The glucagon-like peptide-1 receptor agonist exenatide restores impaired pro-islet amyloid polypeptide processing in cultured human islets: implications in type 2 diabetes and islet transplantation.

AIMS/HYPOTHESIS: Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), is associated with beta cell death in type 2 diabetes as well as in cultured and transplanted human islets. Impaired prohIAPP processing due to beta cell dysfunction is implicated in hIAPP aggregation. We examined whether the glucagon-like peptide-1 receptor (GLP-1R) agonist exenatide can restore impaired prohIAPP processing and reduce hIAPP aggregation in cultured human islets and preserve beta cell function/mass during culture conditions used in clinical islet transplantation. METHODS: Isolated human islets (n = 10 donors) were cultured with or without exenatide in normal or elevated glucose for 2 or 7 days. Beta cell apoptosis, proliferation, mass, function, cJUN N-terminal kinase (JNK) and protein kinase B (PKB) activation and amyloid formation were assessed. ProhIAPP, its intermediates and mature hIAPP were detected. RESULTS: Exenatide-treated islets had markedly lower JNK and caspase-3 activation and beta cell apoptosis, resulting in higher beta/alpha cell ratio and beta cell area than non-treated cultured islets. Exenatide improved beta cell function, manifested as higher insulin response to glucose and insulin content, compared with non-treated cultured islets. Phospho-PKB immunoreactivity was detectable in exenatide-treated but not untreated cultured islets. Islet culture caused impaired prohIAPP processing with decreased mature hIAPP and increased NH(2)-terminally unprocessed prohIAPP levels resulting in higher release of immature hIAPP. Exenatide restored prohIAPP processing and reduced hIAPP aggregation in cultured islets. CONCLUSIONS/INTERPRETATION: Exenatide treatment enhances survival and function of cultured human islets and restores impaired prohIAPP processing in normal and elevated glucose conditions thereby reducing hIAPP aggregation. GLP-1R agonists may preserve beta cells in conditions associated with islet amyloid formation.

Mutation spectrum and predicted function of laforin in Lafora's progressive myoclonus epilepsy.

BACKGROUND: Lafora's disease is a progressive myoclonus epilepsy with pathognomonic inclusions (polyglucosan bodies) caused by mutations in the EPM2A gene. EPM2A codes for laforin, a protein with unknown function. Mutations have been reported in the last three of the gene's exons. To date, the first exon has not been determined conclusively. It has been predicted based on genomic DNA sequence analysis including comparison with the mouse homologue. OBJECTIVES: 1) To detect new mutations in exon 1 and establish the role of this exon in Lafora's disease. 2) To generate hypotheses about the biological function of laforin based on bioinformatic analyses. METHODS: 1) PCR conditions and components were refined to allow amplification and sequencing of the first exon of EPM2A. 2) Extensive bioinformatic analyses of the primary structure of laforin were completed. RESULTS: 1) Seven new mutations were identified in the putative exon 1. 2) Laforin is predicted not to localize to the cell membrane or any of the organelles. It contains all components of the catalytic active site of the family of dual-specificity phosphatases. It contains a sequence predicted to encode a carbohydrate binding domain (coded by exon 1) and two putative glucohydrolase catalytic sites. CONCLUSIONS: The identification of mutations in exon 1 of EPM2A establishes its role in the pathogenesis of Lafora's disease. The presence of potential carbohydrate binding and cleaving domains suggest a role for laforin in the prevention of accumulation of polyglucosans in healthy neurons.

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.

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 accommodated 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.

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.

Proportions of diether, macrocyclic diether, and tetraether lipids in Methanococcus jannaschii grown at different temperatures.

Growth of Methanococcus jannaschii over a wide temperature range (47 to 75 degrees C) is correlated with an ability to alter dramatically the proportions of three ether lipid cores. These lipids shifted from predominantly diether (2,3-di-O-phytanyl-sn-glycerol) at the lower growth temperatures to macrocyclic diether and tetraether at near optimal growth temperatures. Lipid head groups varied as well, especially with respect to an increase in phosphate at the higher temperatures.

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.

Generation of allospecific cytolytic T-lymphocytes stimulated by pure pancreatic beta-cells in absence of Ia+ dendritic cells.

A murine mixed islet-lymphocyte coculture system (MILC) was used to quantitate the immunogenicity of a pure population of pancreatic beta-cells to more clearly define whether stimulator major histocompatibility complex (MHC) class II-positive dendritic cells are a major component leading to islet immunogenicity. Pancreatic beta-cells express MHC class I antigen but not class II antigen. These experiments compared the in vitro immunogenicity of fluorescence-activated cell sorted (FACS-IV) pure beta-cells (MHC class I-positive cells only) relative to unpurified dispersed islet cells (MHC class I-positive cells and class II-positive cells). The results demonstrated the surprising finding that pure DBA/2J (H-2d) pancreatic beta-cells stimulated a strong cytotoxic T-lymphocyte (CTL) response when exposed to C57BL/6 (H-2b) allosplenocytes in the MILC, similar to DBA/2J nonpurified dispersed islet cells. Furthermore, the stimulation of CTL by both purified beta-cells and nonpurified dispersed islet cells was blocked by addition of MHC-specific anti-class I monoclonal antibody directed against stimulator MHC antigen. The data imply that the highly immunogenic MHC class II-positive passenger leukocytes present in the islets were not necessary for the generation of the immune response in the presence of MHC class I-positive beta-cells. Although most of the pretreatment regimens attempting to decrease islet immunogenicity have been directed at eliminating the MHC class II-positive passenger leukocytes from the islets, this work suggests that modulation of MHC class I antigen may be an important approach.

In vitro generated allospecific cytolytic T lymphocytes injure pancreatic islets.

The role of cytotoxic T lymphocytes in the rejection of pancreatic islet allografts remains poorly defined. The present study was designed to assess the ability of in vitro generated cytolytic T lymphocytes to produce allospecific functional and structural damage of mouse pancreatic islets. A mixed lymphocyte-islet coculture model (MLIC) has been developed, in which islets from DBA/2J mice (H-2d) stimulate the generation of allospecific cytolytic T lymphocytes (C57B1/6, H-2b), as measured by lysis of allospecific chromium-labeled tumor targets. Responder C57B1/6 splenocytes sensitized to DBA/2J islets were harvested from the MLIC on Day 5 and cocultured with either freshly isolated DBA/2J or B10.BR (H-2K) islets. Islet injury was determined by assessment of beta cell function after 8 hr (as measured by insulin release in response to a glucose challenge) and islet destruction after 24 hr of coculture with the sensitized splenocytes. Whereas coculture of third party B10.BR islets with MLIC-sensitized C57B1/6 anti-DBA splenocytes had no effect on insulin release or structure, incubation of allospecific DBA/2J islets with these splenocytes resulted in inhibition of insulin release after 8 hr and disintegration of the islets by 24 hr. The depletion of MLIC-sensitized C57B1/6 anti-DBA splenocytes with anti-Lyt2 monoclonal antibody, but not anti-L3T4 monoclonal antibody, prevented the allospecific destruction of fresh islets by the splenocytes in culture. This study suggests that allospecific, cytotoxic T lymphocytes may play an important role in the effector mechanism of pancreatic islet allograft destruction.

The patient and the dying psychiatrist.

In the last few years a great deal of attention has been paid to the dying patient and his relatives, but very little to the illness of the psychiatrist, particularly when it is of a terminal nature. This paper is about Jean Caron, M.D., a psychiatrist who suffered from leukemia, and his patients some of whom came to learn about it in an indirect fashion and others in a more open way. The knowledge of his illness and oncoming death, while first experienced as severe shock, became therapeutically useful in relieving previous losses and abandonments and completing unfinished mourning. It appears possible to mourn a person who is still living and through his help, mourn previous losses while this person is mourning his own life.

[To lock up madness.]. / Enfermer la folie.

In New France, Madness has initially been considered according to European traditions : lunatics had to be excluded, taken out of sight. Very soon though, a distinction was made between the function of protection, attributed to general hospitals and later to asylums, and the function of treatment, undertaken by the Hôtel-Dieu (fore-shadowing the role played by general hospitals in the twentieth century). In 1960, changing concepts in the fields of the aetiology and treatment of mental illness, the hugeness of mental hospitals and the transformation of Quebec society from rural to urban hasten the re-organization of psychiatric services, without eliminating the risk of excluding the mentally ill. This paper also describes briefly psychiatric hospitals and their dependencies in the early sixties in Quebec.