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Objective: Understanding the regulation of adipocyte differentiation by cellular and extracellular factors is crucial for better management of chronic conditions such as obesity, insulin resistance and lipodystrophy. Experimental infection of rats with a human adenovirus type 36 (Ad-36) improves insulin sensitivity and promotes adipogenesis, reminiscent of the effect of thiozolinediones. Therefore, we investigated the role of Ad-36 as a novel regulator of the adipogenic process. Design and Results: Even in the absence of adipogenic inducers, infection of 3T3-L1 preadipocytes and human adipose-derived stem cells (hASC) by Ad-36, but not Ad-2 that is another human adenovirus, modulated regulatory points that spanned the entire adipogenic cascade ranging from the upregulation of cAMP, phosphatidylinositol 3-kinase and p38 signaling pathways, downregulation of Wnt10b expression, and increased expression of CCAAT/enhancer binding protein-b and peroxisome proliferator-activated receptor g2 and consequential lipid accumulation. Next, we identified that E4 open reading frame (orf)-1 gene of the virus is necessary and sufficient for Ad-36-induced adipogenesis. Selective knockdown of E4 orf-1 by RNAi abrogated Ad-36-induced adipogenic signaling cascade in 3T3-L1 cells and hASC. Compared to the null vector, selective expression of Ad-36 E4 orf-1 in 3T3-L1 induced adipogenesis, which was abrogated when the PDZ-binding domain of the protein was deleted. Conclusion: Thus, Ad-36 E4 orf-1 is a novel inducer of rodent and human adipocyte differentiation process.

Objective. Glucocorticoid-induced leucine zipper (GILZ) has effects on inflammatory pathways that suggest it to be a key inhibitory regulator of the immune system, and its expression is exquisitely sensitive to induction by glucocorticoids. We undertook this study to test our hypothesis that GILZ deficiency would exacerbate experimental immune-mediated inflammation and impair the effects of glucocorticoids on inflammation and, correspondingly, that exogenous GILZ would inhibit these events.Methods. GILZ ؊/؊ mice were generated using the Cre/loxP system, and responses were studied in delayedtype hypersensitivity (DTH), antigen-induced arthritis (AIA), K/BxN serum-transfer arthritis, and lipopolysaccharide (LPS)-induced cytokinemia. Therapeutic expression of GILZ via administration of recombinant adeno-associated virus expressing the GILZ gene (GILZ-rAAV) was compared to the effects of glucocorticoid in collagen-induced arthritis (CIA).Results. Increased T cell proliferation and DTH were observed in GILZ ؊/؊ mice, but neither AIA nor K/BxN serum-transfer arthritis was affected, and GILZ deficiency did not affect LPS-induced cytokinemia. Deletion of GILZ did not impair the effects of exogenous glucocorticoids on CIA or cytokinemia. In contrast, overexpression of GILZ in joints significantly inhibited CIA, with an effect similar to that of dexamethasone. Conclusion. Despite effects on T cell activation, GILZ deficiency had no effect on effector pathways of arthritis and was unexpectedly redundant with effects of glucocorticoids. These findings do not support the hypothesis that GILZ is central to the actions of glucocorticoids, but the efficacy of exogenous GILZ in CIA suggests that further evaluation of GILZ in inflammatory disease is required.

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cytokine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAVdelivered transgenes did not interfere with islet cell insulin production and were expressed in both ␤-and non-␤-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance longterm graft survival. Diabetes 50:515-520, 2001A ttempts to use islet cell transplantation for reversing type 1 diabetes have been documented for more than two decades; however, the procedure has been largely unsuccessful (1,2). Concurrent mechanisms believed to underlie this lack of success include rejection, recurrence of anti-islet cell autoimmunity, and nonspecific islet loss because of perturbation of the graft microenvironment (e.g., inflammation, ischemia/reperfusion). A number of candidate gene products may prevent immune-mediated destruction and extend graft survival (e.g., interleukin [IL]-4, manganese superoxide dismutase, Bcl-2) (3). Furthermore, these genes may prove safer and more effective than systemic pharmacological immunosuppression because some agents are themselves potentially prodiabetogenic (e.g., cyclosporine, FK506, steroids) through imposition of increased metabolic demand. However, such studies have been limited by the lack of gene transfer vectors that are safe, efficient, and long lasting (4). Recombinant adeno-associated virus (rAAV) vectors have recently demonstrated some superiority to other viral and nonviral systems with regard to their in vivo safety, efficiency, and duration of action both in animal models and in early persistent infections in humans without known pathology and with only modest immune responses (5-10). rAAV retains these beneficial properties and therefore has the potential to be an ideal vector for in vivo gene transfer. However, previous studies have failed to demonstrate rAAV transdu...