In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells.

AIMS/HYPOTHESIS: Transplantation of insulin-producing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulin-producing cells from adult rat exocrine cells cultured in the presence of growth factors. METHODS: Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15. RESULTS: In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulin-immunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft. CONCLUSIONS/INTERPRETATION: Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.

Netrin-1 expression in fetal and regenerating rat pancreas and its effect on the migration of human pancreatic duct and porcine islet precursor cells.

AIMS/HYPOTHESIS: We investigated the expression and function of netrin-1, a diffusible laminin-like protein known to regulate neuronal-cell migration in the pancreas. We questioned whether this factor regulates migration of pancreatic epithelial cells and whether this could be involved in islet neogenesis. METHODS: We studied fetal and adult rat pancreas wherein duct ligation induced islet neogenesis. Netrin-1 expression was analysed by RT-PCR, western blot and immunohistochemistry. In vitro cell migration was measured with a human pancreatic duct cell line (CAPAN-2) and with fetal porcine islet cells. We also studied the expression of two netrin-receptors, neogenin and deleted in colorectal cancer. RESULTS: We found a transient expression of netrin-1 mRNA and protein in fetal pancreas from E15 to E18, and in adult pancreas after duct ligation. In normal adult pancreas there was very little netrin-1 expression. Netrin-1 expression was observed both in endocrine and exocrine cells. At the immunohistochemical level, it was expressed by islet cells during tissue regeneration. We could show that netrin-1 increases the migration of fetal islet cells and of a ductal cell line, mainly via a chemokinetic effect. From the two well-established netrin receptors, DCC and neogenin, we only found neogenin to be expressed in the pancreas. Neogenin expression coincided with the period of netrin-1 up-regulation. CONCLUSION/INTERPRETATION: Netrin-1 is involved in pancreatic morphogenesis and tissue remodelling and plays a role in the regulation of duct-cell and fetal-islet cell migration. This can be of importance in islet regeneration, where migration of islet precursors takes place.

Interleukin (IL)-6, IL-8 and IL-10 in serum and CSF of Trypanosoma brucei gambiense sleeping sickness patients before and after treatment.

Serum and cerebrospinal fluid (CSF) concentrations of interleukin (IL)-6, IL-8, IL-10, tumour necrosis factor-alpha and interferon-gamma were determined in 46 Trypanosoma brucei gambiense sleeping sickness patients in DR Congo, before and after treatment. According to their CSF cell number before treatment, patients were classified as early-stage (0-5 cells/microL), intermediate-stage (6-20 cells/microL) or late-stage patients (> 20 cells/microL). In serum, slightly higher IL-8 concentrations were found in early-stage patients compared to intermediate- or late-stage patients. These high IL-8 levels dropped after treatment. Higher IL-10 concentrations were detected in serum of patients in intermediate or late stage compared to early-stage patients. In both intermediate- and late-stage groups, serum IL-10 decreased after treatment. In CSF, elevated concentrations of IL-6, IL-8 and especially of IL-10 were observed in late-stage T. b. gambiense patients. After treatment, these concentrations dropped to levels similar to those of the other patients. Tumour necrosis factor-alpha was detected only in a few serum and CSF samples, which were scattered over the different patient groups. Interferon-gamma was detected in serum of 5 patients and remained undetectable in CSF.

Mitogenic effect of gastrin and expression of gastrin receptors in duct-like cells of rat pancreas.

BACKGROUND & AIMS: Ductular metaplastic cells are observed during pancreas injury. Growth control by gastrin and expression of gastrin/cholecystokinin (CCK) B receptors were evaluated in these cells. METHODS: Acinoductal transdifferentiation was induced in vitro by culturing of acinar cells, and ductular metaplasia was obtained in vivo by ligation of the pancreatic ducts. Mitogenic effects of gastrin I on ductal complexes in vivo and of tetragastrin, pentagastrin, and gastrin I and II, with or without the CCK-B receptor antagonist L-365,260, on duct-like cells in vitro were analyzed by 5-bromo-2'-deoxyuridine labeling. Immunocytochemistry, Western blotting, and reverse-transcription polymerase chain reaction were applied for detection of the CCK-B receptor. RESULTS: Gastrin analogues induced a mitogenic stimulus in the duct-like cells in vitro and in ductal complexes in duct-ligated rat pancreas. Immunocytochemistry showed expression of CCK-B receptors in these models and in fetal but not normal adult exocrine pancreas. Additionally, up-regulation of CCK-B receptors during ductular metaplasia was shown by Western blotting and reverse-transcription polymerase chain reaction. CONCLUSIONS: Duct-like pancreatic epithelial cells in vitro and ductal complexes in vivo express gastrin/CCK-B receptors and proliferate in response to gastrin.

Factors affecting caries experience in French adolescents.

Three hundred French 14-15-yr-old adolescents were randomly selected. They were examined clinically and caries experience was determined by using the DMFS index. The aim of this investigation was to evaluate children's habits using a self-administered questionnaire, to clarify the actual influence of well-known factors such as fluoride exposure, diet, oral hygiene and socioeconomic factors on caries experience and to stress those factors of primary importance. A multiple regression analysis revealed the variables which significantly contributed to explain DMFS scores in a final model: Age, sex, frequency of sweet consumption, use of standard or high fluoride toothpastes, bleeding during toothbrushing, living in St Yorre (F- = 0.45 mg/l). At a time when caries experience is decreasing, it seems that fluoride supply, snacking and oral hygiene are still independent and significant determinants of caries experience in French adolescents.