No abstract

We report an 18-yr-old youth with a metastatic foregut carcinoid tumor, Cushing's syndrome, and hypersomatotropic gigantism. Administration of cyproheptadine caused a dramatic fall in urinary cortisol excretion and plasma ACTH levels associated with clinical remission of the Cushing's syndrome. GH secretion was not affected by cyproheptadine administration. Ectopic ACTH secretion was confirmed by RIA of tumor extracts and immunohistochemical demonstration of ACTH-containing cells in hepatic metastases. There were two sources of GH production demonstrated in this patient. Ectopic secretion of GH by the carcinoid hepatic metastases was documented by both RIA and immunohistochemical techniques. A somatotrophic pituitary tumor was also present. The histological characteristics of this tumor suggest adenomatous hyperplasia rather than de novo neoplastic change as the likely mechanism of its pathogenesis. GH releasing factor-like activity was demonstrated in extracts of plasma and in extracts of the carcinoid tumor. We conclude that cyproheptadine exerted an effect on the ectopic ACTH-producing cells but not on the ectopic GH-producing cells or on adenohypophyseal GH secretion. Production of a GH releasing factor-like activity by the carcinoid tumor may have caused the pituitary somatotrophic tumor.

Beta granules isolated from rat islets of Langerhans and subjected only to phosphotungstic acid had, in negatively stained images, a 50-A periodicity . This periodicity was also observed in thin-section profiles of beta granules in intact cells . In shadowed preparations, the granules were spherical in shape and had irregular edges and surface structure . The presence of such a periodicity in the beta granule indicates that its matrix may be composed of a crystalline material .The isolation of a fraction of secretory granules from isolated islets of the rat pancreas (Howell, Fink, and Lacy, 1969) has made possible further characterization of the morphological properties of the isolated granules by methods not applicable to granules retained within the intact cell . MATERIALS AND METHODSIn most experiments, the washed pellet of the secretory granule fraction, prepared by density gradient centrifugation , was resuspended in 0.2 ml of a cold medium containing 160 mM KCI, 5 mm NaCl, 0 .5 MM MgC1 2 , and 5 mm sodium phosphate, pH 6 .0, and maintained at ice-bath temperature while samples were withdrawn . Grids were prepared as soon as the pellet had been resuspended, since the insulin content of the granules becomes progressively solubilized with time after suspension (Howell, Young, and Lacy, 1969) . All grids were prepared within 10 min of resuspension . In a single experiment, the pellet was resuspended in 2 .5% glutaraldehyde in 0 .15 M phosphate buffer, pH 7 .5 . In a further experiment, the pellet was resuspended in 0 .3 M sucrose, buffered at pH 6 .0 with 0 .005 M phosphate buffer .For negatively stained preparations, a small drop of suspension was placed on a collodion-carboncoated 400-mesh copper grid, allowed to sit for 1 min, then drained by contact with filter paper . A drop of 2% % phosphotungstic acid, pH 6 .0, was placed on the grid and, after a minute, was drained in the same way . The first grid thus prepared was immediately placed in the vacuum chamber of the electron microscope ; other grids were prepared and allowed to dry in a Petri dish containing a desiccant .For shadow-casted preparations, after a drop of the suspension had been drained from the grid, the grid was flushed several times with distilled water for removal of salts . Some of the grids, while still wet, were subjected to osmium vapors for several minutes ; others were allowed to dry in a Petri dish containing a desiccant . The grids were shadowed with platinum at an angle of 10°, or alternatively shadowed with platinum during continuous rotation of the grid (rotational shadowing) .Secretory granule pellets, isolated islets, and pancreatic tissue were fixed in 2 .5%/%, glutaraldehyde followed by osmium tetroxide solution, dehydrated in ethyl alcohol, and embedded in an epoxy resin according to Lockwood's procedure (Lockwood, 1964) . The thin sections were stained with uranyl on

The lack of a technique that allows mass isolation of intact, viable human islets is part of the reason that islet transplantation has not become available to the human diabetic. This report outlines the history of islet isolation and presents two new technical modifications that have been developed in the dog. Many of the current problems in islet isolation are presented, including the difficulty in obtaining enough human pancreatic tissue with minimal warm-ischemia time; inadequate distention of the pancreas to provide sufficient disruption for maximal enzymatic reaction to release intact islets; inefficient chopping methods; the use of collagenase of variable composition; different digestion methods for obtaining isolated islets; and inefficient methods for separating and purifying the islets from the ductal, acinar, and fibrous components. The first new modification involves distention of the dog pancreas through the venous system of the gland rather than the ductal system. This results in improved intralobular disruption, which improved the yield of isolated dog islets by permitting more efficient collagenase digestion. The second new modification eliminates the concept of isolating intact islets: the dog pancreas is digested by trypsin to a single-cell preparation that is partially purified by Ficoll gradients; further purification of the endocrine cells results from selective aggregation using rotational culture. This process produces pseudoislets that contain all the islet cell types and can be kept in culture for up to 4 wk, releasing their hormones in response to appropriate stimuli. These modifications may assist in the struggle to isolate the elusive human islet for safe and effective islet transplantation in the diabetic patient.

No abstract