Factors influencing isolation of islets of Langerhans.

More efficient methods of islet isolation must be developed for islet transplantation to become clinically routine. During collagenase dispersal of human pancreas, an amorphous, viscous, gellike material often develops and entraps large numbers of islets, thereby reducing the yield. When donor human pancreas is minced and treated with collagenase, the gel forms most abundantly if the digestion temperature is less than 35 degrees C and if pH falls below 7.2 +/- 0.2. Gel formation appears to be proportional to warm- or cold-ischemia time and may be related to tissue trauma during collection. Once gel has formed, trapped islets cannot be released by filtration, dilution, DNase, incubation temperature, or pH adjustment. These characteristics suggest that the material is gelatin derived from collagen released enzymatically from pancreatic stroma. We demonstrate that gelation is greatly reduced or eliminated when 1) the incubation medium includes glycerol--a common gelatin solvent--at 5% (vol/vol), 2) the minced tissue-to-total incubation volume ratio is greater than or equal to 1:10, 3) free-islet exposure to pancreatic digestion products is minimized by frequent separation of islets, and 4) collagenase concentration is optimized by titration. Gelation is also minimized by maintaining 5) incubation temperature at 38 +/- 1 degree C and 6) pH in the range 7.7-7.9. Variations in these physical and chemical conditions were analyzed by determining islet yields (stereoscopic microscope counts of serially diluted samples) and by insulin radioimmunoassay of acid alcohol extracts of isolated islets after separation through discontinuous Ficoll gradients. When isolation conditions are optimized as stated, we typically recover 3.3 +/- 1.0 x 10(4) islets/g pancreas, corresponding to greater than 10(6) islets per donor.

Model of associations between psychosocial variables and health-outcome measures of adolescents with IDDM.

The purpose of this study was to develop a model that describes the contributions of key psychosocial variables to the health outcome of adolescents with insulin-dependent diabetes mellitus (IDDM). Subjects were 93 adolescents with IDDM and their parents. Health-outcome measures included adherence and metabolic control (HbA1c). Psychosocial variables included adolescent age, chronic life stress, social competence, family relations, and family knowledge about IDDM. Multiple regression analyses showed that adherence (P less than .029) and stress (P less than .052) were directly related to metabolic control and that knowledge about IDDM (P less than .029), family relations (P less than .099), and adolescent age (P less than .086) had direct effects on adherence. Combined, the independent variables accounted for 14.5% of the variance in predicting HbA1c and 18.5% of the variance in predicting adherence. In general, these findings are consistent with extant theory. The direct link between stress and metabolic control, however, contrasts with the current view that psychosocial variables affect metabolic control indirectly through their influence on adherence behavior. The methodological limitations of the findings are noted, directions for future research are suggested, and the implications for clinical interventions are described.

Race and sex differences in metabolic control of adolescents with IDDM: a function of psychosocial variables?

Race and sex differences have been reported in the prevalence of complications from insulin-dependent diabetes mellitus (IDDM). Because metabolic control is assumed to be associated with the probability of developing future complications, race and sex differences in metabolic control were assessed in a group of adolescents. Subjects were 27 Black and 27 White adolescents who were similar in age, age at diagnosis, and social class. Girls did not differ on number of days since their last menses. A 2-by-2 (race-by-sex) analysis of variance revealed that the Black female group had worse HbA1c than each of the other groups (P less than .012). Multivariate analyses of variance were conducted to determine whether the poor metabolic control of the Black girls was associated with pertinent psychosocial variables. The analyses revealed that their poor control was not associated with a deficiency in the girls' knowledge about IDDM, adherence to treatment, self-concept, coping patterns, family functioning, stress, social support, or involvement in the health-care system. If the poor control of the Black girls is not associated with psychosocial deficiencies, biological variables or psychosocial variables not assessed in this study are probably operative.

Evaluation of a portable insulin infusion pump for outpatient management of brittle diabetes.

Three "brittle" diabetic patients were given constant subcutaneous insulin infusion with a portable battery-driven pump, and their plasma glucose and hemoglobin A1 were measured at frequent intervals during inpatient or outpatient periods. Mean plasma glucose decreased significantly in all inhospital patients and remained significantly less than before pump therapy in two of three as outpatients during the 8--12 wk of follow-ups; however, complete normalization of glucose metabolism was not accomplished in any. All three demonstrated a progressive decline in hemoglobin A1 levels to normal or near-normal values. The need for long-term studies of open loop infusion systems in a research setting before their adaptation to routine care is emphasized.

Comparison of high-dose and low-dose insulin by continuous intravenous infusion in the treatment of diabetic ketoacidosis in children.

We studied the efficacy of low-dose (0.1 U/kg/h) and high-dose (1..0 U/kg/h) insulin, given randomly to children with diabetic ketoacidosis (DKA) by continuous intravenous infusion without a loading dose. Plasma glucose reached 250 mg/dl in 3.4 +/- 0.4 h with the high-dose insulin group compared with 5.4 +/- 0.5 h with the low-dose insulin group (P < 0.01). During the first 12 h of therapy, plasma glucose fell below 100 mg/dl in 2 of 16 in the low-dose compared with 12 of 16 in the high-dose patients. The decrement of ketone bodies, cortisol, and glucagon was similar in both groups. The number of hours required for HCO3(-) greater than or equal to meq/l and arterial blood pH greater than or equal to 7.30 were not significantly different in the two groups. Hypokalemia (K < 3.4 meq/L) occurred in 3 of 16 low-dose and 10 of 16 high-dose patients. The data show that low-dose insulin, with a slower rate of glucose decrease, is as effective as a high dose for the treatment of DKA in children with less incidence of hypokalemia and decreased potential for hypoglycemia.

Correlation of hyperandrogenism with hyperinsulinism in polycystic ovarian disease.

We evaluated basal plasma total immunoreactive insulin (insulin), androstenedione, and testosterone in 14 obese women: 8 with polycystic ovarian disease (PCOD) and 6 obese controls. All 3 hormones were significantly elevated (P less than 0.02 to P less than 0.001) in PCOD patients. A significant correlation among basal levels of plasma insulin, androstenedione, and testosterone was demonstrated. The PCOD group had significantly higher levels of glucose at 1, 2, and 3 h, with similar significant increases in plasma insulin levels at 0, 2, and 3 h. A significant correlation was found between plasma insulin response areas and plasma testosterone (P less than 0.001) in the control and PCOD patients. These studies demonstrate that hyperandrogenism correlates with hyperinsulinism.

Glucose intolerance and insulin resistance in aplastic anemia treated with oxymetholone.

Because of a suspected association between the drug oxymetholone and abnormal glucose metabolism, we determined immunoreactive insulin (IRI) and plasma glucose during oral glucose tolerance testing in seven patients with aplastic anemia, six of whom received oxymetholone therapy. All patients receiving oxymetholone therapy had abnormal glucose and/or IRI values. This finding was independent of GH, cortisol, and glucagon. In one patient, glucose and IRI levels were normal before oxymetholone but became abnormally elevated after the use of this drug. Furthermore, normal glucose and IRI values were present in the single patient not receiving oxymetholone. Thus, a positive relationship was demonstrated between oxymetholone administration and the presence of glucose intolerance and insulin resistance.

Diagnosis of hidden central hypothyroidism in survivors of childhood cancer.

To determine how often central hypothyroidism remains undetected by routine out-patient tests of thyroid hormone, we studied 208 pediatric cancer survivors referred for evaluation because of signs of subtle hypothyroidism or hypopituitarism. Of the 208 (68 females and 140 males), 110 had brain tumors, 14 had other head/neck tumors, 11 had solid tumors remote from head and neck, and 73 had leukemia. Patients were evaluated 1-16 yr (mean, 6.1+/-4.1 yr) after tumor diagnosis. The nocturnal TSH surge and response to TRH were measured. Of 160 patients with free T4 in lowest third of normal, 34% had central hypothyroidism (blunted TSH surge or low/delayed TSH peak or delayed TSH decline after TRH); 9% had central hypothyroidism with mild TSH elevation (mixed hypothyroidism). Another 16% had mild primary hypothyroidism (TSH, 5-15 mU/L). Of 48 with free T4 in the upper two thirds of normal, 14% had central hypothyroidism; 17% had mild primary hypothyroidism. Incidence of central, mixed, and mild primary hypothyroidism 10 yr after tumor diagnosis was significantly related to total cranial radiation dose (P < 0.0001). Of 62 patients with central hypothyroidism, 34% had not developed GH deficiency. TSH surge identified 71%, and response to TRH identified 60% of those with central hypothyroidism. More than half of the slowly growing patients who have received cranial or craniospinal radiation for childhood cancer develop subtle hypothyroidism. In our study group, 92% of patients with central hypothyroidism and 27% with mixed hypothyroidism would have remained undiagnosed using baseline thyroid function tests alone. Both TSH surge and response to TRH must be evaluated to identify all of these patients.

Neonatal progeroid (Wiedemann-Rautenstrauch) syndrome: report of five new cases and review.

The neonatal progeroid syndrome (NPS), or Wiedemann-Rautenstrauch, is a rare autosomal recessive disorder comprised of generalized lipoatrophy except for fat pads in the suprabuttock areas, hypotrichosis of the scalp hair, eyebrows, and eyelashes, relative macrocephaly, triangular face, natal teeth, and micrognathia. We report on 5 new patients who demonstrate phenotypic variability and who represent the single largest series of NPS reported to date. Two of the patients are from an African-American kindred, an ethnic occurrence not reported previously. The fact that there are 2 pairs of sibs among the 5 patients further supports that NPS is an autosomal recessive condition. This report also includes a review of the previously reported 16 patients and compares them with the 5 new patients. Abnormalities in endocrine and lipid metabolism were found in 3 of 5 patients. Skeletal findings in 2 of our patients demonstrated some new findings as well as the typical radiological abnormalities previously noted in NPS. It is apparent, based on the 21 cases, that mild to moderate mental retardation is common in NPS. Long term follow-up of patients with NPS should provide more information relative to their ultimate psychomotor development. NPS is usually lethal by 7 months; however, on rare occasions, patients have survived into the teens. Our 3 surviving patients range in age from 16-23 months. Variability in the phenotype of NPS is clear; however, the phenotype remains distinct enough to allow a secure diagnosis.

Genetic and immunological markers of insulin dependent diabetes in Black Americans.

ICA and GAD65 autoantibody profiles and HLA-DR and DQ analysis were performed on 43 Black juvenile onset IDDM patients and 34 unrelated Black controls from Tennessee, USA. 75% of patients were positive for GAD65 autoantibodies but only 53% had ICA; 39% both ICA and GAD65 antibodies. The strongest HLA association was with the DR3 haplotype DRB1*03 DQA1*0501 DQB1*0201 (63% of patients v 12% of controls RR = 13.0, p < 0.00002). DRB1*04 DQA1*0301 DQB1*0302, associated with IDDM in Caucasians but rare in Negroids, occurred in 27% of patients and 6% of controls (RR = 5.9, p < 0.04). All patients carried DQB1*0302 or DQB1*0201. DQB1*0602 was significantly reduced in patients (2.4% v 41%, RR = 0.036, p < 0.008) and DRB1*1501 was absent in patients (0% v 35%). The frequency of GAD65 autoantibodies in Black American IDDM patients is comparable to that in Caucasians; however ICA positivity is reduced. GAD65 antibodies may therefore be a more sensitive serological test to identify individuals in the Black American general population for markers associated with increased risk of developing IDDM. Current screening methods for predicting preclinical IDDM in Caucasians relies on a combination of immune and HLA markers of IDDM; studies of these markers in the Black Americans will make it possible to extend these options to additional genetically diverse populations.

An integral membrane protein form of brain L-glutamate decarboxylase: purification, characterization and its relationship to insulin-dependent diabetes mellitus.

A new and novel form of L-glutamate decarboxylase (GAD; EC 4.1.1.15) was purified from whole porcine brain to apparent homogeneity by a combination of column chromatographies on DE-52, ultragel AcA 34, hydroxylapatite and Sephadex G-200, and native gel electrophoresis. The purified GAD was established as an integral membrane protein based on hydrophobic interaction chromatography and membrane extraction studies. This membrane GAD (MGAD) has a native molecular weight of 120 +/- 5 kDa and is a homodimer of 60 +/- 2 kDa. Immunoprecipitation and immunoblotting tests using the sera from insulin-dependent diabetes mellitus (IDDM) patients revealed the presence of antibodies against this newly identified MGAD in IDDM. The role of MGAD in the pathogenesis of IDDM and related autoimmune disorders is also discussed.

Formation of pseudoislets from human pancreatic cultures.

We have successfully developed a technique for culturing human islet cells obtained from the cadaveric pancreata of children. Within 24-48 h of in vitro culture, collagenase-digested human pancreatic tissue formed epithelioid monolayers. Scattered within these monolayers were insulin-positive cells, as detected by immunocytochemical and dithizone staining. Treatment of the beta cell-containing epithelioid-cell monolayers with EDTA resulted in the formation of spherical cellular clusters, i.e., pseudoislets. These pseudoislets differed from isolated islets of Langerhans in that they showed a more peripheral distribution of insulin-positive cells. Our studies have demonstrated that insulin-positive cells can be detected in monolayers obtained from human pancreata 3-4 weeks after culture. When exposed to varying concentrations of glucose, these cells secreted insulin. The development of this in vitro technique for culturing human pancreatic islet tissue could provide a model for systematically studying in vitro islet function.

Family system variables and the health status of adolescents with insulin-dependent diabetes mellitus.

This study investigated the associations between important dimensions of family relations and the metabolic control of adolescents with insulin-dependent diabetes mellitus (IDDM). Participants were 94 intact families (mother, father, and adolescent) of adolescents with IDDM. Metabolic control was determined by the adolescents' level of HbA(1c), and family relations were assessed with the Marital Adjustment Scale and the Family Adaptability and Cohesion Evaluation Scales-II. Although zero-order correlations showed that good metabolic control was associated with high family cohesion, family flexibility, and high marital satisfaction, hierarchical regression analyses revealed that these associations were considerably attenuated when participant demographic variables, especially the duration of IDDM, were controlled. Follow-up analyses showed that the associations between family relations and metabolic control were mediated by the duration of IDDM. Under conditions of short duration, there were strong associations between family relations and metabolic control. As duration lengthened, however, these associations decreased substantially. These results suggest that the associations between family relations and health are relatively complex in this sample of adolescents with IDDM.

Coping styles in youths with insulin-dependent diabetes mellitus.

The relationships between two coping styles (i.e., use of personal and interpersonal resources; ventilation and avoidance) and two health outcomes (i.e., adherence and metabolic control) were evaluated in 135 youths with insulin-dependent diabetes mellitus (IDDM). Individual characteristics (i.e., age, duration of illness) and contextual variables (i.e., stress, family relations) were used to predict coping styles. Poor adherence to treatment, older adolescent age, and long duration of IDDM were correlated with ventilation and avoidance coping. Youths with short duration of IDDM were more likely to cope through the use of personal and interpersonal resources, although this strategy was not associated with health outcomes. A multiple regression analysis indicated that high ventilation and avoidance coping was predicted by high stress, low family cohesion, and older adolescent age. In addition, the interaction between family adaptability and duration of IDDM significantly predicted ventilation and avoidance coping.

Laparoscopic pancreatectomy for persistent hyperinsulinemic hypoglycemia of infancy.

BACKGROUND: A 4-week-old male infant (4.9 kg) with persistent hyperinsulinemic hypoglycemia of infancy (PHHI) underwent a laparoscopic pancreatectomy to evaluate its feasibility. Preoperative medications included diazoxide and glucagon to maintain adequate blood glucose levels. METHODS: Laparoscopic pancreatectomy was performed using a 5-mm cannula at the umbilicus, external fixation, transcutaneous suture-assisted gastric retraction to expose the lesser sac, and three additional 3.5-mm cannula sites. The pancreas was resected from the splenic hilum to the mesenteric vessels. The splenic vein was dissected from the under surface of the pancreas using electrocautery, and the spleen was easily preserved. Surgery time was 75 min, and minimal blood loss occurred. RESULTS: The child required no narcotic medication and tolerated a regular diet immediately after surgery. Serum glucose levels did decrease postoperatively, and the child required diazoxide, dextrose infusion, glucagon, and octreotide. On postoperative day 7, the child underwent an open near-total pancreatectomy, after which he remained asymptomatic. Essentially no scarring was found in the lesser sac, and the remaining pancreatic remnant was resected without difficulty. CONCLUSIONS: Laparoscopic pancreatectomy can be performed safely, even in a newborn patient, without prolonged operative time or unnecessary risk. The technique using external fixation and transcutaneous suture-assisted gastric retraction provides excellent exposure to the pancreas and lesser sac. In patients with PHHI, in whom reoperative additional pancreatectomy is very likely, this technique is the ideal initial surgical approach.

Caloric restriction for 24 hours increases mean night growth hormone.

UNLABELLED: In obesity, serum growth hormone (GH) is usually low, confounding GH assessment of short obese children. We evaluated whether 24-h caloric restriction would permit better discrimination between normal GH secretion and GH deficiency (GHD) by elevating night GH levels. DESIGN AND PATIENTS: Serum was obtained every 20 minutes 2000-0800 h before and 2200-0400 h after 24 hours of caloric restriction (8% of usual calories) in 24 normal height children [14 normal (weight for height 10-90th percentile); 10 obese (weight for height > 95th percentile)] and in 31 short children (height shorter than -2.0 SD below mean for age). All samples from both nights per child were assayed for GH simultaneously to eliminate interassay variability. RESULTS: Mean GH increased significantly in all groups after caloric restriction (P < 0.01). Obese children had lower baseline mean GH and GH amplitude compared to normal (P < 0.01); GH increased into normal range after restriction. Basal GH studies in short children were not significantly below normal. Surprisingly, some with low stimulated GH increased their night GH into the normal range after caloric restriction. CONCLUSIONS: Caloric restriction for 24 h enhances night GH similarly in short and in normal children, and thus does not increase the diagnostic utility of night GH studies in non-obese short children. Caloric restriction reverses suppressed GH secretory state of obese children, perhaps by decreasing diet-dependent somatostatin inhibition of GH secretion.

Histologic appearance of the islet organoid.

The authors have attempted to develop a hybrid organoid capable of supporting the engraftment of human pancreatic islet tissue transplants. In this report, they we report the histologic appearance of this organoid structure for implantation periods up to 16 days. Both pancreatic duct-like and glandular cells were present inside the organoid. Insulin positive cells were identified within the pancreatic tissue. Although polymorphonuclear (PMN) cells were seen in association with tissue necrosis, the authors observed no lymphocyte infiltration in the area of the xenografted human pancreatic islet tissue in the implanted organoid. Endothelial cell growth factor induced neovascularization inside the organoid sufficient to support the engraftment of transplanted pancreatic islet tissue. This organoid model may provide an alternative for clinical pancreatic transplantation.

Post-traumatic cutaneous mucormycosis in diabetes mellitus. Short-term antifungal therapy.

Mucormycosis is infrequently encountered in the pediatric population in any of its forms (nasopharyngeal, disseminated, pulmonary, or cutaneous) and generally is associated with the immunocompromised host. We present an adolescent with poorly controlled diabetes mellitus who developed a progressive skin lesion 3 weeks after a motor vehicle accident. Rhizopus species was isolated from the lesion, and the biopsy revealed a fungal vasculopathy. Control of her diabetes, aggressive surgical intervention and a 10-day course of antifungal therapy (amphotericin B) resulted in a favorable outcome. This article illustrates the importance of considering cutaneous fungal infections, especially those in the class zygomycetes, in the diabetic patient with unusual, severe or persistent skin lesions. Early recognition is essential in order to avoid morbidity and mortality from this unusual opportunistic infection.