Over-expression of AMP-activated protein kinase impairs pancreatic {beta}-cell function in vivo

Treatment of type 1 diabetes by islet transplantation is currently limited by loss of functional beta-cell mass after transplantation. We investigated here whether adenovirus-mediated changes in AMP-activated protein kinase (AMPK) activity, previously shown to affect insulin secretion in vitro, might affect islet graft function in vivo. In isolated mouse and rat islets, insulin secretion stimulated by 17 (vs 3) mmol/l glucose was inhibited by 36.5% (P<0.01) and 43% (P<0.02) respectively after over-expression of constitutively-active AMPK- (AMPK CA) versus null (eGFP-expressing) viruses, and glucose oxidation was decreased by 38% (P<0.05) and 26.6% (P<0.05) respectively. Increases in apoptotic index (terminal deoxynucleotide transferase-mediated deoxyuridine trisphosphate biotin nick end-labelling) (TUNEL)) were also observed in AMPK CA- (22.8 +/- 3.6% TUNEL-positive cells, P<0.001), but not AMPK DN- (2.72 +/- 3.9%, positive cells, P=0.05) infected islets, versus null adenovirus-treated islets (0.68 +/- 0.36% positive cells). Correspondingly, transplantation of islets expressing AMPK CA into streptozotocin-diabetic C57 BL/6 mice improved glycaemic control less effectively than transplantation with either null (P<0.02) or AMPK-DN-infected (P<0.01) islets. We conclude that activation of AMPK inhibits beta-cell function in vivo and may represent a target for therapeutic intervention during islet transplantation.     

Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet {beta}-cells from pancreatic duct cells and an increase in functional {beta}-cell mass

Pancreatic islet transplantation is a viable treatment for type 1 diabetes, but is limited by human donor tissue availability. The combination of epidermal growth factor (EGF) and gastrin induces islet beta-cell neogenesis from pancreatic exocrine duct cells in rodents. In this study we investigated whether EGF and gastrin could expand the beta-cell mass in adult human isolated islets that contain duct as well as endocrine cells. Human islet cells were cultured for 4 wk in serum-free medium (control) or in medium with EGF (0.3 mug/ml), gastrin (1.0 mug/ml), or the combination of EGF and gastrin. beta-Cell numbers were increased in cultures with EGF plus gastrin (+118%) and with EGF (+81%), but not in cultures with gastrin (-3%) or control medium (-62%). After withdrawal of EGF and gastrin and an additional 4 wk in control medium, beta-cell numbers continued to increase only in cultures previously incubated with both EGF and gastrin (+232%). EGF plus gastrin also significantly increased cytokeratin 19-positive duct cells (+678%) in the cultures. Gastrin, alone or in combination with EGF, but not EGF alone, increased the expression of pancreatic and duodenal homeobox factor-1 as well as insulin and C peptide in the cytokeratin 19-positive duct cells. Also, EGF plus gastrin significantly increased beta-cells and insulin content in human islets implanted in immunodeficient nonobese diabetic-severe combined immune deficiency mice as well as insulin secretory responses of the human islet grafts to glucose challenge. In conclusion, combination therapy with EGF and gastrin increases beta-cell mass in adult human pancreatic islets in vitro and in vivo, and this appears to result from the induction of beta-cell neogenesis from pancreatic exocrine duct cells.     

Early administration of keratinocyte growth factor improves {beta}-cell regeneration in rat with streptozotocin-induced diabetes

The aim of our study was to investigate the ability of keratinocyte growth factor (KGF; palifermin) in regulating beta-cell growth in normal newborn rats and in rats with neonatal diabetes. Wistar rats were injected with streptozotocin (STZ) to induce diabetes on the dayof birth. From days 2 to 6 after birth, animals received a daily s.c. injection of KGF (STZ/KGF group) and at the dose of 3 mg/kg body weight or saline solution (STZ groups). A group of non-diabetic Wistar rats was treated either with saline (Wistar group) or with KGF from days 2 to 6 after birth at the dose of 3 mg/kg body weight (Wistar/KGF group). beta-cell mass was measured at day 7 after birth in all groups. beta- and ductal cells replication were measured in all groups and apoptosis was assessed in the pancreas of 2-, 4-, and 7-day-old STZ and STZ/KGF rats. The total beta-cell mass of the 7-day-old KGF/STZ neonates was significantly increased compared with that of age-matched STZ rats. beta-cell replication rate was decreased at day 2 in the STZ/KGF group and was similar in the 4- and 7-day-old rats from STZ and STZ/KGF groups. Duct cell replication was significantly increased in the pancreas of 2- and 4-day-old KGF/STZ neonates when compared with that of age-matched rats from STZ control group. The rate of apoptosis in the neonatal pancreases of STZ and KGF/STZ groups was not significantly different. In non-diabetic Wistar rats, KGF treatment led to a slight but significant increase in duct cell proliferation at day 2 without significant changes in the total beta-cell mass in the 7-day-old rats. We provide evidence for a growth-promoting effect of KGF during beta-cell regeneration in neonatal diabetic rats. KGF exerts strong mitogenic effect on the pancreatic duct cells, thus expanding the population of precursor cells that subsequently differentiate into insulin-producing beta-cells.     

Nonimmunoglobulin (non-Ig)/BCL6 gene fusion in diffuse large B-cell lymphoma results in worse prognosis than Ig/BCL6

Chromosomal translocation involving the BCL6 gene affects not only immunoglobulin (Ig) genes but also a number of non-Ig genes as partners. The molecular anatomy of the BCL6 gene rearrangements in 39 cases with diffuse large B-cell lymphoma (DLBCL) by long-distance polymerase chain reaction-based assays was determined. The results showed that Ig genes were affected in 21 cases; non-Ig genes, 15 cases; a deletion of more than a 1-kb segment, 2 cases; and a point mutation, 1 case. Comparative studies between the 21 cases with Ig gene partners and the 17 cases with non-Ig gene partners, including 2 cases with the deletion, showed that the overall survival of the latter group of patients was significantly inferior to that of the former (P = .0440), and the estimated 2-year overall survival rates were 58.3% vs 17.6% (P = .005). Non-Ig/BCL6 fusion is a poor prognostic indicator of DLBCL, and DLBCL with BCL6 translocation could be subclassified according to the individual partner locus and/or gene. (Blood. 2000;96:2907-2909)     

Murine B-cell leukemia lymphoma (BCL1) cells as a target for NK cell-mediated immunotherapy

Natural killer (NK) cells are important for their ability to recognize and lyse tumor cells and virus infected cells. NK cells express triggering receptors that are specific for non-MHC ligands. This article describes the 35S release cytotoxic assay, which measures the ability of NK cells derived from spleen cells taken from polyIC-treated mice to lyse B-cell leukemia (BCL1) cells. BCL1 cells express ligands for NKp46 on the cell surface membrane and they are sensitive to allogeneic but not syngeneic IL-2 activated natural killer cells.     

Rearrangement of the BCL6 locus at 3q27 is an independent poor prognostic factor in nodal diffuse large B-cell lymphoma

Diffuse large B-cell lymphomas (DLBCL) are a heterogeneous group of tumours, varying in clinical features, immunophenotype and cytogenetics. The aim of this study was to investigate the prognostic significance of BCL6 gene rearrangement at the 3q27 locus in patients with primary nodal disease, and to examine interrelationships with immunophenotype and International Prognostic Index (IPI). We have developed a fluorescent in situ hybridization (FISH)-based technique for the retrospective analysis of the effect of BCL6 gene rearrangements on survival, using nuclei extracted from paraffin-embedded tissue. FISH results were obtained in 111 presentation cases of nodal DLBCL. The IPI was calculated and each case was stained immunocytochemically for BCL6, BCL2 and CD10. 3q27 rearrangements were detected in 25% of cases. BCL2 protein and a germinal centre (GC) phenotype (defined as CD10+, BCL6+) were expressed in 56% and 41% of cases respectively. In multivariate analysis, rearrangement of 3q27 and BCL2 expression and the absence of a GC phenotype were associated with a poor prognosis. These factors can be used in conjunction with the IPI to improve risk stratification in nodal DLBCL.     

Transforming growth factor-{beta}1 modulates responses of CD34+ cord blood cells to stromal cell-derived factor-1/CXCL12

Disruption of stromal cell-derived factor-1 (SDF-1/CXCL12 [CXC chemokine ligand 12]) interaction leads to mobilization of stem/progenitor cells from bone marrow to circulation. However, prolonged exposure of CD34+ cells to SDF-1 desensitizes them to SDF-1. So how do cells remain responsive to SDF-1 in vivo when they are continuously exposed to SDF-1? We hypothesized that one or more mechanisms mediated by cytokines exist that could modulate SDF-1 responsiveness of CD34+ cells and the desensitization process. We considered transforming growth factor-beta1 (TGF-beta1) a possible candidate, since TGF-beta1 has effects on CD34+ cells and is produced by stromal cells, which provide niches for maintenance and proliferation of stem/progenitor cells. TGF-beta1 significantly restored SDF-1-induced chemotaxis and sustained adhesion responses in cord blood CD34+ cells preexposed to SDF-1. Effects of TGF-beta1 were dependent on the dose and duration of TGF-beta1 pretreatment. Phosphorylation of extracellular signal-regulated kinase 1 (Erk1)/Erk2 was implicated in TGF-beta1 modulation of migratory and adhesion responses to SDF-1. Our results indicate that low levels of TGF-beta1 can modulate SDF-1 responsiveness of CD34+ cells and thus may facilitate SDF-1-mediated retention and nurturing of stem/progenitor cells in bone marrow.     

Exenatide (exendin-4) improves insulin sensitivity and {beta}-cell mass in insulin-resistant obese fa/fa Zucker rats independent of glycemia and body weight

The effects of the incretin mimetic exenatide (exendin-4) on metabolic parameters, insulin sensitivity, and beta-cell mass were examined in nondiabetic, insulin-resistant obese fa/fa Zucker rats. After 6 wk of treatment, ad libitum-fed exenatide-treated (EX) and pair-fed vehicle control (PF) rats had comparable food intake, body weight, hemoglobin A(1c) (HbA(1c)), and fasting plasma concentrations of glucose, insulin, and lipids. Concurrent decreases in food intake and weight gain were observed in EX and PF rats, compared with ad libitum-fed vehicle control (CON) rats (P < 0.001). The increases in HbA(1c) and fasting plasma insulin concentrations that occur during the normal progression of this disease model were significantly reduced in EX and PF rats, compared with CON rats (P < 0.001). The insulin sensitivity index (ISI; glucose infusion rate to plasma insulin concentration) measured during a hyperinsulinemic euglycemic clamp was 224% higher in EX rats than CON rats (P < 0.001) and 61% higher in EX rats than PF rats (P < 0.004). The latter difference was despite comparable HbA(1c), fasting glucose, fasting insulin, total cholesterol, high-density lipoprotein, and daily food consumption between EX and PF animals. In the absence of exenatide, beta-cell mass was hyperbolically related to ISI (beta-cell mass * ISI was constant). Analogous to the disposition index, the beta-cell mass * ISI product was 63% greater in EX than PF rats (P < 0.05). Thus, exenatide increased beta-cell mass to a greater extent than would be expected in animals of comparable insulin resistance, suggesting a direct trophic effect on islet neogenesis in obese fa/fa rats independent of body weight and glycemia.     

Isolation and characterization of growth factor(s) from a human B-cell lymphoma

We demonstrate that human neoplastic B cells (Br cells) contain a cytoplasmic protein of molecular mass 60 Kd that exhibits B-cell growth factor (BCGF) activity on growth factor-dependent long-term human B cells as well as on autochthonous tumor cells. This 60-Kd protein is recognized by antibodies against a similar intracellular 60-Kd protein derived from normal human lymphocytes. These results demonstrate that the two proteins share epitope homology. Microculture bioassays indicate that neoplastic and normal 60-Kd proteins are capable of driving neoplastic B cells through S-phase. Western immunoblot analysis indicates that neoplastic B cells secrete 60- as well as 14-Kd protein. Immunoaffinity-purified proteins secreted by Br cells exhibit BCGF activity in anti-IgM or dextran sulfate-preactivated human B cells. In addition, a double-antibody immunofluorescence staining technique was used to demonstrate that Br cells express cell surface receptors for BCGF molecule(s). These studies provide support for the autocrine growth model for neoplastic human B cells and suggest that the autocrine growth factor derived from such tumor cells is similar if not identical to normal BCGF molecules.