In order to design a feasible somatic cell gene delivery sysgogues. While glucose responsiveness commenced at a tem for the treatment of type I diabetes, a suitable cell type lower concentration than normal islets, a secretion curve needs to be determined. We have previously shown that approaching normal physiological conditions was generthe stable transfection of the full-length insulin cDNA into ated. Immunoelectron microscopy revealed the presence the human liver cell line, (HEP G2ins) resulted in synthesis, of insulin-containing granules, similar in size and appearstorage and acute regulated release of insulin to analogues ance to those of the normal beta cell. These results demof cAMP, but not to the physiological stimulus glucose. In onstrate that while it is most likely that the HEP G2ins/g attempting to explain the lack of glucose responsiveness cell line predominantly secretes insulin via the constitutive of the HEP G2ins cells we have stably transfected these pathway, significant acute regulated release was seen in cells with the human islet glucose transporter GLUT 2 response to glucose, and thus represents significant pro-(HEP G2ins/g cells). The HEP G2ins/g cell clones exhibit gress in the creation of a genetically engineered 'artificial glucose-stimulated insulin secretion and glucose potentibeta cell' from a human hepatocyte cell line. ation of the secretory response to nonglucose secreta-
We describe a 21 month old male infant who presented with failure to thrive associated with severe hypokalaemia and metabolic acidosis, together with hypomagnesaemia. Evaluation revealed marked renal and probable faecal potassium wasting, distal renal tubular acidosis, mild urinary magnesium wasting, and a normal gastric pH ( (Arch Dis Child 2001;84:504-507)
Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14-20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 +/- 13 and 43 +/- 8%, respectively, with the number of beta-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6-1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of beta-cells for clinical transplantation.
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