High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program.

Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, beta-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased beta-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of beta-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days' culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5+MAN5). We also analyzed by reverse transcriptase-polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in beta-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring beta-cell death.

Primary endocrine carcinoma of the parotid salivary gland associated with a lung carcinoid: a possible new association.

An endocrine carcinoma of the left parotid salivary gland in a 58-year-old woman is reported. The tumour displayed a large argyrophilic cell-component and at ultrastructural level endocrine-like granules (ELG) were evident. As endocrine-paracrine cells are not normally present in the parotid, it is suggested that the endocrine elements may have been derived from an anomalous differentiation of the ductal epithelial stem cells. A bronchial carcinoid, removed seven years previously, proved structurally, cytologically and histochemically different from the tumour of the parotid salivary gland. It is proposed that the occurrence of the two tumours might be an as yet undescribed association which is more than fortuitous.

Well-differentiated endocrine tumours of the middle ear and of the hindgut have immunocytochemical and ultrastructural features in common.

The immunocytochemical analysis of two cases of well-differentiated endocrine tumours (carcinoids) of the middle ear revealed predominant cell populations producing pancreatic polypeptide (PP)-related peptides, glucagon-related peptides, and serotonin (the latter only in one case). In consecutive sections PP- and glucagon-related immunoreactivities mainly colocalized in the same tumour cells. Ultrastructurally tumour cells were characterized by medium-sized to large granules of moderate to high density, on which PP and glicentin were localized by the immunogold technique. No amphicrine cells were found. These features are consistent with those of similar tumours in the rectal mucosa that are mainly composed of L cells coexpressing both PP-related and glucagon-related peptides. Additional tumour antigens of hindgut type detected immunohistochemically were prostatic acid phosphatase and CAR-5 mucin. Expression of the CAR-5 antigen was also found in samples of normal middle ear mucosa, in which endocrine cells have not been identified. In case 1 peritumoral mucosal invaginations showed a proliferation of endocrine cells identical immunophenotypically to tumour cells, possibly representing a precursor lesion. It is concluded that well-differentiated endocrine tumours of the middle ear are a distinct pathological entity characterized by multiple hormone production, typically involving three classes of hormones (pancreatic polypeptide-related peptides, glucagon-related peptides, and serotonin) of the hindgut endocrine system.

Tryptophyllin-like immunoreactivity in rat adenohypophysis.

A new amphibian peptide family has been isolated from the skin of a South American frog Phyllomedusa rhodei and named Tryptophyllins (TPH) because of their content in tryptophyl residue. Using an antiserum against one of these peptides, namely the pentapeptide Met-5-TPH-5-amide (PHE-PRO-PRO-TRP-MET-NH2), we observed the presence of a set of immunoreactive cells in rat adenohypophysis. These cells were far more numerous in pregnant than in normal male and non-pregnant female rats. Dual immunostainings demonstrated that, with some exceptions, almost all the TPH-like immunoreactive cells were gonadotrophs. At electron microscope both types of gonadotroph cells displayed immunoreactivity and the gold particles strongly labelled both types of granules. The Aa. advance the hypothesis that, besides the hormones themselves, the secretory granules might contain some TPH-like sequence.

Glucagon- and PP-related peptides of intestinal L cells and pancreatic/gastric A or PP cells. Possible interrelationships of peptides and cells during evolution, fetal development and tumor growth.

The immunohistochemical detection of six distinct sequences of proglucagon and its derivatives (GRPP, glicentin, glucagon-37, glucagon-29, GLP1, GLP2 and MPGF) in both intestinal L cells and pancreatic or gastric A cells of some mammals (dog, man, guinea pig) confirms that the two cell types produce the same proglucagon molecule, although the final step of its post-translational processing differs in the two cells. Immunohistochemical and ultrastructural patterns of glucagon/glicentin cells in the pancreas of lower vertebrates and early human fetuses, as well as tumor cell studies, suggest an evolution of gastropancreatic A cells from L cells. On the contrary, the PP-related peptide PYY of intestinal L cells, and PP with its C-terminal icosapeptide extension of pancreatic PP cells, likely originate from different prohormones. Although intermediate patterns of peptide expression can be observed, including some F-type PP cells of the dog pancreas (uncinate process) and pyloric mucosa showing PYY immunoreactivity or rare PYY and/or HPP immunoreactive cells of the human rectum lacking glicentin reactivity, no obvious relationship can be established between L cells and pancreatic (F-type) PP cells. However, some evolutionary, embryogenetic and oncogenetic link may exist between L cells and human D1-type PP cells, a minor population of PP cells scattered in the pancreatic tissue of dorsal pouch origin and a major fraction of tumor PP cells.

Neoplastic cells containing lysozyme in gastric carcinomas.

A case of gastric carcinoma mostly composed of cells with histological, immunohistochemical and ultrastructural features of Paneth cells prompted a comparative investigation of the occurrence of similar cells in gastric, colorectal and mammary carcinomas. Cells containing lysozyme were demonstrated by the immunoperoxidase-PAP technique in 34.9% of 83 gastric carcinomas. They were found in 38% of intestinal-type and in 30% of diffuse-type tumours. Paneth-type granules were demonstrated ultrastructurally in 4 of 7 carcinomas in which lysozyme had been demonstrated immunohistochemically. No lysozyme was demonstrated in a series of 30 breast carcinomas and in only 1 of 27 cases of colorectal neoplasm. The possibility of using lysozyme as a marker for some carcinomas of gastric origin is considered.

Regulatory peptides in neuronal neoplasms of the central nervous system.

Neuronal tumors of CNS were examined immunohistochemically for regulatory peptides. Thirteen ganglion cell neoplasms, one cerebellar ganglioneuroblastoma, one cerebellar neuroblastoma, and four medulloblastomas were studied. Sixteen non-neuronal intracranial neoplasms were examined as controls. Immunoreactive vasoactive intestinal polypeptide (VIP) was observed in seven cases of ganglion cell neoplasm and in the one cerebellar ganglioneuroblastoma. The cerebellar neuroblastoma, all of the medulloblastomas, and all of the non-neuronal intracranial neoplasms were negative. Four additional ganglion cell neoplasms were tested for the presence of neurotensin and somatostatin. Two contained neurotensin. The results suggest that CNS ganglion cell neoplasms share with their extracranial counterparts the production of certain hormonal polypeptides. Since these peptides are presumed to be specific markers for neurons, the immunohistochemical detection of these substances may provide diagnostically useful technique in the diagnosis of such lesions.

The contribution of immunohistochemistry to the diagnosis of neuroendocrine tumors.

The use of specific secretory products, hormones, and neuroregulators as diagnostic tools for neuroendocrine tumors is illustrated. Results of extensive immunohistochemical and cytologic investigations are discussed in the light of other pathologic and clinical findings to serve as a basis for tumor classification and as a help in prognostic evaluation. Endocrine tumors of the pancreas, gut, lung and urogenital tract are dealt in some detail.

Endocrine tumors of the duodenum and upper jejunum. A study of 33 cases with clinico-pathological characteristics and hormone content.

Thirty duodenal and three upper-jejunal endocrine tumors are reported. Clinical manifestations included: a) the Zollinger-Ellison syndrome (10 cases); b) peptic ulcer disease in which hypergastrinemia was not documented (3 cases); c) cholestasis or cholelithiasis (4 cases); d) abdominal pain (4 cases); e) gastro-intestinal bleeding (1 case); f) celiac sprue (1 case). Ten further tumors were discovered incidentally, at autopsy or in pathological specimens after gastrectomy or duodenopan-createctomy. Histological pattern was trabecular in 19 cases, insular in 2 and mixed in ten cases. Two cases were typical ganglioneuromatous paragangliomas. All tumors were examined immunohistochemically. Twelve tumors contained gastrin, four somatostatin, six both of these peptides, one serotonin, two both gastrin and serotonin, and two tumors contained gastrin, serotonin and somatostatin. Ganglioneuromatous paragangliomas combined somatostatin and/or pancreatic polypeptide containing endocrine cells with protein-S100-positive Schwann cells. In four tumors no peptide or amine was demonstrated. Gastrin cell tumors (63.6% of our cases), both functionally active (gastrinomas) and clinically silent, predominated in the proximal duodenum, while somatostatin cell tumors (15.1%) and paragangliomas were mostly found in the periampullary region. Two tumors were classified as malignant on the basis of lymph node metastases, and both were jejunal gastrinomas associated with Zollinger-Ellison syndrome. Two somatostatin cell tumors had manifestations of von Recklinghausen's disease.

The diffuse endocrine-paracrine system of the gut in health and disease: ultrastructural features.

At least 16 types of endocrine-paracrine cells have been identified ultrastructurally in the gastrointestinal mucosa. The production of hormones and local messengers such as 5-hydroxytryptamine, gastrin, cholecystokinin, somatostatin, secretin, gastric inhibitory peptide (GIP), enteroglucagon (glicentin, GLI), motilin, neurotensin, substance P and the enkephalins, by these cells, has been established. Progress has also been made in cytological studies of gut and pancreatic endocrine tumours. Argentaffin EC cell carcinoids, gastrinomas (of several ultrastructurally different varieties of gastrin cells), L-cell tumours and D-cell tumours are among those cytologically and functionally defined in the gut. Functionally undefined tumours include the so-called non-argentaffin carcinoids arising in various parts of the gut, some of which have been characterised cytologically as gastric ECL cell tumours and gastroduodenal P-D1-cell tumours. Gastrinomas, vipomas and rare argentaffin carcinoids are among gut-related pancreatic endocrine tumours. Non-functional paragangliomas, usually with some neuromatous component, occur in the duodenal wall. Extrapancreatic vipomas display ultrastructural features of ganglioneuroblastomas with peptidergic granules.

[Malignant myoepithelioma associated with in situ and invasive ductal carcinoma. Description of a case and review of the literature]. / Mioepitelioma maligno associato a carcinoma duttale in situ ed invasivo NAS. Descrizione di un caso e revisione della letteratura.

A case of malignant myoepithelioma of the breast, associated with in situ and invasive carcinoma NOS is described. Myoepithelial differentiation was demonstrated with immunohistochemistry and electron microscopy. The tumour affected the left breast of a 72 year old lady. The patient had been treated with quadrantectomy with axillary dissection, followed by radiotherapy. At the time of diagnosis no local or distant metastases were found. Bone, pulmonary and cerebral metastases appeared 28 months after treatment. Malignant myoepitheliomas share histological and immunohistochemical features with monophasic sarcomatoid carcinomas. Comparison and and possible relationship with monophasic sarcomatoid carcinomas is discussed.

Ultrastructural localization of motilin in endocrine cells of human and dog intestine by the immunogold technique.

Motilin-immunoreactivity has been localized by two electron immunocytochemical techniques, using gold-labelled protein A or IgG as second layer, in a specific type of endocrine cell scattered in the epithelium of human and canine upper small intestine. The motilin (M) cell is characterized by relatively small (180 nm in man; 200 nm in the dog), solid granules with homogeneous core and closely applied membrane, round in man, round to irregularly-shaped in the dog. Perinuclear microfilaments are prominent in human motilin cells.

Ultrastructural localization of cholecystokinin in endocrine cells of the dog duodenum by the immunogold technique.

Cholecystokinin (CCK) has been localized by the immunogold technique in a type of endocrine cell of the dog duodenum characterized by small (166 +/- 38 nm) secretory granules with fairly dense, homogeneous core separated from its enveloping membrane by a thin clear space. The CCK cell is immunocytochemically distinct and cytologically different from other types of endocrine cells, as the secretin, GIP and motilin cells, already identified in the dog duodenum.

Ultrastructural localization of secretin in endocrine cells of the dog duodenum by the immunogold technique. Comparison with ultrastructurally characterized S cells of various mammals.

Secretin has been localized by the immunogold technique in endocrine cells of the dog duodenum--previously described as "K cells"--characterized by secretory granules with double structure consisting of a secretin-containing osmiophilic core surrounded by an argyrophil halo. Granules resembling those of dog secretin cells were also found in some ultrastructurally characterized S cells of the cat, pig, rat and rabbit duodenum, thus confirming in these species the identification of S cells with secretin cells. Conversely, the cells previously described as "S cells" in the dog lacked secretin immunoreactivity.

Ultrastructural localization of gastric inhibitory polypeptide (GIP) in a well characterized endocrine cell of canine duodenal mucosa.

The polypeptide hormone GIP has been localized ultrastructurally by using specific, monoclonal GIP antibodies and an immunogold technique on aldehyde-osmium fixed specimens of dog duodenal mucosa. A single type of cell showing round, homogeneous, fairly osmiophilic granules with closely applied membrane and a mean size of 188 nm +/- 34 SD has been identified as the GIP cell.

Types of endocrine cells in the human colon and rectum.

At least four types of endocrine-like cells have been detected histochemically in the mucosa of the human colon and rectum, i.e. argentaffin cells storing 5-hydroxytryptamine (5HT) and non-argentaffin cells reacting with glucagon, somatostatin and bovine pancreatic peptide (BPP) antibodies. Ultrastructurally, four main types and three rare types of endocrine-like cells have been identified. Among the former cells were: (1) argentaffin EC1 cells, known to store 5HT and substance P, (2) poorly argyrophil L cells, corresponding to the glucagon-immunoreactive cells storing enteroglucagon or glucagon-like immunoreactivity (GLl), (3) inconstantly argyrophil F-like cells, possibly corresponding to BPP-immunoreactive cells, and (4) fairly argyrophil H cells of unknown function. Rare D cells, corresponding to somatostatin cells, N cells, corresponding to neurotensin cells, and P cells, of unknown function, have been also found.

Argyrophil pituitary tumors showing TSH cells or small granule cells.

Among 74 histochemically and ultrastructurally studied pituitary adenomas, 12 apparently chromophobe tumors were characterized by the presence of numerous argyrophil cells. All these argyrophil adenomas failed to reveal presence of GH, prolactin or ACTH cells. Two tumors were found to consist of well granulated cells reacting intensely with anti-TSH antibodies and resembling TSH cells of the normal pituitary. The remaining argyrophil adenomas did not show TSH immunostaining and, with one exception, failed to react with an anti-HCG serum staining gonadotroph cells of human pituitary. They were composed of small, closely apposed cells with small compact or vesicular granules. These tumor cells seem to correspond to some small argyrophil cells found in non-neoplastic pituitary, which differ from TSH cells and from all other types of functionally identified adenohypophyseal cells.

Ultrastructural features of neuroendocrine differentiated carcinomas of the breast.

The ultrastructural patterns of neuroendocrine (NE) differentiated breast carcinomas are analyzed and discussed. Reports in the literature describe wide variations in the size of observed dense-core membrane-bound granules and discrepancies in their interpretation. In the present study 24 cases of breast carcinoma with recognized morphologic, histochemical, and immunocytochemical features of NE tumors were investigated. Five different types of dense-core granules of neurosecretory (NS) type (confirmed by the ultrastructural localization of chromogranin A) and five different cell types were recognized. Some amphicrine cells were found to contain both mucin and NS granules. Another notable ultrastructural feature of breast NE carcinomas was the presence of clear vesicles of presynaptic type, which correlated with expression of synaptophysin.

The endocrine cells of the pancreas and related tumours. Ultrastructural study and classification.

Up to seven endocrine cell types have been identified ultrastructurally in the pancreas, including glucagon A cells, insulin B cells, somatostatin D cells, pancreatic peptide F cells and 5-hydroxytryptamine EC cells. In addition, D1 cells, which have been proposed as the cell type producing VIP and possible P cells of unknown function are seen. Various patterns of endocrine cell differentiation have been found in 20 endocrine pancreatic tumours. Well and poorly differentiated B cells have been identified in 6 insulinomas, diagnostic G cells in 3 out of 7 gastrinomas, D1 and/or F cells in 7 diarrheogenic tumours. Moreover, cells apparently unrelated to the prevalent clinical syndrome have been noted in 8 of the 20 tumours. Granular non diagnostic cells (poorly diagnostic gastrin cells? D1 cells?) were particularly frequent in gastrinomas; agranular or poorly granular cells, either by "active" or "Stem cell" type, were present in nearly all tumours, particularly in diarrheogenic tumours, gastrinomas and malignant insulinomas. A cytological classification of pancreatic endocrine tumours is proposed.