Ronald C. Merrell

Isolating the Elusive Islet

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.

Telemedicine and international disaster response: Medical consultation to Armenia and Russia via a telemedicine spacebridge

UNLABELLEDnThe Telemedicine Spacebridge, a satellite-mediated, audio-video-fax link between four United States and two Armenian and Russian medical centers, permitted remote American consultants to assist Armenian and Russian physicians in the management of medical problems following the December 1988 earthquake in Armenia and the June 1989 gas explosion near Ufa.nnnMETHODSnDuring 12 weeks of operations, 247 Armenian and Russian and 175 American medical professionals participated in 34 half-day clinical conferences. A total of 209 patients were discussed, requiring expertise in 20 specialty areas.nnnRESULTSnTelemedicine consultations resulted in altered diagnoses for 54, new diagnostic studies for 70, altered diagnostic processes for 47, and modified treatment plans for 47 of 185 Armenian patients presented. Simultaneous participation of several US medical centers was judged beneficial; quality of data transmission was judged excellent.nnnCONCLUSIONnThese results suggest that interactive consultation by remote specialists can provide valuable assistance to on-site physicians and favorably influence clinical decisions in the aftermath of major disasters.

The Independence of Insulin Release and Ambient Insulin In Vitro

The effect of insulin on its own secretion was tested in three independent experimental models using insulin concentrations that approached physiologic values. The collected secretions from glucose-stimulated islet tissue had no effect on insulin release from other islets. Perifused insulin had no effect on the release of endogenous insulin even when the assay was completely controlled for dilutional effects. Perifused insulin had no effect on the release of prelabeled insulin from glucose-stimulated islets. Similarly, proinsulin did not affect insulin release. Porcine insulin did not affect the function of porcine or canine islets. These studies strongly support the independence of glucose-stimulated insulin secretion and ambient insulin.

Allotransplantation of Islet Endocrine Aggregates

Dissociated pancreatic islets form endocrine aggregates from single-cell suspension by rotation culture. Islet cell aggregates, or neoislets, can provide endocrine reconstitution for diabetic rats and enjoy prolonged graft survival when neoislets are transplanted across a major histocompatibility barrier (Lewis to ACI). Long-term survival of grafted neoislets was obtained in 71% of recipients without any immunosuppression and in 100% of recipients with minimal immunosuppression. As predicted by cell-cell recognition in rotation-mediated aggregation, neoislets apparently exclude mesenchymal cells that bear la antigens. Therefore, reduced immunogenicity is accomplished.

Suppression, Stress, and Accommodation of Transplanted Islets of Langerhans

Successful intrasplenic islet autotransplantation in dogs requires an islet cell mass considerably greater than what might be expected based on studies of subtotal pancreatectomy. Grafts of marginal function ultimately fail, suggesting severe limitations in the capacity of an islet graft to adapt. Accommodation was tested in established intrasplenic grafts by either chronically stressing the graft with mild carbohydrate intolerance induced by exogenous corticosteroids or chronically suppressing the graft with exogenous insulin. After these manipulations, insulin output into the portal vein in response to intravenous (i.v.) glucose was measured and compared with that of normal dogs and dogs receiving islet autografts with no further treatment with either steroids or insulin. Transplanted islets tolerated the two manipulations well in that neither exogenous steroid nor insulin led to failure of the graft as a consequence of either stress or protracted diminished demand. The major determinant of successful islet grafting is the endocrine competence of the initial graft. If that competence is provided at the outset, the graft can adapt to a considerable range of demand for insulin secretion.

Beta cell contact and insulin release

Insulin secretion by intact islets, dispersed islet cells and dispersed cells allowed to reaggregate was compared in perifusion. Although single cells and aggregates showed basal insulin secretion and a prompt response to glucose challenge, basal secretion, peak insulin secretion and total insulin secretion during a 60 minute stimulation were profoundly less than those activities of intact islets. These results suggest that dispersed beta cells are responsive to glucose as a secretagogue, but the magnitude of the response is greatly diminished and not restored by simple cell contact.

Effect of cyclosporine on established islet autografts

Cyclosporine (CyA) is toxic to the function of isolated islets and this toxicity may, in part, explain the failure of islet allografts as well as autografts with CyA immunosuppression. Not only do canine allografts fail despite CyA immunosuppression, but control autografts given CyA from the day prior to transplantation have a very high failure rate. In this study, we investigated CyA effects on established islet autografts. Twenty mongrel dogs underwent total pancreatectomy and successful intrasplenic islet autotransplantation. Ten served as control autografts (Group 1); five were started on oral CyA on the 5th postoperative day (Group 2) and 5 dogs were given CyA from the 10th day after grafting (Group 3). Intravenous glucose tolerance tests were performed before operation, before starting CyA and after 3 weeks. Plasma insulin was determined by radioimmunoassay. Control dogs remained normoglycemic throughout the study as did Group 3 animals. In Group 2, 2 of 5 dogs failed, both on the 4th day of CyA, while the other 3 were normoglycemic throughout the study. No significant difference was shown among the K values, fasting blood glucose and peak plasma insulin values following IVGTT before and after treatment with CyA. CyA begun the day before autografting gravely compromises graft success. However, after the graft is well established, an adverse effect of CyA on islet cell function is not evident.

Donor-specific antigen and cyclosporine in rat islet allografts

Combination therapy with one dose of 3 M KCl extracted donor-soluble antigen (Ag) and a short course of cyclosporine (CsA) has proven to prolong the survival of kidney allografts by enhancing specific T-suppressor populations. This regimen is tested in rat islet allografts in this study (Lewis to ACI). A 3-day perioperative course of 10 mg/kg/day CsA on Days -1, 0, and 1 did not prolong graft survival (MST = 10.7 +/- 2.5 days vs 9.4 +/- 1.2 days in controls). When this course of CsA therapy was combined with a single dose of donor antigen on Day -1, the survival time was prolonged slightly but significantly (MST = 14.0 +/- 5.8 days). Three cycles of a 3-day course of CsA therapy at 7-day intervals, a total of nine doses of 10 mg/kg/day CsA, were effective in delaying rejection of islet allografts (MST = 26.4 +/- 30.3). Moreover, combined therapy with donor antigen and three cycles of a 3-day course of CsA prolonged the survival of islet allografts (MST = 57.7 +/- 51.4 days) with 50% of recipients still normoglycemic at 60 days after transplantation. These findings indicate that the combination therapy of donor antigen with a short course of CsA has a powerful effect to prevent the rejection of islet allografts, as shown in kidney allografts, in rats.

The endocrine function of heterotopic islets of Langerhans

Islets of Langerhans may be removed from the pancreatic environment and transferred as autografts to a heterotopic site such as the spleen or liver. The success of such grafts has been equated with normoglycemia but success in dogs has been variable. We have developed a model of canine islet autografting that entails total pancreatectomy, preparation of a pancreatic suspension by perfusion of the pancreatic duct with collagenase, and delivery of the autograft to the spleen by reflux through tributaries of the splenic vein. In this study, fasting insulin response to intravenous glucose (0.5 g/kg), tolbutamide (0.5 g), glucagon (1.0 mg), and arginine (460 mg/kg) was measured before and 3 weeks after intrasplenic islet autografts. The 5 animals studied achieved fasting normoglycemia immediately; therefore, they were successful grafts. The response to glucose, however, was distinctly abnormal with statistically different peripheral insulin levels at 1, 3, 5, and 10 minutes. The insulin response was graphed, the area under the curve determined, and the integrated response was only 20% of preoperative controls. This diminished insulin response was reflected in impaired glucose clearance with a K value of 1.95 ± 0.24 in grafted animals compared to 3.12 ± 0.74 in preoperative controls. Peak insulin responses, however, to tolbutamide (70.4% of normal), glucagon (85.4%), and arginine (94.7%) were not statistically different in grafted animals compared to controls. The disparity of beta cell response to these various secretagogues suggests significant alteration of beta cell function either as a consequence of heterotopic placement, grafting injury, or accommodation to reduced islet number. Comparison of these data with those obtained in other systems supports some sort of accommodation to explain these beta cell changes.RésuméLes îlots de Langerhans peuvent être extraits de leur environnement pancréatique et transplantés comme autogreffe sur un site hétérotypique tel que la rate ou le foie. La normoglycémie est assimilable au succès de la greffe mais ces résultats varient chez le chien. Les auteurs ont développé un modèle dautogreffe des îlots chez le chien comprenant une pancréatectomie totale, une préparation de suspension pancréatique obtenue par perfusion du canal pancréatique avec de la collagènase. La transplantation splénique est obtenue par reflux à travers les affluents de la veine splénique. La réponse insulinique rapide à linjection de glucose intraveineux (0.5 mg/kg), tolbutamide (0.5 g), glucagon (1.0 mg), et arginine (460 mg/kg) est mesurée avant et 3 semaines après la greffe. Les 5 animaux étudiés montrent immédiatement une normalisation rapide de la glycémie; la greffe est donc un succès. Par contre, la réponse à linjection de glucose est distinctement anormale avec des niveaux périphériques dinsuline statistiquements différents à 1, 3, 5, et 10 minutes après injection. La réponse insulinique est représentée sur un graphe. Laire située sous la courbe obtenue et la réponse intégrée représente seulement 20% du témoin préopératoire. Cette diminution de la réponse insulinique reflète une altération de la clearance du glucose avec une valeur K de 1.95 ± 0.24 chez les animaux greffés comparée aux 3.12 ± 0.74 des témoins préopératoires. Par contre le pic de réponse insulinique au tolbutamide (70.4%), au glucagon (85.4%), et à larginine (94.7%) ne sont pas statistiquement différents entre les animaux greffés et les témoins. La disparité de réponse des cellules béta à ces stimulateurs de la sécrétion suggère une altération significative de la fonction de ces cellules. Cette altération pourrait être la conséquence de la situation hétérotypique, dun traumatisme lors de la greffe ou de la mise en place dun nombre réduit dilots. La comparaison de ces résultats avec ceux obtenus dans dautres systèmes doit tenir compte dune telle mise en place pour expliquer les modifications des cellules béta.ResumenLos islotes de Langerhans pueden ser removidos del ambiente pancreático y transferidos en forma de autoinjertos a un lugar heterotópico tal como el bazo o el hígado. El éxito de tales injertos o trasplantes ha sido determinado por la conversión a normoglicemia, pero el éxito ha sido variable en perros. Hemos desarrollado un modelo de autoinjerto canino de islotes, el cual comprende pancreatectomía total, preparación de una suspensión pancreática mediante perfusión del canal pancreático con colagenasa y ubicación del autoinjerto en el bazo por reflujo a través de la vena esplénica. En este estudio se hizo la medición de la respuesta insulínica a glucosa intravenosa (0.5 gm/kg), tolbutamida (0.5 g), glucagón (1.0 mg), y arginina (460 mg/kg) antes y 3 semanas después del trasplante intraesplénico de islotes. Los 5 animales estudiados lograron inmediatamente normoglicemia de ayuno, lo cual es indicativo de éxito. Sinembargo, la respuesta a la glucosa apareció claramente anormal con niveles estadísticamente diferentes de insulina periférica a los 1, 3, 5, y 10 minutos. La respuesta insulínica fue colocada en forma de gráfica para la determinación del área bajo la curva, lo cual mostró que la respuesta integrada fue de solo 20% de los controles preoperatorios. Esta disminuida respuesta insulínica se reflejó en una alterada depuración de glucosa con un valor K de 1.95 ±0.24 en los animales trasplantados contra 3.12 ±0.74 en los controles preoperatorios. Sinembargo, los valores pico de la respuesta insulínica a la tolbutamida (70.4% del normal), glucagón (85.4%), y arginina (94.7%) no fueron estadísticamente diferentes en los animales trasplantados en comparación con los controles. La disparidad de la respuesta de las células beta a estos diversos secretagogos sugiere una alteración de la función de las células beta, bien como consecuencia de la ubicación heterotópica, como lesion del injerto, o como acomodación a un reducido numéro de islotes. La comparación de estos datos con los que han sido obtenidos en otros sistemas da apoyo a la hipótesis de algún tipo de acomodación o adaptación que explique estos cambios en las células beta.

Regulation of the glucose enhanced insulin pool.

Exposure of islets to high levels of glucose at a critical time leads to enhanced insulin release when later stimulated by glucose. Newly synthesized insulin is preferentially released with subsequent stimulation, implying the creation or enlargement of a separately regulated pool of insulin in response to the initial stimulus. Epinephrine via beta adrenergic receptors can trigger the discharge of the enhanced insulin pool via a beta adrenergic receptor response. Raising intracellular cAMP levels or stimulation by arginine also discharge the marked pool. The enhanced pool is accessible by several independent mechanisms.