drug response and prognosis of individual patients.     

Electron Microscopic Analysis of Myeloma Cells in Relation to Drug Response and Prognosis

Myeloma cells obtained from 33 patients were analyzed by electron microscopy to determine whether there is any correlation among ultrastructural characteristics of myeloma cells, drug response, and prognosis. The median survival time after diagnosis of responders (18 cases) and nonresponders (15 cases) was more than 423 days and 139 days, respectively. Nuclear immaturity and three cyto-plasmic abnormalities —scattered pattern of mitochondria, single-sac looplike structures, and numerous intramitochondrial granules —were found to be associated with poor outcome. Although the cytoplasm of almost all the myeloma cells examined was judged to be mature or intermediate, the degree of nuclear immaturity was considered to correspond closely to the grade of nuclear-cytoplasmic asynchrony. Thus the electron microscopic examination of myeloma cells is of use to assess accurately their immaturity and abnormality and might provide clues for the prediction of drug response and prognosis of individual patients.     

小鼠胰岛内分泌细胞的构成及其与Ⅱ型糖尿病的关系

目的观察和分析II型糖尿病动物模型db/db小鼠病程进展中胰岛内分泌细胞的构成变化及与II型糖尿病病因的关系。方法分别选取3、5、8、10和12月龄的尾静脉空腹血糖高于10.0mmol/L且肥胖的db/db自发性糖尿病小鼠,每组5只,作为糖尿病组;选取相应年龄段的尾静脉空腹血糖低于6.0mmol/L体重正常的db/+m表型正常小鼠,每组5只,作为对照组。于相应年龄段取胰尾,用于免疫组织化学观察和比较。结果各月龄糖尿病组胰岛B细胞(胰岛素阳性细胞)阳性率低于对照组(p<0.05),而A细胞(胰高血糖素阳性细胞)和D细胞(生长抑素阳性细胞)阳性率分别高于对照组(p<0.05)。糖尿病组胰岛B细胞阳性率随自发性db/db糖尿病小鼠病程进展呈降低趋势(p<0.05),而A细胞和D细胞阳性率则呈增高趋势(p<0.05);对照组3种细胞阳性率无变化(P>0.05)。结论II型糖尿病动物模型db/db小鼠胰岛内分泌细胞的构成变化与糖尿病病情的轻重有关,胰岛内分泌细胞之间的比例失衡可能与II型糖尿病的发病有关。     

Metastatic Progression of Pancreatic Cancer: Changes in Antioxidant Enzymes and Cell Growth

Pancreatic cancer has a dismal prognosis due to the fact that patients present late when metastatic disease is already present. Previous studies have demonstrated that pancreatic cancer cells have decreased levels of MnSOD, which correlates well with increased rates of tumor cell proliferation. Recently, we have found that nude mice injected with MIA PaCa-2 human pancreatic cancer cells in the flank occasionally develop ascites and intra-abdominal metastatic deposits. Mice that developed ascites were sacrificed and the ascites cultured. Necropsy demonstrated metastatic tumors in the retroperitoneum, which were excised, digested, and cultured. Western blots, enzyme activity and enzyme activity gels were performed for manganese superoxide dismutase (MnSOD), copper/zinc (CuZnSOD), catalase, and glutathione peroxidase (GPx) in the ascites cell line, metastatic tumor cell line, MIA PaCa-2 primary pancreatic cancer cell line, and the Capan-1, a metastatic pancreatic cancer cell line. Cell growth, plating efficiency, growth in soft agar and growth in nude mice were determined in the ascites, metastatic tumor, and MIA PaCa-2 cell lines. MnSOD, CuZnSOD, and GPx protein and activity were increased in the ascites, metastatic tumor, and Capan-1 cell lines compared to MIA PaCa-2. The ascites and metastatic tumor cell lines had decreased cell growth, plating efficiency, and growth in soft agar, but the ascites cell line had increased cell growth in 4 and 1% O₂ concentrations in vitro and more rapid growth in vivo. Metastatic disease is associated with changes in the content and activity of antioxidant enzymes with an associated change in growth characteristics depending on the O₂ concentrations.     

Immunoelectron Microscopic Localization of Plasminogen Activator Inhibitor Type 1 (PAI-1) in Smooth Muscle Cells from Morphologically Normal and Atherosclerotic Human Arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

Immunoelectron Microscopic Localization of Plasminogen Activator Inhibitor Type 1 (PAI-1) in Smooth Muscle Cells from Morphologically Normal and Atherosclerotic Human Arteries

Vascular wall fibrinolytic system proteins are believed to play a pivotal role in atherogenesis. Tissue-type plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA) influence persistence of luminal thrombi and proteolysis of extracellular matrix, respectively. The major physiologic inhibitor of t-PA and u-PA is plasminogen activator inhibitor type 1 (PAI-1). All three of these fibrinolytic system proteins have been detected in vascular endothelial cells, smooth muscle cells, and macrophages by light microscopic immunohistochemistry. This study was undertaken to delineate, by immunoelectron microscopy, the loci of PAI-1 in smooth muscle cells from intact morphologically normal and atherosclerotic human arteries as well as in isolated and cultured smooth muscle cells from arteries. In intact vessels, PAI-1 immunoreactivity was associated with contractile filaments in cells in both normal and atherosclerotic tissues. Lipid-laden smooth muscle cells in atherosclerotic vessels were mainly of the synthetic phenotype and displayed lesser amounts of PAI-1 associated with rough endoplasmic reticulum and contractile filaments. Isolated smooth muscle cells exhibited either a contractile or synthetic phenotype. In the cells with a contractile phenotype, PAI-1 was associated with the contractile elements, whereas in the cells with a synthetic phenotype, the PAI-1 was associated predominantly with elements of the endoplasmic reticulum. Because PAI-1 is associated predominantly with contractile filaments in smooth muscle cells, the net amount of immunodetectable PAI-1 appears to be greater in contractile compared with synthetic phenotype cells.     

Emperipolesis in a Case of Malignant Lymphoma: Electron Microscopic and Immunohistochemical Investigation

In the tissue sections of axillary lymph nodes surgically removed from a case of malignant lymphoma (non-Hodgkin's, diffuse, large cell type), many large cells were observed to contain lymphoma cells in their cytoplasm. From the findings of light microscopy in serial sections and electron microscopy, this phenomenon was confirmed to be “emperipolesis.” By immunohistochemistry, the large cells that contained lymphoma cells possessed most of the monocyte/macrophage markers, whereas the lymphoma cells revealed some B-cell markers, suggesting that they were of germinal center cell origin. In a survey of the literature, we found no report describing emperipolesis in the tissue sections of malignant lymphoma. Although the precise mechanisms and biological significance of emperipolesis in the present case are not fully understood, the existence of some interactions between macrophages and lymphoma cells is suggested.