Andy McShea

Nuclear Factor-κB Regulates β-Cell Death

Apoptotic β-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of β-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized β-cells to tumor necrosis factor (TNF)-α–induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in β-cells. Regulation of A20 was nuclear factor-κB (NF-κB)–dependent, two NF-κB sites within the A20 promoter were found to be necessary and sufficient for A20 expression in β-cells. Activation of NF-κB by TNF receptor–associated factor (TRAF) 2, TRAF6, NF-κB–inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect β-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in β-cells. Further, A20 expression is NF-κB dependent, thus linking islet proinflammatory gene responses with protection from apoptosis.

Nuclear factor-kappaB regulates beta-cell death: a critical role for A20 in beta-cell protection.

Apoptotic β-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of β-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized β-cells to tumor necrosis factor (TNF)-α–induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in β-cells. Regulation of A20 was nuclear factor-κB (NF-κB)–dependent, two NF-κB sites within the A20 promoter were found to be necessary and sufficient for A20 expression in β-cells. Activation of NF-κB by TNF receptor–associated factor (TRAF) 2, TRAF6, NF-κB–inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect β-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in β-cells. Further, A20 expression is NF-κB dependent, thus linking islet proinflammatory gene responses with protection from apoptosis.

Integrated microfluidic biochips for DNA microarray analysis

A fully integrated and self-contained microfluidic biochip device has been developed to automate the fluidic handling steps required to perform a gene expression study of the human leukemia cell line (K-562). The device consists of a DNA microarray semiconductor chip with 12,000 features and a microfluidic cartridge that consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. A single-color transcriptional analysis of K-562 cells with a series of calibration controls (spiked-in controls) was performed to characterize this new platform with regard to sensitivity, specificity and dynamic range. The device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than 3 orders of magnitude. Experiments also demonstrated that chip-to-chip variability was low, indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis.

Nuclear factor-κB regulates β-cell death : A critical role for a20 in β-cell protection

Apoptotic β-cell death is central to the pathogenesis of type 1 diabetes and may be important in islet graft rejection. Despite this, genetic control of β-cell apoptosis is only poorly understood. We report that inhibition of gene transcription sensitized β-cells to tumor necrosis factor (TNF)-α–induced apoptosis, indicating the presence of a regulated antiapoptotic response. Using oligonucleotide microarrays and real-time PCR, we identified TNFAIP3/A20 as the most highly regulated antiapoptotic gene expressed in cytokine-stimulated human and mouse islets. Cytokine induction of A20 mRNA in primary islets and insulinoma cells was rapid and observed within 1 h, consistent with A20 being an immediate early response gene in β-cells. Regulation of A20 was nuclear factor-κB (NF-κB)–dependent, two NF-κB sites within the A20 promoter were found to be necessary and sufficient for A20 expression in β-cells. Activation of NF-κB by TNF receptor–associated factor (TRAF) 2, TRAF6, NF-κB–inducing kinase, or protein kinase D, which transduce signals downstream of Toll-like receptors, TNF receptors, and free radicals, respectively, were all potent activators of the A20 promoter. Moreover, A20 expression was induced in transplanted islets in vivo. Finally, A20 expression was sufficient to protect β-cells from TNF-induced apoptosis. These data demonstrate that A20 is the cardinal antiapoptotic gene in β-cells. Further, A20 expression is NF-κB dependent, thus linking islet proinflammatory gene responses with protection from apoptosis.

Integrated Microfluidic CustomArray Device for Bacterial Genotyping and Identification

The ongoing threat of the potential use of biothreat agents (such as Bacillus anthracis) as a biochemical weapon emphasizes the need for a rapid, miniature, fully automated, and highly specific detection assay. An integrated and self-contained microfluidic device has been developed to rapidly detect B. anthracis and many other bacteria. The device consists of a semiconductor-based DNA microarray chip with 12,000 features and a microfluidic cartridge that automates the fluid handling steps required to carry out a genotyping assay for pathogen identification. This fully integrated and disposable device consists of low-cost microfluidic pumps, mixers, valves, fluid channels, reagent storage chambers, and DNA microarray silicon chip. Microarray hybridization and subsequent fluid handling and reactions were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. The genotyping results showed that the device was able to identify and distinguish B. anthracis from the other members of the closely related Bacillus cereus group, demonstrating the potential of integrated microfluidic and microarray technology for highly specific pathogen detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluid handling steps and allows the detection and identification of biological warfare agents in a rapid and automated fashion.