Daniel H. Mintz

Islet isolation assessment in man and large animals

SummaryRecent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Pancreatic islet transplantation after upper abdominal exenteration and liver replacement

Nine patients who became diabetic after upper-abdominal exenteration and liver transplantation were given pancreatic islet-cell grafts obtained from the liver donor (eight cases), a third-party donor (one), or both (four). Two patients were diabetic when they died of infections after 48 and 109 days, as was a third patient who died of tumour recurrence after 178 days. The other 6 are alive 101-186 days postoperatively, and five are insulin-free or on insulin only during night-time parenteral alimentation. C-peptide increased 1.7 to 3.3 fold in response to intravenous glucose in these five patients who have had glycosylated haemoglobin in the high normal range. However, the kinetics of the C-peptide responses to intravenous glucose in all eight patients tested revealed an absent first-phase release and a delayed peak response consistent with transplantation and/or engraftment of a suboptimal islet cell mass. The longest survivor, who requires neither parenteral alimentation nor insulin, is the first unequivocal example of successful clinical islet-cell transplantation.

Human islet isolation and allotransplantation in 22 consecutive cases

This report provides our initial experience in islet isolation and intrahepatic allotransplantation in 21 patients. In group 1, 10 patients underwent combined liver-islet allotransplantation following upper-abdominal exenteration for cancer. In group 2, 4 patients received a combined liver-islet allograft for cirrhosis and diabetes. One patients had plasma C-peptide greater than 3 pM and was therefore excluded from analysis. In group 3, 7 patients received 8 combined cadaveric kidney-islet grafts (one retransplant) for end-stage renal disease secondary to type 1 diabetes mellitus. The islets were separated by a modification of the automated method for human islet isolation and the preparation were infused into the portal vein. Immunosuppression was with FK506 (group 1) plus steroids (groups 2 and 3). Six patients in group 1 did not require insulin treatment for 5 to greater than 16 months. In groups 2 and 3 none of the patients became insulin-independent, although decreased insulin requirement and stabilization of diabetes were observed. Our results indicate that rejection is still a major factor limiting the clinical application of islet transplantation in patients with type 1 diabetes mellitus, although other factors such as steroid treatment may contribute to deteriorate islet engraftment and/or function.

Impaired water excretion in myxedema

Abstract The response to acute water ingestion (20 ml/kg body weight) was determined in sixteen patients with myxedema and eighteen control subjects. Comparison of the results in the two groups indicated an abnormality in water excretion in the patients with myxedema. This impaired water diuresis in the patients with myxedema, including those with significant hyponatremia, appeared to be related to decreased volume delivery to the distal diluting segment of the nephron rather than to persistent secretion of antidiuretic hormone. The reevaluation of eleven patients with myxedema after thyroid replacement therapy revealed significant improvement in the response to water ingestion.

Natural history of intrahepatic canine islet cell autografts.

We have serially followed the function of intrahepatic canine islet autografts in 15 beagle dogs for up to 24 mo. Of these, only 20% sustained normal levels of fasting blood glucose for greater than 15 mo posttransplant. Failure of autograft function was accompanied by a preferential loss of well-granulated beta cells in the engrafted islets. The chronic stimulation of an initially marginal intrahepatic beta-cell mass ultimately resulted in metabolic deterioration and loss of beta cells below the minimal threshold required to maintain normal fasting blood glucose levels. It is possible that transplantation of a larger mass of islets would result in indefinite graft function in dogs. However, it remains to be demonstrated in larger mammals, including humans, whether an islet cell mass that is initially adequate in a heterotropic site such as the liver can remain functionally competent over a prolonged period.

In vivo effect of FK506 on human pancreatic islets.

The purpose of this study was to evaluate the in vivo effect of FK506 on human pancreatic islets. Twenty-five nude mice were made diabetic by one intravenous injection of streptozotocin. Approximately 600 islets were administered in the renal subcapsular space 3-5 days following streptozotocin administration. One week after transplantation, the mice were divided in four groups. In group 1, the animals received 1 injection of 0.5 ml of diluent i.p. daily for one week. In groups 2, 3, and 4 the treatments were daily i.p. injection of 0.3, 1, and 3 mg/kg FK506, respectively. After treatment, the functional integrity of the transplanted human islets was tested by measuring the plasma glucose and human C-peptide response to intraperitoneal glucose injection in groups 1 and 4. IPGTT alone was assessed in groups 2 and 3. The results indicate that i.p. administration of FK506 for one week at a dose 0.3 mg/kg/day did not result in any significant alteration of glucose disappearance and C-peptide response to IPGTT. Higher doses of FK506 produced a significant delay in glucose disappearance in groups 3 and 4, and a significant inhibition of glucose-mediated C-peptide response in group 4.

Metabolic clearance and production rates of human growth hormone

The metabolic clearance rate (MCR) of human growth hormone (HGH) was determined by the constant infusion to equilibrium technique utilizing HGH-(125)I. 22 control subjects had a MCR of 229 +/-52 ml/min (mean +/-SD). No difference was evident between sexes, or between various age groups. Patients with acromegaly demonstrated normal MCRs. Moreover, acute elevations of plasma growth hormone concentrations in normal subjects did not alter the MCR of HGH. The MCR was relatively constant from day to day and within the day when subjects were evaluated in the supine position. In contrast, the assumption of the upright position was associated with a mean 24% decrease in the MCR. These results were contrasted with the MCR of HGH observed in a small number of patients with altered thyroid function or diabetes mellitus. In six patients with hypothyroidism the MCR (131 +/-36 ml/min) was significantly decreased (P < 0.001); whereas the MCR in eight patients with hyperthyroidism (240 +/-57 ml/min) did not differ from control subjects. The MCR in eight patients with insulin-independent diabetes mellitus (IID) (185 +/-41 ml/min) and in eight patients with insulin-dependent diabetes mellitus (IDD) (136 +/-31 ml/min) were significantly different from control subjects (P = < 0.05 and P = < 0.001, respectively). These data were interpreted to indicate that the plasma HGH-removing mechanism(s) is not saturated at physiologic plasma HGH levels, that plasma HGH levels alone may not permit distinction between variations in pituitary release of the hormone and its rate of clearance from the plasma, and that the estimation of the MCR of HGH may help clarify the mechanism of abnormal plasma HGH responses to various stimuli. Production rates of HGH (PR) in control subjects (347 +/-173 mmug/min) were contrasted with hyperthyroid patients (529 +/-242 mmug/min, P < 0.05), hypothyroid patients (160 +/-69 mmug/min, P < 0.02), IID (245 +/-100 mmug/min, NS), and IDD (363 +/-153 mmug/min, NS). Considerable variability in the determination of the concentrations of immunoprecipitable HGH-(125)I and endogenous plasma HGH concentrations was encountered at apparent equilibrium conditions. Since both factors are necessary for the PR calculations, the wide 95% confidence limits of the PRs did not permit a clear interpretation of the significance of these observations.

Insulin and Multiplication Stimulating Activity (an Insulin-like Growth Factor) Stimulate Islet β-Cell Replication in Neonatal Rat Pancreatic Monolayer Cultures

A possible role for insulin in stimulating islet β-cell replication was examined in neonatal rat pancreatic monolayer cultures. Addition of insulin to serum-free medium increased the mitotic index and stimulated dose-dependent increases in [3H]-thymidine incorporation in nuclei of islet β-cells in aldehyde-thionine-stained autoradiographs. The effects of insulin were not associated with any significant changes in glucagon or somatostatin levels in the culture media. Multiplication stimulating activity (MSA), an insulin-like growth factor, was about 100-fold more potent than insulin: 3 ng/ml MSA stimulated a half-maximal increase in thymidine labeling of β-cell (+63%, P < 0.005), whereas 300 ng/ml insulin was required for a similar effect. The maximal effects of insulin and MSA were similar, and the combination of maximal stimulatory concentrations of MSA (30 ng/ml) and insulin (3000 ng/ml) was not more effective than either substance added alone, suggesting that both peptides act on the same mechanism(s) regulating β-cell replication. Furthermore, an antibody to the insulin receptor did not prevent the stimulatory effects of either insulin or MSA on thymidine labeling of β-cells. These results demonstrate that insulin can stimulate islet β-cell replication directly, possibly through a receptor for MSA or another insulin-like growth factor.

An Electronic Case Manager for Diabetes Control

OBJECTIVE To evaluate the usage and safety of an electronic case manager (ECM) system designed to facilitate the task of glycemic control. Sustained improvement in blood glucose control is the proven treatment outcome that will reduce or eliminate the long-term complications of diabetes. RESEARCH DESIGN AND METHODS A customized microcomputer system served as the ECM. Located at the clinic, this voice-interactive system required the remote patient to need only a touch-tone telephone. Patients accessed the system to report daily selfmeasured glucose levels or hypoglycemic symptoms together with associated lifestyle events. System beta-testing was in an open-case series (n = 184) in an academic diabetes center with the goal of evaluating the ECM in terms of utilization, frequency of crises, and fiscal matters. RESULTS Of the patients, 58% (n = 107) actively used the ECM for their daily diabetes care, accumulating 788 patient-months of follow-up. Over 45,000 telephone calls were received by the ECM during the start-up year. Each call was processed instantly and automatically. Patients benefited from having 24-h access to the ECM. Prevalence of diabetes-related crises (hyperglycemia =400 mg/dl [22 mmol/1] or hypoglycemia <50 mg/dl [2.8 mmol/1]) decreased approximately threefold (P < 0.05), with a concomitant statistically significant decrease in HbA1c of 0.8% at 6 months (n = 45, P = 0.024) and 0.9% at 12 months (n = 30, P = 0.044). The ECM provided 24-h on-line assistance in adjusting daily insulin and/or tablet therapy, automatic generation of standardized medical reports, electronic medical-legal documentation, as well as a marked reduction in the time spent on the phone with patients. Clinic visits in managing complex diabetes were reduced approximately twofold (P < 0.0001), and the effort spent by case managers was estimated. CONCLUSIONS Patients with diabetes who accessed the ECM system received timely, cost-effective, and reliable medical intervention. This reduced the incidence of diabetic crises and the need for frequent clinic visits. The ECM empowers case managers to provide safer and superior diabetes care.

Home Blood Glucose Monitoring As an Aid in Diabetes Management

Home blood glucose monitoring, using Dextrostix and an Eyetone meter, has been utilized in several subcategories of patients with insulin-dependent diabetes mellitus. These include pregnant patients, anephric patients, patients undergoing weight reduction, patients with altered renal threshold for glucose reabsorption, and patients in whom diabetic regulation is difficult. Patients monitored home blood glucose continually (on a daily basis), intermittently, or only for particular problems or symptoms. Such monitoring can be practically accomplished in a manner acceptable to patients. Motivation, compliance with protocol, and an understanding of the objectives of the program are essential on the part of the patient. Home blood glucose monitoring, however, can provide an insight diabetes regulation that cannot be attained in any other way and can greatly facilitate regulation of diabetes. Such home blood glucose monitoring may increase the likelihood of achievement of a degree of control approximating euglycemia.