Mark Meloche

Expression of the calcium-sensing receptor on human antral gastrin cells in culture.

The presence of the extracellular calcium-sensing receptor on human antral gastrin cells was investigated. Reverse transcription PCR using mRNA isolated from gastrin cell- enriched cell cultures identified a product with a sequence identical to part of the human parathyroid-secreting cell calcium-sensing receptor. Immunocytochemistry with an antibody to the extracellular region of the receptor immunostained all gastrin cells (but not mucin or somatostatin cells), and detected appropriate-sized bands in Western blots of whole cell lysates. Increasing extracellular calcium levels from 0.5 to 9 mM stimulated gastrin release in a concentration-dependent manner, with maximal release obtained at 7.2 mM. A known agonist of the calcium receptor, spermine also stimulated gastrin release. Microfluorimetry of identified gastrin cells demonstrated that increasing extracellular calcium resulted in an initial rapid rise in intracellular calcium followed by a plateau level that returned to basal levels immediately after removal of the elevated calcium. The traces were consistent with activation of a receptor-mediated mechanism rather than a concentration-dependent influx of calcium. In conclusion, these data indicate that G cells express the calcium-sensing receptor, and that activation of the receptor may explain the acid rebound phenomenon associated with calcium-containing antacid preparations.

Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy.

Background. The effect of islet cell transplantation (ICT) on the progression of diabetic microvascular complications is not well understood. Methods. We have conducted a prospective, crossover, cohort study comparing ICT with intensive medical therapy on the progression of diabetic nephropathy, retinopathy, and neuropathy. Results. The rate of decline in glomerular filtration rate is slower after ICT than on medical therapy. There was significantly more progression of retinopathy in medically treated patients than post-ICT. There was a nonsignificant trend for improved nerve conduction velocity post-ICT. Conclusions. ICT is associated with less progression of microvascular complications than intensive medical therapy. Multicenter, randomized trials are needed to further study the role of ICT in slowing the progression of diabetic complications.

Effect of exenatide on beta cell function after islet transplantation in type 1 diabetes.

Background. Islet transplantation can reduce or eliminate the need for insulin in patients with type 1 diabetes. Exenatide is a long acting analogue of Glucagon-like peptide-1 (GLP-1) that augments glucose induced insulin secretion, and may increase &bgr; cell mass. We evaluated the effect of exenatide on insulin secretion after islet transplantation. Methods. Eleven C-peptide positive islet cell recipients with elevated glucose levels were treated with exenatide for three months. Response was assessed by insulin requirements, meal tolerance tests, and hyperglycemic glucose clamps. Results. Ten patients responded to exenatide. Two patients who had not restarted insulin achieved good glycemic control and one patient who had received 5500 IE/kg in first islet infusion was able to stop insulin. Seven other patients decreased their insulin dose by 39% on exenatide. Hyperglycemic clamp studies showed a rise in second phase insulin release (before exenatide: 246±88 pM; during exenatide: 644±294 pM, P<0.01). Meal tolerance studies before and one month after stopping exenatide did not show a difference in glucose or C-peptide values. Nausea and vomiting were the major side effects. Conclusions. Exenatide stimulates insulin secretion in islet transplant recipients. It reduces insulin dose in some patients and may delay the need to resume insulin in others. We did not find any evidence of a trophic effect on islets.

The effect of medical therapy and islet cell transplantation on diabetic nephropathy : An interim report

Background. The effect of islet cell transplantation (ICT) on renal function in type 1 diabetes is uncertain and some recent studies report a significant decline in estimated glomerular filtration rate (GFR) and worsening of albuminuria. Methods. We are conducting a prospective crossover study comparing medical treatment with islet transplantation on the progression of diabetic complications, including renal function. The primary endpoint is change in GFR measured by 99mTc-diethylenetriaminepentaacetate with secondary endpoints including estimated GFR and albumin excretion. Results. We have followed 21 patients after islet transplantation a median of 29 months (range 13–45) and compared their results with medically treated patients followed a median 29.5 months (range 13–56). There is no difference in the rate of decline in measured GFR between medically treated patients (–0.35±0.89; 95% CI: –0.57 to –0.13 mL/min/month/1.73 m2) and those after ICT (–0.31±1.18; 95% CI: –0.61 to –0.01) and neither is significantly different from that expected for the general population. The rate of decline in our estimated GFR results is lower than that reported in other studies and we did not find any worsening of albuminuria. Conclusions. We do not find evidence of worsening of renal function after islet transplantation compared with medically treated patients.

Liposomal cyclosporine : characterization of drug incorporation and interbilayer exchange

A number of previous studies have examined the application of liposomes as carriers for the immunosuppressive agent cyclosporine. These studies, however, have generated equivocal results, particularly with regard to the therapeutic properties of such systems. In the present work, we have characterized cyclosporine incorporation into well defined liposomes, large unilamellar vesicles, and have examined the stability of drug association. Contrary to some earlier reports, we show that only modest levels of cyclosporine can be accommodated in the liposomal membrane and that the extent of drug incorporation is greatly reduced as the bilayer cholesterol content is increased. Furthermore, we demonstrate that cyclosporine, despite its hydrophobic character, can rapidly exchange between vesicles. This raises the possibility that, after i.v. administration, drug migration to other blood components might negate the potential benefits arising from liposomal delivery. In a companion paper, therefore (Choice et al., Transplantation, 1995, this issue), we have followed the pharmacokinetics and biodistribution of liposomal cyclosporine in a study that examined the behavior of both the drug and the liposomal carrier.

Liposomal Cyclosporine: Comparison Of Drug And Lipid Carrier Pharmacokinetics And Biodistribution

In a preceding paper (Ouyang et al., 1995, this issue), we have characterized cyclosporine incorporation into well-defined liposomal systems, large unilamellar vesicles. This study demonstrated that only modest drug levels could be accomodated within the membrane, particularly for cholesterol-containing liposomes, and that rapid drug exchange could occur between vesicles. This raised the possibility that following intravenous administration, drug migration to other blood components might negate the potential benefits arising from liposomal delivery. We have, therefore, examined the pharmacokinetics and biodistribution of both cyclosporine and its liposomal carrier. We show that whereas liposomes, as expected, are only slowly cleared from the blood, redistribution of cyclosporine occurs much more rapidly. Further we have shown that liposomal loss of cyclosporine in blood results from drug migration to the lipoproteins and, to a lesser extent, the erythrocytes. As a result, while liposomes accumulate preferentially in organs of the reticuloendothelial system after intravenous administration, tissue cyclosporine levels, in general, do not reflect the distribution profile obtained for the liposomal carrier.

B7-H4 expression in normal and diseased human islet β cells.

Objectives B7-H4 is a negative coregulatory molecule known to be involved in immune response. We study here B7-H4 expression and its possible role in diabetes and cancer development. Methods Formalin-fixed, paraffin-processed pancreas samples from patients with type 1 diabetes (T1D), insulinoma, pancreatic ductal adenocarcinoma (PDAC), and normal organ donors were studied by bright-field and multifluorescence immunohistochemistry to examine B7-H4 expression and its colocalization with islet endocrine hormones. Quantitative RT-PCR and Western blot assay were used to examine B7-H4 mRNA and protein expression in the islet and exocrine tissues from normal donors and pancreatic cancer cell lines. Results B7-H4 protein expression in islet &bgr; cells is decreased in T1D and PDAC, but increased in insulinoma patients when compared to normal controls; the changes in B7-H4 expression are concomitant with insulin expression on the islet &bgr; cells. The insulin/B7-H4 colocalization on the &bgr; cells, expressed in colocalization coefficient Pearson r, is also changed in these islets. Conclusions Our observation of altered B7-H4 expression, concomitant with insulin expression, in the pancreatic islets of T1D, PDAC, and insulinoma patients when compared to normal controls suggests that B7-H4 pathway might play an important role in maintenance of &bgr;-cell function, but its exact role remains to be explored.

B7-H4 Pathway in Islet Transplantation and β-Cell Replacement Therapies

Type 1 diabetes (T1D) is a chronic autoimmune disease and characterized by absolute insulin deficiency. β-cell replacement by islet cell transplantation has been established as a feasible treatment option for T1D. The two main obstacles after islet transplantation are alloreactive T-cell-mediated graft rejection and recurrence of autoimmune diabetes mellitus in recipients. T cells play a central role in determining the outcome of both autoimmune responses and allograft survival. B7-H4, a newly identified B7 homolog, plays a key role in maintaining T-cell homeostasis by reducing T-cell proliferation and cytokine production. The relationship between B7-H4 and allograft survival/autoimmunity has been investigated recently in both islet transplantation and the nonobese diabetic (NOD) mouse models. B7-H4 protects allograft survival and generates donor-specific tolerance. It also prevents the development of autoimmune diabetes. More importantly, B7-H4 plays an indispensable role in alloimmunity in the absence of the classic CD28/CTLA-4 : B7 pathway, suggesting a synergistic/additive effect with other agents such as CTLA-4 on inhibition of unwanted immune responses.

Improving Islet Engraftment by Gene Therapy

Islet cell transplantation is currently the only feasible long-term treatment option for patients with type 1 diabetes. However, the majority of transplanted islets experience damage and apoptosis during the isolation process, a blood-mediated inflammatory microenvironment in the portal vein upon islet infusion, hypoxia induced by the low oxygenated milieu, and poor-revascularization-mediated lack of nutrients, and impaired hormone modulation in the local transplanted site. Strategies using genetic modification methods through overexpression or silencing of those proteins involved in promoting new formation of blood vessels or inhibition of apoptosis may overcome these hurdles and improve islet engraftment outcomes.

Inhibition of Bombesin-Stimulated Gastrin Release from Isolated Human G Cells by Bombesin Analogs

This study investigated the ability of 6 putative bombesin (BN) antagonists to inhibit BN-stimulated gastrin release from human antral G cells maintained in culture for 48 h. The analogs studied comprised different sequence changes based around a constant 6-amino-acid sequence from the C-terminal of the peptide. At concentrations of 1.0 mumol/l, analogs 1 and 2 stimulated gastrin release 3-fold above basal. The remaining 4 analogs showed no agonistic activity. After the addition of concentrations of 1.0 mumol/l against a BN concentration of 10.0 nmol/l the following levels of inhibition were obtained: analog 3, 90 +/- 1.4%; analog 4, 95 +/- 0.5%; analog 5, 99 +/- 2.4%, and analog 6, 85 +/- 3.8%. The 2 most effective analogs were analog 3, which was 9 amino acids in length with substitutions of two D-phenylalanine residues and a psi-leucine bond [D-Phe6-psi-Leu13-D-Cpa14-BN(6-14)NH2], and analog 5, which was 8 amino acids in length with a methyl ester at the C-terminus and a single D-phenylalanine substitution at the N-terminus [D-Phe6-BN(6-13)OMe]. These results suggest that the BN receptor present on the human antral G cells differs from that on guinea pig acinar cells and canine G cells, being less sensitive to C-terminal structural modifications.