Beginning of pancreatic islet isolation by collagenase digestion (personal reminiscences)

Successful laboratory communication in removable prosthodontics will always be more complex than in fixed prosthodontics. However, the unique characteristics of natural denture teeth (SR-Antaris/SR-Postaris), Ivoclar Williams, Amherst, NY) are enabling clinicians to overcome functional and aesthetic challenges. Specifically, the characterized anatomy of these anterior and posterior teeth, and their translucency and natural layering, facilitate the reestablishment of a natural smile. This article describes the benefits of SR-Antaris/SR-Postaris denture teeth and provides guidelines for communication with the laboratory to ensure predictable results.     

Factors controlling pancreatic islet neogenesis

We have established a model in which cellophane wrapping induces reiteration of the normal ontogeny of beta-cell differentiation from ductal tissue. The secretion of insulin is physiologic and coordinated to the needs of the animal. Streptozotocin-induced diabetes in hamsters can be 'cured' at least half the time. There appears to be activation of growth factor(s) within the pancreas acting in an autocrine, paracrine or juxtacrine manner to induce ductal cell proliferation and differentiation into functioning beta-cells. Given the results of our studies to date, it does not seem premature to envisage new approaches to the treatment of diabetes mellitus. Identification of the factor(s) which regulates islet cell proliferation and differentiation in our model may permit proto-undifferentiated cells and islets to be grown in culture. This concept could be extended to induce endocrine cell differentiation in vitro as well. Furthermore, islet cell growth factors could be used to provide 'trophic support' to islet transplants as a means of maintaining graft viability. There may also be greater scope for gene therapy when the growth factor(s) has been isolated, purified, sequenced and cloned.     

Sensitivity and specificity for detection of islet cell cytoplasmic antibodies using rat pancreatic sections

We determined islet cell cytoplasmic antibodies (ICA) using rat pancreatic sections as a test substrate substitutive for human pancreatic sections by indirect immunofluorescent technique. ICA were measured in sera from 58 patients with insulin-dependent diabetes mellitus (IDDM), 456 with non-insulin-dependent diabetes mellitus (NIDDM), 50 patients with autoimmune diseases, and 110 healthy controls. Seventeen of 58 patients with IDDM showed recent-onset (within 3 months). ICA were also measured in some samples using blood group O human pancreatic sections, and the ICA titers were compared with those measured using rat pancreatic sections. The prevalence of ICA was 55.2% (32/58) in patients with IDDM, 1.5% (7/456) in those with NIDDM, 0% (0/50) in those with autoimmune diseases, and 0.9% (1/110) in the healthy controls. Of the 17 recent-onset IDDM ICA were positive in 14 (82.3%). In comparative study of titers for ICA using rat pancreatic sections or human pancreatic sections, rat pancreatic sections yielded ICA titers as high as human pancreatic sections did. These results demonstrate that ICA assay using rat pancreatic sections was disease-specific, and that antigenicity of the substrate was favorable to ICA. Rat pancreas presents the advantage of greater availability, while providing an identical substrate for ICA. In conclusion, rat pancreatic sections are useful substrate for detecting ICA.     

Targeted oncogenesis of hormone-negative pancreatic islet progenitor cells

Transgenic mice containing an upstream glucokinase (betaGK) promoter- simian virus 40 T antigen (Tag) fusion gene develop neuroendocrine tumors primarily in the pancreas, gut, and pituitary. Pancreatic tumors from a line with delayed tumorigenesis were of two different types: insulinomas and noninsulinomas. The noninsulinomas are often periductal in location, express none of the four major islet peptide hormones, Glut-2, Pdx1, tyrosine hydroxylase, Pax4, Pax6, or Nkx6.1, but do express glucokinase, Sur1, Isl1, Hnf3beta, Hnf6, Beta2/NeuroD, and Nkx2.2. Cells from two different noninsulinoma tumors, when adapted to culture, began to express either insulin, glucagon, or somatostatin. Given the partial gene expression repertoire of the noninsulinoma tumors, their apparent periductal origin, and the ability of these cells to partially cytodifferentiate in culture, we suggest that these tumors are derived from islet progenitor cells. Thus, betaGK-Tag transgenic mice provide a new model system for studying the events that occur during both islet cell neogenesis and normal embryonic development.     

Porcine islet preservation during isolation in University of Wisconsin solution

OBJECTIVE: To report a triplet pregnancy that occurred after intracytoplasmic injection of sperm into cryopreserved oocytes. DESIGN: Case report. SETTING: Instituto de Ginecología y Fertilidad (IFER), Buenos Aires, Argentina. PATIENT: A 36-year-old infertile patient with premature ovarian failure and a previous term pregnancy with fresh donated oocytes. INTERVENTION(S): We administered leuprolide acetate for pituitary down-regulation followed by E2 valerianate in incremental doses until an endometrial lining of >8 mm was observed by ultrasound. Thawing of frozen donated oocytes, intracytoplasmic sperm injection (ICSI), and translaparoscopic fallopian tube ET also were performed. Natural micronized progesterone was administered intravaginally (600 mg/d) before ET. MAIN OUTCOME MEASURE(S): Ultrasound at the 8th week of gestation revealed a triplet pregnancy with active fetal heartbeats. RESULT(S): A triple intrauterine gestation was achieved with the use of microinjection into cryopreserved oocytes. CONCLUSION(S): This case illustrates the feasibility of oocyte cryopreservation for clinical use in the era of ICSI.     

Expression of major histocompatibility antigens on pancreatic islet cells

Insulin-dependent diabetes mellitus is often accompanied by manifestations of autoimmunity and is frequently associated with certain HLA haplotypes, predominantly DR3 and DR4. Because the major histocompatibility antigens are important determinants of the immune response in various tissues, we have investigated their expression on the pancreatic islet cells. Human, mouse, or rat islets of Langerhans, as well as lymphocytes or other differentiated cells, were biosynthetically labeled with radioactive amino acids, lysed in detergent, and immunoprecipitated with several antisera specific for major histocompatibility antigenic groups. The immunoprecipitates were analyzed by NaDodSo4/polyacrylamide gel electrophoresis under reducing conditions followed by autoradiography. The major histocompatibility antigens corresponding to the H-2 K,D molecules in mice, the H1-A in rats, and the HLA-A, -B, and -C in humans were precipitated from both islet and lymphocyte lysates and were accompanied by beta 2-microglobulin. Binding of H-2 antibodies to islet cells was also confirmed by a radioligand assay using 125I-labeled protein A and by indirect immunofluorescence. Analyses in the fluorescence-activated cell sorter revealed that greater than 95% of the cells in the beta-cell-rich fraction were fluorescent, providing further evidence that the pancreatic beta cells express the major histocompatibility antigens. Monoclonal antibodies or mouse alloantisera against HLA-DR or Ia antigens did not react with labeled pancreatic islet cell proteins.     

Initiation of autoimmune diabetes by developmentally regulated presentation of islet cell antigens in the pancreatic lymph nodes

Little is known about the events triggering lymphocyte invasion of the pancreatic islets in prelude to autoimmune diabetes. For example, where islet-reactive T cells first encounter antigen has not been identified. We addressed this issue using BDC2.5 T cell receptor transgenic mice, which express a receptor recognizing a natural islet beta cell antigen. In BDC2.5 animals, activated T cells were found only in the islets and the lymph nodes draining them, and there was a close temporal correlation between lymph node T cell activation and islet infiltration. When naive BDC2.5 T cells were transferred into nontransgenic recipients, proliferating cells were observed only in pancreatic lymph nodes, and this occurred significantly before insulitis was detectable. Surprisingly, proliferation was not seen in 10-day-old recipients. This age-dependent dichotomy was reproduced in a second transfer system based on an unrelated antigen artificially expressed on beta cells. We conclude that beta cell antigens are transported specifically to pancreatic lymph nodes, where they trigger reactive T cells to invade the islets. Systemic or extrapancreatic T cell priming, indicative of activation via molecular mimicry or superantigens, was not seen. Compromised presentation of beta cell antigens in the pancreatic lymph nodes of juvenile animals may be the root of a first "checkpoint" in diabetes progression.     

Porcine neonatal pancreatic cell clusters (NPCCs): a potential source of tissue for islet transplantation

The issue of third-party reimbursement for advanced nurse practitioners is a policy issue at both the state and federal levels. The article reports on a study that explored the perception of certified nurse midwives (CNMs) on the impact of current reimbursement policies on their ability to practice. The issues and problems identified by CNM Practice Directors related to obtaining payment and contracts. In turn, these issues were seen to influence the number of potential clients that can be seen.     

Stem cell isolation

High-dose chemotherapy with autologous hematopoietic progenitor cell support is increasingly used to treat a variety of malignant diseases. A drawback of this technique is the potential for infusing clonogenic tumor cells with the autograft, producing relapse of the disease in the patient. The use of positive selection techniques to isolate stem cells and thus reduce or eliminate tumor cell contamination has been extensively studied over the past few years. Preliminary clinical results have demonstrated that these procedures deplete 2 to 7 logs of tumor cells and do not impair engraftment. It is too early to assess the ultimate clinical benefit of this strategy. Additional applications of CD34-selection include ex vivo expansion of and gene transfer into hematopoietic progenitor cells and T-cell depletion of allogeneic grafts to reduce the incidence of graft-versus-host disease.     

Voltage dependence of rhythmic plateau potentials of pancreatic islet cells

The origin and control of glucose-induced rhythmic plateau potentials of pancreatic islet cells have been studied with intracellular microelectrodes in isolated mouse islets. Rapid changes of extracellular potassium concentration and direct electrical stimulation via a suction electrode were used to perturb islet cell membrane potentials. We show that brief depolarizing stimuli trigger permature plateau potentials, and brief hyperpolarizing currents abort endogenous plateaus. Both responses occur in an all-or-none manner, show a reciprocal relationship between stimulus strength and stimulus duration, have stimulus thresholds that approach zero at the time of the endogenous event, and completely reset the endogenous plateau rhythm. These results indicate that the plateau potentials are due to voltage-dependent regenerative mechanisms as in other electrically excitable tissues and implicate membrane potential or membrane ionic fluxes in the glucose-dependent pacemaker system that triggers their onset and offset.     

Diagnosis and treatment of insulin-secreting pancreatic islet cell tumors in ferrets: 57 cases (1986-1994)

OBJECTIVE: To evaluate clinical, laboratory, radiographic, ultrasonographic, surgical, and histologic findings in ferrets with insulinoma and to determine their long-term outcome. DESIGN: Retrospective study. ANIMALS: 57 ferrets with a histopathologic diagnosis of pancreatic islet cell tumor. PROCEDURE: Medical records of ferrets with pancreatic islet cell tumors were reviewed. RESULTS: Lethargy, weakness, and collapse were the most common clinical signs. All ferrets had hypoglycemia, and hyperinsulinemia was documented in 39 of 47 (83%) ferrets. Ultrasonographic examination of the abdomen revealed pancreatic nodules in 5 of 23 ferrets. Surgical treatment was performed in 50 ferrets, 3 were treated by medical management alone, and 4 did not have treatment. At the time of surgery, 1 pancreatic nodule was found in 13 (26%) ferrets and multiple nodules were found in 37 (74%) ferrets. Pancreatic carcinoma alone was found in 34 ferrets. Whereas a combination of carcinoma and either hyperplasia or adenoma was found in 23 ferrets; 4 ferrets had metastasis to regional lymph nodes or liver. In 26 (53%) ferrets, hypoglycemia persisted after surgery, necessitating medical treatment with prednisone, diazoxide, or both. Sixteen (33%) ferrets had redevelopment of hypoglycemia at 1 to 23.5 months (median, 10.6 months) after surgery. Only 7 of the 50 (14%) ferrets remained euglycemic after surgery. CLINICAL IMPLICATIONS: In ferrets, surgical removal of insulin-secreting pancreatic islet cell tumors is recommended as definitive treatment; however, multiple pancreatic nodules are common, making complete excision of all tumor tissue difficult. Persistent hypoglycemia after surgical treatment indicates that lifelong medical management with prednisone or diazoxide or both may be necessary in many ferrets. Finally, because the insulin-secreting tumors are malignant, long-term cure and survival are not likely.     

Leptin regulation of islet amyloid polypeptide secretion from mouse pancreatic islets

Leptin receptors are expressed in pancreatic beta-cells. However, leptin's role in islet hormone secretion is essentially unknown. In the present study, we aimed to elucidate leptin's effect on isolated pancreatic NMRI mouse islets by examining islet amyloid polypeptide (IAPP) and insulin secretion in acute experiments and after 48-hr exposure to leptin (1-100 nM). It was also examined whether a putative effect of leptin was affected by the glucose concentration. Islets were cultured in medium RPMI 1640 + 10% fetal calf serum, and the effects of leptin on islet cell replication, glucose metabolism, and hormone content were subsequently examined. Glucose-stimulated IAPP secretion was reduced both acutely and after 48-hr exposure to leptin, whereas only minor effects were found on insulin release, i.e. an inhibition in islets cultured with 1 nM leptin. An acute inhibitory effect by 10 nM leptin was observed on the ratio of IAPP/insulin release at 5.6-11.1 mM glucose, but this was overcome by 16.7 mM glucose. The islet glucose oxidation rate was enhanced by 1 nM leptin, but decreased at higher concentrations of leptin in acute experiments. In contrast, glucose metabolism was not affected in long-term experiments. Moreover, leptin did not influence islet (pro)insulin synthesis or the cell replication rate after culture. In conclusion, we show that islet IAPP release seems to be more sensitive to leptin than is insulin release. The effect of leptin on islet hormone secretion is dependent on the glucose concentration. The regulation of hormone secretion seems to be dissociated from glucose metabolism, an effect previously described in islets after exposure to certain cytokines. Our data necessarily suggest that a previously proposed negative feedback loop between leptin and insulin can be counteracted by IAPP.     

Intact pancreatic islet function despite humoral xenorecognition in the pig-to-monkey combination

BACKGROUND: The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture. METHODS: Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed. RESULTS: Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered. CONCLUSIONS: Despite the fact that pig beta-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.     

Albuminuria after fetal pancreatic islet transplantation: a 10-year follow-up

AIM OF THE STUDY: The prevention of diabetic nephropathy is as yet an unresolved issue. The aim of our study was to assess the effects of transplantation of long-term cultured and cryopreserved fetal pancreas islets on metabolic control and the development of diabetic nephropathy. METHODS: Serum C-peptide, glucose, HbA1c, insulin requirements, urinary albumin excretion rate, and blood pressure of 10 insulin-dependent diabetic patients after transplantation were compared with a group of 27 insulin-dependent diabetic controls on insulin therapy only during a 10-year follow-up. RESULTS: In the first year after transplantation mean insulin requirement decreased from 53.6+/-2.2 to 35.8+/-1.2 units. C-peptide levels appeared (0.55+/-0.08 ng/ml) and remained detectable throughout the follow-up. Blood glucose and HbA1c were significantly (P<0.05) lower than in the controls. Mean albumin excretion rates of the transplant and the control groups during the follow up were 18.8+/-8.5 and 11.7+/-2.0, 16.6+/-6.6 and 14.0+/-2.3, 15.0+/-5.0 and 15.1+/-2.7, 15.3+/-7.5 and 20.4+/-4.2, 19.8+/-6.2 and 36.7+/-11.1, 11.7+/-3.6 and 51.3+/-14.6, 14.1+/-4.2 and 71.4+/-23.1, 22.7+/-8.6 and 92.0+/-28.1, 18.0+/-5.9 and 107.6+/-35.6, 21.7+/-11.0 and 101.5+/-29.3 microg/min respectively. From the 6th year the difference between the two groups was significant (P<0.001). In the transplant group initial mean systolic and diastolic blood pressure values were 132.0+/-3.3 and 81.5+/-1.5 mmHg, in the controls 130.4+/-3.4 and 79.6+/-1.6 mmHg respectively. Significant changes (P<0.05) of blood pressure during the follow-up or differences between the two groups were not observed. CONCLUSIONS: We conclude that fetal islet transplantation is effective in achieving good long-term diabetes control and in the prevention of diabetic nephropathy.     

Glucose turnover and insulin sensitivity in rats with pancreatic islet transplants

To study the metabolic effects of insulin derived from islet grafts, oral glucose tolerance (OGT) and glucose turnover were examined in streptozotocin-induced diabetic Lewis rats rendered normoglycemic by syngeneic islet grafts in the renal subcapsular space (REN), in REN with renal vein-to-mesenteric vein anastomosis (REN-RMA), in the liver (intrahepatic [IH]), or in a parahepatic omental pouch (POP) and compared with normal rats. Normal OGT was found at 1 month posttransplant in all animals receiving approximately 3,000 islets, with hyperinsulinemic responses in the REN group compared with the other groups, and with higher C-peptide responses in the IH group than in the other groups (P < 0.05 by one-way analysis of variance). Glucose turnover studies in the insulin-stimulated steady state (INS-SS; infusion of insulin at 10 pmol x kg(-1) x min(-1)) at 2 months posttransplant showed that whole body glucose disappearance rates (Rd) were similar in all groups, but the REN group had higher steady-state insulin levels than the other groups. Glucose infusion rates (GIRs) were lower in the REN and IH groups than in the other groups. Apparent endogenous glucose production (EGP) was not completely inhibited in the REN and IH groups, while complete inhibition was observed in the other groups. When INS-SS insulin levels were matched to the level in REN rats by increasing the insulin infusion rate to 20 pmol x kg(-1) x min(-1) in REN-RMA, IH, and normal rats, GIR and Rd were elevated, exceeding those values in REN rats, but GIR in IH rats was still lower than in REN-RMA and normal rats. Thus, 1) in the REN group, impairment of inhibition of EGP and of stimulation of Rd by exogenous insulin contribute to insulin resistance; 2) in the IH group, incomplete inhibition of EGP is the major determinant of insulin resistance; and 3) with portal delivery of insulin in the REN-RMA and POP groups, normal insulin sensitivity is preserved. The present study confirms that hepatic portal delivery of islet secretions is necessary for physiological regulation of glucose metabolism. The study also suggests the IH grafts do not provide physiological regulation of glucose metabolism, raising the question of whether the liver is an appropriate site for insulin-secreting tissue replacement therapy in diabetes.     

Use of islet cells in cell therapy

The results of our previous work [Hibino et al., Biochim. Biophys. Acta 1174 (1993) 162-170] suggested that a highly repetitive DNA component facilitates bending of the helix axis to be recognized by the nuclear scaffold proteins from rat liver, P123 and P130. In the present experiment, it was shown that binding of these proteins to such a repetitive DNA component from rat liver nuclei (370-bp XmnI fragment) is based on a cooperative mode of interaction, although the binding activity of P130 is much higher than that of P123. The immunoblot analysis with anti-phosphoamino acid antibodies suggested that phosphorylation of serine and threonine residues occurs on P123 and P130, but also of tyrosine residue(s) on P130. The phosphatase assay showed that phosphoryl groups on these proteins may be involved in altering the DNA binding activities of the proteins. Thus, the results in the present study imply that phosphorylation of a nuclear scaffold protein in addition to the degree of bending of the DNA helix axis plays an important role in anchoring chromatin to the nuclear scaffold and in construction of a higher-order chromatin structure.