Transplantation of purified islet cells in diabetic rats. I. Standardization of islet cell grafts.

A standardized procedure was developed for the preparation of rat islet cell grafts with selected cell number and composition. After collagenase digestion of pancreases and elutriation of tissue fragments, islets were isolated and dissociated, and cells were purified by autofluorescence-activated cell sorting. Approximately 30% of the initial beta-cell mass was lost during digestion and elimination of small mostly exocrine particles. Fifty percent was recovered in isolated islet preparations and 30% in the purified beta-cell suspensions of greater than 95% purity and viability. Sorting according to cellular flavin adenine dinucleotide content discriminated islet beta-cells from islet endocrine non-beta-cells, fibroblasts, leukocytes, and exocrine cells. Purified endocrine islet cell grafts were prepared by aggregating 10(6) pure beta-cells with or without 8 x 10(5) pure endocrine non-beta-cells. In contrast to intact islets, the purified aggregates were devoid of nonendocrine and damaged cells. Intraportal implantation of a pure beta-cell graft rapidly and permanently normalized the diabetic state of streptozocin-administered animals. The standardized preparation of purified beta-cell grafts allows us to address several metabolic and immunological questions concerning islet cell transplantation in diabetes.

Metastasizing neuroendocrine carcinoma of the larynx with calcitonin and somatostatin secretion and CEA production, resembling medullary thyroid carcinoma.

A 55-year-old man presented with a metastasizing moderately differentiated neuroendocrine carcinoma of the larynx (atypical carcinoid). Immunocytochemical demonstration of neuroendocrine markers (neuron-specific enolase and chromogranin-A) and presence of membrane-bound neurosecretory granules in the cells established the neuroendocrine nature of the tumour. In addition, the tumour was found to produce calcitonin, somatostatin and carcino-embryonic antigen (CEA). Calcitonin and somatostatin were also secreted. On the basis of this particular marker constellation the tumour closely resembles medullary thyroid carcinoma. Review of the recent literature on carcinoids of the larynx reveals immunoreactivity for calcitonin and CEA in a high percentage of cases.

Diffuse and focal nesidioblastosis. A clinicopathological study of 24 patients with persistent neonatal hyperinsulinemic hypoglycemia.

The morphological abnormalities of the endocrine pancreas that underlie persistent neonatal hyperinsulinemic hypoglycemia (PNHH) and are included under the heading "nesidioblastosis" appear to be heterogeneous. We studied pancreatic specimens of 24 patients by light microscopy and immunocytochemistry in order to classify the changes of the endocrine pancreas and relate them to the therapy applied. Two main forms of nesidioblastosis were recognized: a focal and a diffuse type. Both types occurred with equal frequency. Focal nesidioblastosis was characterized by nodular hyperplasia of islet-like cell clusters, including ductuloinsular complexes and hypertrophied insulin cells with giant nuclei. In nine patients, this lesion was unifocal (including an adenoma-like nodule), while two patients each had two separate foci, and one patient had at least three foci. Diffuse nesidioblastosis involved the entire pancreas; it was distinguished by irregularly sized islets and ductulo-insular complexes, both of which contained distinctly hypertrophied insulin cells. Three pancreases lacked these diagnostic features and thus posed difficult diagnostic problems. From the follow-up data, we conclude that partial pancreatectomy with excision of the diseased focus is the treatment of choice for most patients with focal nesidioblastosis whereas diffuse nesidioblastosis requires near-total pancreatectomy. In two patients who had multifocal and diffuse nesidioblastosis and were treated at the age of 6 and 8 years, respectively, the changes of the endocrine pancreas were comparable with those of the young age group and failed to reveal any signs of maturation.

Ontogeny of hormone-secreting cells of the rat pituitary gland: an immunocytochemical study on dissociated cells.

An immunocytochemical study was undertaken in foetal, prepubertal and mature rats to determine the time of differentiation of various types of adenohypophyseal cells during development. Freshly dissociated pituitary cells from foetal (18-21 days postconception), neonatal (from birth up to 30 days) and adult rats (more than 8 weeks) were characterized using immunocytochemical methods. All types of hormone-producing cells were present at day 18 postconception, although only 20% of the cells were immunolabelled. Adrenocorticotropin (ACTH)-secreting cells accounted for the highest number of hormone-positive cells. Growth hormone-secreting cells increased remarkably from day 18 postconception onwards. Prolactin-secreting cells were not seen in the foetal adenohypophysis and did not start to increase until 10 days after birth, whereas by that time the number of ACTH, thyrotropin, follicle-stimulating and luteinizing hormone-secreting cells had stopped increasing. By day 30 after birth, 80-95% of the cells were immunoreactive.

[Evaluation of diagnostic and therapeutic methods in hyperinsulinism in newborn infants and infants. Apropos of a retrospective study of 26 cases]. / Evaluation des méthodes diagnostiques et thérapeutiques de l'hyperinsulinisme du nouveau-né et du nourrisson. A propos de l'étude rétrospective de 26 enfants.

This report concerns 26 cases of hyperinsulinism in neonates and infants. Clinically, the syndrome beginning in the neonatal period seems to differ by several aspects from the disease with a later onset: diagnostic difficulties, treatment modalities and long term prognosis. Insulinemia determined when the patient presents with hypoglycemia is often (75 to 80% of cases) normal. The diagnosis of hyperinsulinism has to be based on the clinical characteristics of hypoglycemia (timing of the episodes, response to glucagon) and on specific biological data (C-peptide, plasma ketone bodies and branched-chain amino acids at the time of hypoglycemia induced by fasting). The pathogenesis of hyperinsulinism is still poorly understood and has several etiologies.

An unexpected cause of pathologic hip fracture. Malignant fibrous histiocytoma.

We describe a case of a 68-year-old patient presenting with pathological hip fracture and multiple pulmonary metastases, who has been operated without a prior histological diagnosis. The hip lesion was thought to be a metastasis of an unknown primary tumor. The proximal part of the femur was resected and replaced by a Müller mega-prosthesis. Histological analysis of the resected bone revealed a malignant fibrous histiocytoma, a rare but very aggressive bone tumor. The patient died three weeks after operation of widespread metastases. By presenting this case report, we want to stress the importance of pretreatment histological diagnosis of osteolytic bone lesions in older patients with metastases.

Interaction of sulfonylureas with pancreatic beta-cells. A study with glyburide.

In this study on purified rat pancreatic beta-cells, we show that the second-generation sulfonylurea glyburide stimulates insulin release through a direct interaction with the beta-cells. During static incubations, 2 microM glyburide releases 0.16 pg insulin per beta-cell, which corresponds to a half-maximal glucose stimulation. This effect occurs independently from the glucose-recognition unit, being detectable at both nonstimulatory and stimulatory glucose concentrations and proceeding without alterations in the rate of glucose oxidation. The secretagogue action of glyburide appears not to be mediated through cAMP but is potentiated by cAMP-generating substances such as glucagon (10(-8) M; 0.31 pg insulin released per beta-cell). Its 10-fold higher potency in isolated islets is attributed to the markedly higher cAMP levels that are maintained in islet beta-cells under the influence of locally released glucagon. Perifused pancreatic beta-cells respond to glyburide with a biphasic insulin release. After removal of the drug, the cells continue to secrete insulin at the same rate for greater than or equal to 30 min. This prolonged secretory activity coincides with a cellular accumulation of the drug, primarily in association with membranes of secretory vesicles and mitochondria. Tolbutamide also stimulates insulin release from pure beta-cells, but it is less powerful on a molar basis and does not lead to a sustained hormone release after its removal from the extracellular medium. We conclude that the hypoglycemic action of glyburide is at least partly the result of a direct interaction with pancreatic beta-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical and ultrastructural findings in a mature retroperitoneal teratoma.

Report is made of a mature retroperitoneal teratoma in a 32-year-old man. Investigation of the tumor revealed cells immunoreactive for ACTH, Met-enkephalin, beta-LPH, serotonin, FSH, BPP, S100, Neuron-specific-enolase. These cells were mainly present in the glandular epithelium, lining the cysts of the tumor. Ultrastructurally, neuro-secretory granules were demonstrated in the cytoplasm of the tumoral endocrine cells. At no time did the patient display endocrine symptoms.

Presence of pancreatic hormones in islet cells with MHC-class II antigen expression.

In the normal rat pancreas, only few islet cells express MHC-class II antigens. Their nature and function has not yet been elucidated. We report a method for the purification of Ia-positive islet cells by fluorescence-activated cell sorting. The isolated mononuclear cells appear of nonendocrine origin but contain vacuoles with partially digested secretory vesicles. Both insulin- and glucagon-immunoreactive granules were identified in these vacuoles of varying size and composition. It is suggested that at least part of the rat islet cells with class II antigen expression can exhibit phagocytotic properties leading to the uptake of fragments from damaged endocrine cells. This functional characteristic may implicate this particular islet cell type in the pathology of the endocrine pancreas in type I diabetes.

Melanotic neuroectodermal tumor of infancy with high serum levels of alpha-fetoprotein. Ultrastructural study and immunological evidence of glial fibrillary protein and alpha-fetoprotein.

We report an infant melanotic neuroectodermal tumor in the anterior part of the upper gingiva, which was associated with high urinary excretion of vanilmandelic acid. Serum levels of alpha-fetoprotein were abnormally high (200 ng/ml). They returned to normal after removal of the tumor (10 ng/ml). The tumor was characterized histologically and ultrastructurally by the presence of melanocytic and astrocytic cells containing glial fibrillary protein. Intracellular alpha-fetoprotein was demonstrated by the immunoperoxidase method (PAP) in scattered foci of interstitial cells.

Postnatal development of growth hormone and prolactin cells in male and female rat pituitary. An immunocytochemical light and electron microscopic study.

Localization and ultrastructural maturation of prolactin (PRL) and growth hormone (GH) cells were studied in pituitaries from neonatal, immature (4-6 weeks old), and adult rats (2-3 months old) by light and electron microscopic immunocytochemistry. The distribution pattern of these cells did not change with age. Both cell types were concentrated laterodorsally, with PRL cells adjacent to the intermediate lobe and GH cells nearer the center of the pars distalis. Labeling density of the immunogold reaction was highest for both hormones in immature rats. In neonatal and immature rats, one PRL cell type with granules 200 nm in diameter was present. In adult rats, two types of PRL cells were present: one containing polymorphous granules measuring about 500 nm (prevalent in female rats), the other with spherical granules about 200 nm (prevalent in male rats). No changes were detected in GH cells during maturation.

Glucose alters configuration of gap junctions between pancreatic islet cells.

In rat pancreatic islets, gap junctional subunits (GJS) occur under two different configurations, namely in linear single strands and in polygonal particle aggregates. The present freeze-fracture study demonstrates that GJS can rapidly (dis)assemble into one of these membrane specializations without changes in their total number. Isolation of the pancreatic gland and its perfusion at 2.8 mM glucose is accompanied by a decrease in polygonally packed GJS from 46 to 16%. A rise in medium glucose concentration is followed, within 10 min, by a dose-dependent increase in the percent polygonal particles. This glucose effect on gap junction configuration is calcium dependent and reversible upon glucose removal; it is still entirely detectable when protein synthesis is blocked by cycloheximide. These results indicate that islet gap junctions are dynamic structures that rapidly adjust their configuration to extracellular regulators of beta-cell function. In the light of previous observations, it is suggested that this rapid (dis)assembly of gap junctional structures be considered as a component in the ionic and metabolic coupling between insulin-containing beta-cells of the pancreas.

Detection of cell surface antigens in cryostat sections with immunogold-silver staining.

Immunogold-silver staining was used for the detection of lymphocyte cell surface antigens in cryostat sections of lymphoid tissues. The sections were incubated with monoclonal mouse antibodies and then with colloidal gold-labeled goat anti-mouse antibodies. They were then immersed in a physical developer, counterstained, and mounted. In light microscopy, the tissue architecture was well preserved, and a dark labeling was seen on the positive cells. Optimal labeling conditions were determined. The distribution of the lymphocyte subsets, as defined by a panel of monoclonal antibodies in tonsil and reactive lymph nodes, was similar to that found with a biotin-avidin-horseradish peroxidase method. The monoclonality of the neoplastic cells in lymph nodes of B-cell non-Hodgkin's lymphomas clearly could be demonstrated. The sensitivity of the technic was comparable with that of the biotin-avidin-horseradish peroxidase labeling method. In addition, immunogold-silver labeling was combined with acid phosphatase cytochemistry.

Selective complement fixation by pancreatic B-cells after binding to islet cell surface antibodies.

A two wavelength immunofluorescence system was used to demonstrate islet cell surface antibodies (ICSA) from juvenile diabetics and complement on the same viable rat islet cell. When incubated with rat islet cells at 4 C and at 37 C, ICSA specific for B-cells proved capable of binding complement. In contrast, ICSA specific for both B and non-B cells were able to fix complement on all cell types at 4 C but only on B-cells at 37 C. This study provides further evidence for the B-cell specific cytotoxic effect of ICSA in the presence of complement at physiological temperature. In addition, the ultrastructural events leading to B-cell destruction after ICSA and complement binding were studied by transmission electron microscopy (TEM).