Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

Functional insulin receptors are known to occur in pancreatic beta cells; however, except for a positive feedback on insulin synthesis, their physiological effects are unknown. Amperometric measurements at single, primary pancreatic beta cells reveal that application of exogenous insulin in the presence or absence of nonstimulatory concentrations of glucose evokes exocytosis mediated by the beta cell insulin receptor. Insulin also elicits increases in intracellular Ca 2؉ concentration in beta cells but has minimal effects on membrane potential. Conditions where the insulin receptor is blocked or cell surface concentration of free insulin is reduced during exocytosis diminishes secretion induced by other secretagogues, providing evidence for direct autocrine action of insulin upon secretion from the same cell. These results indicate that the beta cell insulin receptor can mediate positive feedback for insulin secretion. The presence of a positive feedback mechanism for insulin secretion mediated by the insulin receptor provides a potential link between impaired insulin secretion and insulin resistance.

The effects of extracellular Zn 2؉ and pH and intravesicular pH on insulin and 5-hydroxytryptamine (5-HT) secretion from pancreatic beta cells were investigated. Insulin and 5-HT secretion from single cells was detected by amperometry as a series of current spikes corresponding to detection of multimolecular packets secreted by exocytosis. Spike width was used as a measure of the kinetics of clearance from the cell and the area of spikes as a measure of amount released. Changes in extracellular pH from 6.9 to 7.9 caused insulin spikes to become narrower with no change in area, whereas the same treatments had no effect on 5-HT secretion. Treatment of cells with Bafilomycin A 1 or N-ethylmaleimide, both of which are expected to increase intravesicular pH by inhibiting V-type H ؉ -ATPase, had no effect on 5-HT secretion but caused insulin spikes to become more narrow. These results indicate that exposure to high pH, whether intravesicular or extracellular, accelerates release of insulin during exocytosis without affecting the amount of insulin released. Increasing extracellular Zn 2؉ concentration from 0 to 25 M increased the width and decreased the area of insulin spikes without affecting 5-HT secretion. Zn 2؉ effects were likely exerted through a common-ion effect on Zn 2؉ -insulin dissociation. It was concluded that intravesicular storage conditions and extracellular ions can affect free insulin concentration in the vicinity of beta cells during secretion.

Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet beta-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the beta-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 mug/ml), gastrin (1.0 mug/ml), or the combination of EGF and gastrin. beta-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (-3%) or control medium (-62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, beta-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased beta-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases beta-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of beta-cell neogenesis from pancreatic exocrine duct cells.

Rodent models have been essential to understanding the immune-mediated failure of aortic valve allografts (AVAs). Decellularization has been proposed to reduce the immunogenicity of AVAs. The objective of this study was to determine the most effective method to decellularize AVAs for use in a rat model. Three different decellularization techniques were compared in Lewis aortic valves. Detergent decellularization involved a series of hypotonic and hypertonic Tris buffers at 4 degrees C for 48 h/buffer containing 0.5% Triton X-100 followed by a 72 h washout in phosphate-buffered saline. Osmotic decellularization was performed in similar manner to the detergent-based technique except without the addition of Triton X-100. Enzymatic decellularization consisted of trypsin/EDTA at 37 degrees C for 48 h. Assessment was performed with light microscopy (H&E, Movat's pentachrome), immunohistochemistry for residual cellular elements, and hydroxyproline assays. Detergent-based methodology effected near-complete decellularization of both the leaflets and aortic wall in addition to preservation of the extracellular matrix (ECM). Osmotic lysis was associated with preservation of ECM and moderate decellularization. Enzymatic decellularization resulted in complete decellularization but extensive degeneration and fragmentation of the ECM. When implanted into the infrarenal aorta of allogeneic rats for 1 week, valves decellularized with detergent-based and osmotic methodology failed to stimulate an allogeneic immune response as evidenced by an absence of T cell infiltrates. Osmotic lysis protocols with low dose detergent appear to be most effective at both removing antigenic cellular elements and preserving ECM.

Islet allotransplantation can provide prolonged insulin independence in selected individuals with type 1 diabetes. Whether islet transplantation also restores hypoglycemic counterregulation is unclear. To determine if hypoglycemic counterregulation is restored by islet transplantation, we studied hormone responses and hypoglycemic symptom recognition in seven insulinindependent islet transplant recipients using a 3-h stepped hypoglycemic clamp, and compared their responses to those of nontransplanted type 1 diabetic subjects and nondiabetic control subjects. Glucagon responses of islet transplant recipients to hypoglycemia were significantly less than that observed in control subjects (incremental glucagon [mean ؎ SE]: ؊12 ؎ 12 vs. 64 ؎ 22 pg/ml, respectively; P < 0.05), and not significantly different from that of nontransplanted type 1 diabetic subjects (؊17 ؎ 10 pg/ml). Epinephrine responses and symptom recognition were also not restored by islet transplantation (incremental epinephrine [

A human pancreatic beta-cell line that is functionally equivalent to primary beta-cells has not been available. We established a reversibly immortalized human beta-cell clone (NAKT-15) by transfection of primary human beta-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed beta-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

The combination of pharmacokinetic and pharmacodynamic (measurement of the biological effect) monitoring of immunosuppressive drugs provides a method for the optimization of drug dosing. We chose to investigate this using mycophenolic acid (MPA), an immunosuppressive drug that mediates its effect by the inhibition of inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo biosynthesis of purines. Using an assay developed for measurement of IMPDH activity in whole blood, the concentration required for 50% inhibition of IMPDH activity was approximately 200 mg/L (58 +/- 8.3% for whole blood [n = 6] and 55 +/- 10.0% for isolated lymphocytes). To ascertain the relationship between MPA concentration and IMPDH inhibition in vivo, dogs were administered a single dose of mycophenolate mofetil, the pro-drug of MPA, at 20 or 40 mg/kg orally. Pharmacokinetic analysis revealed that the Cmax of the 40-mg/kg group was statistically greater than that of the 20-mg/kg group (P < 0.05). There were no statistical differences in the other parameters investigated (area under the curve, beta half-life, mean residence time, volume of distribution at steady state, and clearance) between the two treatment groups. The half-life was calculated at approximately 8 hr for both dose groups. There was also substantial variability among the dogs in the absorption and clearance of MPA. An inverse relationship was found between the MPA concentration and IMPDH. Maximal inhibition of IMPDH activity of 30-40% occurs approximately 2-4 hr after dosing, followed by a gradual restoration in enzyme activity. After 24 hr, there is an increase in IMPDH activity that exceeds the pre-dosing levels in some cases by 3-fold. Evaluation of the pharmacokinetic and the pharmacodynamic responses to MPA in the canine model suggests that the drug should be administered ever 8 hr to optimize its immunosuppressive efficacy. This combined approach can be used for optimization of doses of this and other immunosuppressive drugs.