Effects of Imatinib Mesylate (Gleevec) on Human Islet NF-kappaB Activation and Chemokine Production In Vitro

Imatinib Mesylate (Gleevec) is a drug that potently counteracts diabetes both in humans and in animal models for human diabetes. We have previously reported that this compound in human pancreatic islets stimulates NF-κB signaling and islet cell survival. The aim of this study was to investigate control of NF-κB post-translational modifications exerted by Imatinib and whether any such effects are associated with altered islet gene expression and chemokine production in vitro.Human islets were either left untreated or treated with Imatinib for different timepoints. IκB-α and NF-κB p65 phosphorylation and methylation were assessed by immunoblot analysis. Islet gene expression was assessed using a commercial Pathway Finder microarray kit and RT-PCR. Islet chemokine production was determined by flow cytometric bead array analysis.Human islet IκB-α and Ser276-p65 phosphorylation were increased by a 20 minute Imatinib exposure. Methylation of p65 at position Lys221 was increased after 60 min of Imatinib exposure and persisted for 3 hours. Microarray analysis of islets exposed to Imatinib for 4 hours revealed increased expression of the inflammatory genes IL-4R, TCF5, DR5, I-TRAF, I-CAM, HSP27 and IL-8. The islet release of IL-8 was augmented in islets cultured over night in the presence of Imatinib. Following 30 hours of Imatinib exposure, the cytokine-induced IκB-α and STAT1 phosphorylation was abolished and diminished, respectively. The cytokine-induced release of the chemokines MIG and IP10 was lower in islets exposed to Imatinib for 30 hours.Imatinib by itself promotes a modest activation of NF-κB. However, a prolonged exposure of human islets to Imatinib is associated with a dampened response to cytokines. It is possible that Imatinib induces NF-κB preconditioning of islet cells leading to lowered cytokine sensitivity and a mitigated islet inflammation

Assay for high glucose-mediated islet cell sensitization to apoptosis induced by streptozotocin and cytokines

Pancreatic β-cell apoptosis is known to participate in the β-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB)

Perinatal asphyxia: current status and approaches towards neuroprotective strategies, with focus on sentinel proteins

Delivery is a stressful and risky event menacing the newborn. The mother-dependent respiration has to be replaced by autonomous pulmonary breathing immediately after delivery. If delayed, it may lead to deficient oxygen supply compromising survival and development of the central nervous system. Lack of oxygen availability gives rise to depletion of NAD+ tissue stores, decrease of ATP formation, weakening of the electron transport pump and anaerobic metabolism and acidosis, leading necessarily to death if oxygenation is not promptly re-established. Re-oxygenation triggers a cascade of compensatory biochemical events to restore function, which may be accompanied by improper homeostasis and oxidative stress. Consequences may be incomplete recovery, or excess reactions that worsen the biological outcome by disturbed metabolism and/or imbalance produced by over-expression of alternative metabolic pathways. Perinatal asphyxia has been associated with severe neurological and psychiatric sequelae with delayed clinical onset. No specific treatments have yet been established. In the clinical setting, after resuscitation of an infant with birth asphyxia, the emphasis is on supportive therapy. Several interventions have been proposed to attenuate secondary neuronal injuries elicited by asphyxia, including hypothermia. Although promising, the clinical efficacy of hypothermia has not been fully demonstrated. It is evident that new approaches are warranted. The purpose of this review is to discuss the concept of sentinel proteins as targets for neuroprotection. Several sentinel proteins have been described to protect the integrity of the genome (e.g. PARP-1; XRCC1; DNA ligase IIIα; DNA polymerase β, ERCC2, DNA-dependent protein kinases). They act by eliciting metabolic cascades leading to (i) activation of cell survival and neurotrophic pathways; (ii) early and delayed programmed cell death, and (iii) promotion of cell proliferation, differentiation, neuritogenesis and synaptogenesis. It is proposed that sentinel proteins can be used as markers for characterising long-term effects of perinatal asphyxia, and as targets for novel therapeutic development and innovative strategies for neonatal care