Conditional interleukin-12 gene therapy promotes safe and effective antitumor immunity.

We and others have previously demonstrated that (chronic) interleukin (IL)-12 gene therapy delivered intratumorally through ex vivo gene-engineered dendritic cell (DC) is competent to promote the regression of established murine tumors. In this report, we have developed a conditional expression system (rAd.RheoIL12) to determine the temporal requirements of transgenic IL-12p70 production by administered DC on therapeutic outcome in a subcutaneous B16 melanoma model. DCs infected with rAd.RheoIL12 (DC.RheoIL12) secreted IL-12p70 in a tightly regulated fashion in response to a synthetic diacylhydrazine small molecule ligand in vitro, and the treatment benefit of DC.RheoIL12 delivered into B16 lesions was strictly ligand dependent in vivo. Indeed, DC.RheoIL12-based therapy promoted the regression of established day 7 B16 tumor lesions after intratumoral injection, provided that ligand administration occurred within 24 h of DC injection and was sustained for approximately 5 or more days. Treatment efficacy was correlated to the magnitude of systemic anti-B16 CD8(+) T cells cross-primed in vivo, which in turn, appeared dependent on the early enhanced in vivo survival of adoptively transferred DC.RheoIL12 in tumor and tumor-draining lymph nodes. The unique safety feature of DC.RheoIL12 application was emphasized in a combined treatment model with rIL-2, where profound TNF-alpha-associated toxicity could be ameliorated upon discontinuation of activating ligand administration.

Osteopontin deficiency impacts the pancreatic TH1/TH2 cytokine profile following multiple low dose streptozotocin-induced diabetes.

Osteopontin (OPN) is a phosphorylated acidic glycoprotein that causes chemotaxis of macrophages and downregulation of nitric oxide synthesis. OPN has been shown to be involved in the pathogenesis of autoimmune diseases. Here, we tested the hypothesis that increased expression of pancreatic OPN in experimental diabetes has a protective role. The immune response phenotype associated with the induction of diabetes was evaluated in male OPN knockout (KO) and wild type (WT) mice. Multiple low dose streptozotocin (STZ) (MLDS), 40 mg/kg, was injected intraperitoneally for 5 days to establish a model for autoimmune diabetes. Glucose levels and body weight were evaluated in the vehicle and STZ treated groups. ELISA assay was used to monitor OPN serum levels in the WT diabetic mice. Histological studies evaluated insulitis development and Western blot analysis was employed to evaluate the expression levels of Th1 cytokines (TNF-alpha and IFN-gamma) and Th2 cytokines (IL-10 and IL-4). Immunohistochemistry was employed to localize IL-4 in the diabetic WT pancreata. Both WT and KO mice developed diabetes. In the WT, OPN serum levels were significantly upregulated 1 day after STZ injection. Pancreatic islets appeared larger in the KO group. Mild lymphocytic infiltrate and apoptosis were detected in the WT diabetic islets, while no signs of inflammation were detected in the KO group. WT diabetics showed upregulation of both Th1 and Th2 cytokines, whereas in the diabetic KO a mild upregulation of Th1 cytokines was detected with significant downregulation of IL-4. In the diabetic WT mice, IL-4 was localized in the interlobular connective tissue. Our studies show that the pancreatic immune response to MLDS diabetes is balanced between the Th1 and Th2 in the WT animals. KO mice show mild polarization towards the Th1 response. Although OPN is a known promoter for Th1 responses, it appears to have a regulatory control over the Th2 response in MLDS.

Streptozotocin (STZ) mediates acute upregulation of serum and pancreatic osteopontin (OPN): a novel islet-protective effect of OPN through inhibition of STZ-induced nitric oxide production.

Osteopontin (OPN) is a secreted acidic phosphoprotein that binds to a cell-surface integrin-binding motif and is involved in many inflammatory and immune-modulating disorders. There is compelling evidence that soluble OPN can in a variety of situations help cells survive an otherwise lethal insult. In this study we show that OPN is localized in the rat pancreatic islets and ducts. Staining of pancreatic serial sections with islet hormone antibodies showed that all islet cells express OPN. Rats treated with a single dose of streptozotocin (STZ; 50 mg/kg) showed acute upregulation of serum OPN levels and pancreatic OPN mRNA and protein. Serum OPN dropped by the end of day 7 but was still higher than prediabetic levels. Pancreatic mRNA and protein showed a similar pattern. Twenty-four hours after STZ injection, the intensified OPN expression was localized towards the periphery of the islets and surrounded the remaining insulin-positive cells. To explore the significance of OPN acute upregulation, freshly isolated islets were pretreated with OPN (0.15-15 nM) before addition of STZ. OPN significantly reduced the STZ-induced NO levels in the islets through an Arg-Gly-Asp (RGD)-dependent reduction of inducible NO synthase (iNOS) mRNA levels. Addition of OPN to freshly isolated mildly diabetic islets (blood glucose <300 mg/dl) significantly improved their glucose-stimulated insulin secretion and reduced their NO levels. Next we investigated the regulation of OPN in beta-cells. When STZ (5 mM) was added to the beta-cell line RINm5F it significantly increased OPN mRNA levels within 6 h. To distinguish between the effect of STZ and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with STZ (0.05-5 mM) or with glucose (5-25 mM). STZ induced upregulation of OPN promoter activity within 3 h, while high glucose induced upregulation of OPN promoter activity after 48 h. Our data introduce OPN as a novel islet protein that is differentially regulated by STZ and glucose in the islets. OPN initial upregulation after diabetes induction was probably due to STZ-induced toxicity, while maintenance of the high OPN levels might be due to hyperglycemia. The acute induction of OPN after STZ-induced diabetes might represent an endogenous mechanism to protect the islets against STZ-induced cytotoxicity, partly via an RGD-dependent NO regulatory mechanism.