A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. This need for transplantable human islets has stimulated ...efforts to expand existing pancreatic islets and/or grow new ones. To test the hypothesis that human adult duct tissue could be expanded and differentiated in vitro to form islet cells, digested pancreatic tissue that is normally discarded from eight human islet isolations was cultured under conditions that allowed expansion of the ductal cells as a monolayer whereupon the cells were overlaid with a thin layer of Matrigel. With this manipulation, the monolayer of epithelial cells formed three-dimensional structures of ductal cysts from which 50-to 150-μ m diameter islet-like clusters of pancreatic endocrine cells budded. Over 3-4 weeks culture the insulin content per flask increased 10- to 15-fold as the DNA content increased up to 7-fold. The cultivated human islet buds were shown by immunofluorescence to consist of cytokeratin 19-positive duct cells and hormone-positive islet cells. Double staining of insulin and non-β cell hormones in occasional cells indicated immature cells still in the process of differentiation. Insulin secretion studies were done over 24 h in culture. Compared with their basal secretion at 5 mM glucose, cysts/cultivated human islet buds exposed to stimulatory 20 mM glucose had a 2.3-fold increase in secreted insulin. Thus, duct tissue from human pancreas can be expanded in culture and then be directed to differentiate into glucose responsive islet tissue in vitro. This approach may provide a potential new source of pancreatic islet cells for transplantation.
Background & Aims: The multidrug resistance (MDR) gene codes for a drug efflux pump P-glycoprotein 170 (Pgp-170) expressed on the surface of lymphocytes and intestinal epithelial cells. Inflammatory ...bowel disease (IBD) poorly responsive to medical therapy may relate to MDR expression because glucocorticoids are known Pgp-170 substrates.
Methods: Using flow cytometry, we measured peripheral blood lymphocyte (PBL) MDR in 153 IBD patients and 50 healthy volunteers, and assessed the relationship between PBL, mucosal intraepithelial lymphocyte (IEL), and mucosal epithelial cell (EC) MDR expression in a further 20 IBD patients and 19 controls.
Results: Compared with controls, PBL MDR was significantly elevated in patients with Crohn's disease who required bowel resection for failed medical therapy (mean ± SEM, 26.7 ± 2.8 vs. 11.9 ± 1.0;
P <0.0001) and patients with ulcerative colitis who required proctocolectomy for failed medical therapy (20.3 ± 2.5 vs. 11.9 ± 1.0;
P = 0.001). PBL MDR remained stable over time and was not influenced by disease activity or glucocorticoid therapy. Both PBL and mucosal MDR expression appeared independent of disease activity, and there was a significant correlation between PBL MDR expression and both IEL expression (
r = 0.92;
P < 0.0001) and EC expression (
r = 0.54;
P < 0.001).
Conclusions: PBL and mucosal MDR expression may play an important role in determining the response of IBD patients to glucocorticoid therapy.
GASTROENTEROLOGY 2000;118:279-288
Fortification of food with folic acid to prevent neural-tube defects in babies also lowers plasma total homocysteine, which is a risk factor for vascular disease. We investigated the effect of folate ...and vitamin B12 on homocysteine concentrations. 30 men and 23 women received sequential supplementation with increasing doses of folic acid. After supplementation, the usual dependency of homocysteine on folate diminished, and vitamin B12 became the main determinant of plasma homocysteine concentration. This finding suggests that a fortification policy based on folic acid and vitamin B12, rather than folic acid alone, is likely to be much more effective at lowering of homocysteine concentrations, with potential benefits for reduction of risk of vascular disease.
Aims/hypothesis
We assessed whether per-arnt-sim (PAS) domain-containing protein kinase (PASK) is involved in the regulation of glucagon secretion.
Methods
mRNA levels were measured in islets by ...quantitative PCR and in pancreatic beta cells obtained by laser capture microdissection. Glucose tolerance, plasma hormone levels and islet hormone secretion were analysed in C57BL/6
Pask
homozygote knockout mice (
Pask
−/−
) and control littermates. Alpha-TC1-9 cells, human islets or cultured E13.5 rat pancreatic epithelia were transduced with anti-
Pask
or control small interfering RNAs, or with adenoviruses encoding enhanced green fluorescent protein or PASK.
Results
PASK
expression was significantly lower in islets from human type 2 diabetic than control participants.
PASK
mRNA was present in alpha and beta cells from mouse islets. In
Pask
−/−
mice, fasted blood glucose and plasma glucagon levels were 25 ± 5% and 50 ± 8% (mean ± SE) higher, respectively, than in control mice. At inhibitory glucose concentrations (10 mmol/l), islets from
Pask
−/−
mice secreted 2.04 ± 0.2-fold (
p
< 0.01) more glucagon and 2.63 ± 0.3-fold (
p
< 0.01) less insulin than wild-type islets. Glucose failed to inhibit glucagon secretion from PASK-depleted alpha-TC1-9 cells, whereas
PASK
overexpression inhibited glucagon secretion from these cells and human islets. Extracellular insulin (20 nmol/l) inhibited glucagon secretion from control and PASK-deficient alpha-TC1-9 cells. PASK-depleted alpha-TC1-9 cells and pancreatic embryonic explants displayed increased expression of the preproglucagon (
Gcg
) and AMP-activated protein kinase (AMPK)-alpha2 (
Prkaa2
) genes, implying a possible role for AMPK-alpha2 downstream of PASK in the control of glucagon gene expression and release.
Conclusions/interpretation
PASK is involved in the regulation of glucagon secretion by glucose and may be a useful target for the treatment of type 2 diabetes.
Oxidative stress, which is found in pancreatic β-cells in the diabetic state, suppresses insulin gene transcription and secretion, but the signaling pathways involved in the β-cell dysfunction ...induced by oxidative stress remain unknown. In this study, subjecting rat islets to oxidative stress activates JNK, p38 MAPK, and protein kinase C, preceding the decrease of insulin gene expression. Adenovirus-mediated overexpression of dominant-negative type (DN) JNK, but not the p38 MAPK inhibitor SB203580 nor the protein kinase C inhibitor GF109203X, protected insulin gene expression and secretion from oxidative stress. Moreover, wild type JNK overexpression suppressed both insulin gene expression and secretion. These results were correlated with changes in the binding of the important transcription factor PDX-1 to the insulin promoter; adenoviral overexpression of DN-JNK preserved PDX-1 DNA binding activity in the face of oxidative stress, whereas wild type JNK overexpression decreased PDX-1 DNA binding activity. Furthermore, to examine whether suppression of the JNK pathway can protect β-cells from the toxic effects of hyperglycemia, rat islets were infected with DN-JNK expressing adenovirus or control adenovirus and transplanted under renal capsules of streptozotocin-induced diabetic nude mice. In mice receiving DN-JNK overexpressing islets, insulin gene expression in islet grafts was preserved, and hyperglycemia was ameliorated compared with control mice. In conclusion, activation of JNK is involved in the reduction of insulin gene expression by oxidative stress, and suppression of the JNK pathway protects β-cells from oxidative stress.
Transplantation of islet cells is an effective treatment for type 1 diabetes with critically labile metabolic control. However, during islet isolation, blood supply is disrupted, and the transport of ...nutrients/metabolites to and from the islet cells occurs entirely by diffusion. Adequate oxygen supply is essential for function/survival of islet cells and is the limiting factor for graft integrity. Recently, we developed an immunoisolated chamber system for transplantation of human islets without immunosuppression. This system depended on daily oxygen supply. To provide independence from this external source, we incorporated a novel approach based on photosynthetically-generated oxygen. The chamber system was packed sandwich-like with a slab of immobilized photosynthetically active microorganisms (Synechococcus lividus) on top of a flat light source (LEDs, red light at 660 nm, intensity of 8 μE/m(2)/s). Islet cells immobilized in an alginate slab (500-1,000 islet equivalents/cm(2)) were mounted on the photosynthetic slab separated by a gas permeable silicone rubber-Teflon membrane, and the complete module was sealed with a microporous polytetrafluorethylene (Teflon) membrane (pore size: 0.4 μm) to protect the contents from the host immune cells. Upon illumination, oxygen produced by photosynthesis diffused via the silicone Teflon membrane into the islet compartment. Oxygen production from implanted encapsulated microorganisms was stable for 1 month. After implantation of the device into diabetic rats, normoglycemia was achieved for 1 week. Upon retrieval of the device, blood glucose levels returned to the diabetic state. Our results demonstrate that an implanted photosynthetic bioreactor can supply oxygen to transplanted islets and thus maintain islet viability/functionality.
Aims/hypothesis The process of islet isolation can cause chemical and mechanical injury to beta cells. In addition, hyperglycaemia after islet transplantation can compromise beta cell function. The ...aim of this experiment was to evaluate changes in gene expression in endogenous islets using laser-capture microdissection (LCM). Materials and methods Islets from B6AF1 mice were studied in situ in the pancreas as well as those freshly isolated or cultured for 24 h. Fresh islets were transplanted under the kidney capsule of syngeneic diabetic (streptozocin-induced) and non-diabetic mice. Frozen sections from all the samples were prepared for LCM to obtain beta cell-enriched tissue; RNA was extracted and amplified using T7 polymerase. RT-PCR was used to assess expression of selected genes critical for beta cell function (Ins, Ipf1 previously known as Pdx1, Slc2a2 previously known as GLUT2 and Ldha) and the stress response (Hmox1 previously known as HO-1, Gpx1, Tnfaip3 previously known as A20 and Fas). Immunostaining was also performed. Results In freshly isolated and cultured islets, insulin and Ipf1 mRNA levels were decreased by 40% (compared with islets in situ), while stress genes were upregulated. Comparison between in situ pancreatic islets and engrafted beta cells of cured mice showed declines in Ipf1 expression. Conclusions/interpretation Our experiment, the first report to investigate changes in gene expression in endogenous islets using LCM, indicate that beta cells following islet isolation and residing in a foreign graft environment have decreased expression of genes involved in insulin production and increased expression of stress genes. Our data suggest that an islet graft, even in successful transplantation, may be different from endogenous islets in gene expression.
When realising future fusion reactors, their stationary burning must be maintained and the heat flux to the divertor must be reduced. This essentially requires a stationary internal transport barrier ...(ITB) plasma with a fast control system. However, the time scale for determining the position of the foot point of an ITB is not clearly understood even though its understanding is indispensable for fast profile control. In this study, the foot point of the electron ITB (eITB) was observed to be reformed at the vicinity of a magnetic island when the island started to form. In addition, the enhanced confinement region was observed to expand during the eITB formation according to the radial movement of the magnetic island toward the outer region. Compared to the time scales of the local heat transport, the faster time scales of the movement of the eITB foot point immediately after island formation (~0.5 ms) suggest the importance of the magnetic island for plasma profile control to maintain stationary burning.
Evidence is presented that nanosized particles of crystals do not necessarily adopt a periodic atomic structure as their bulk counterparts do and/or as predicted by theory. As an example, 1.6-nm Au ...particles grown inside a dendrimeric host are studied and found to possess a heavily disordered, metallic glass-type structure. The nanoparticle’s structure evolves toward the face-centered-cubic-type lattice of bulk Au only upon removal of solvent. The results show that periodicity, which rules the structure and properties of bulk crystals, is less of a constraint at the nanoscale level and, therefore, may be used as tunable parameter in nanotechnology research.
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