In Vivo Synthesis of Cyclic-di-GMP Using a Recombinant Adenovirus Preferentially Improves Adaptive Immune Responses against Extracellular Antigens.

There is a compelling need for more effective vaccine adjuvants to augment induction of Ag-specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3'-5')-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase into an adenovirus vaccine, fostering production of c-di-GMP as well as proinflammatory responses in mice. In this study, we recombined a more potent diguanylate cyclase gene, VCA0848, into a nonreplicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I IFN-ß and activation of innate and adaptive immune cells early after administration into mice as compared with control vectors. Coadministration of the extracellular protein OVA and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. In addition, we found that coadministration of AdVCA0848 with another adenovirus serotype 5 vector expressing the HIV-1-derived Gag Ag or the Clostridium difficile-derived toxin B resulted in significant inhibitory effects on the induction of Gag and toxin B-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular Ag, which can be used as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers.

Modulation by bexarotene of mRNA expression of genes in mouse lung tumors.

Bexarotene has demonstrated chemopreventive and therapeutic efficacy towards mouse lung tumors. Using specimens from our published study that demonstrated the efficacy of bexarotene, we report herein its ability to modulate mRNA expression of genes in both lung and lung tumors. Strain A/J mice were administered vinyl carbamate to induce lung tumors. This was followed by 200 mg/kg body weight of bexarotene administered by oral gavage during Wks 4-25 or 23-25. The mice were sacrificed at Wk 25. The expression of 26 genes was decreased in lung tumors, whereas only two genes, Apolipoprotein D and CYP26b, had their mRNA expression increased by bexarotene. Genes with increased mRNA expression in untreated lung tumors include: epiregulin and kininogen-1 (increased by more than 40-fold) and Caspase-3, Cyclin D1, DNA methyltransferase 3a (Dnmt-3a), E-prostanoid 3 receptor (EP3), c-myc, surfactant protein-C, and survivin (increased by 1.7- to 3.6-fold). Bexarotene decreased the mRNA expression of Caspase-3, Dnmt-3a, EP3, and survivin, as well as the expression of the Cyclin E1, estrogen receptor-alpha, and iNOS genes. Bexarotene had a greater effect in decreasing the expression of Caspase-3, Cyclin E1, Dnmt-3a, EP3, iNOS, and survivin, when administered to mice with established tumors than when administered to mice while tumors were emerging. In summary, bexarotene modulated mRNA expression of genes in mouse lung tumors, being more effective in established tumors than in emerging tumors, suggesting that modulation of expression could be useful as a biomarker for the therapeutic and chemopreventive activity of the drug, especially in established tumors.

ERAP1 deficient mice have reduced Type 1 regulatory T cells and develop skeletal and intestinal features of Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is a prototypical sero-negative autoimmune disease that affects millions worldwide. Single nucleotide polymorphisms in the Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) gene have been linked to AS via GWAS studies, however, the exact mechanism as to how ERAP1 contributes to pathogenesis of AS is not understood. We undertook µCT imaging and histologic analysis to evaluate bone morphology of the axial skeletons of ERAP1-/- mice and discovered the hallmark skeletal features of AS in these mice, including spinal ankylosis, osteoporosis, and spinal inflammation. We also confirmed the presence of spontaneous intestinal dysbiosis and increased susceptibility to Dextran Sodium Sulfate (DSS)-induced colitis in ERAP1-/- mice, however the transfer of healthy microbiota from wild type mice via cross-fostering experiments did not resolve the skeletal phenotypes of ERAP1-/- mice. Immunological analysis demonstrated that while ERAP1-/- mice had normal numbers of peripheral Foxp3+ Tregs, they had reduced numbers of both "Tr1-like" regulatory T cells and tolerogenic dendritic cells, which are important for Tr1 cell differentiation. Together, our data suggests that ERAP1-/- mice may serve as a useful animal model for studying pathogenesis of intestinal, skeletal, and immunological manifestations of Ankylosing Spondylitis.

Prevention of mouse lung tumors and modulation of DNA methylation by combined treatment with budesonide and R115777 (Zarnestra MT).

Budesonide (an anti-inflammatory glucocorticoid), R115777 (a farnesyl transferase inhibitor, Zarnestra, Tipifarnib) or combinations of them were evaluated for prevention of lung tumors and for modulation of DNA methylation in tumors. Lung tumors were induced by vinyl carbamate in female strain A mice. One week later, mice received 60 or 100 mg/kg R115777 by oral gavage and 5 days/week, 0.8 or 1.6 mg/kg of budesonide in their diet, or their combined treatment until killed at 20, 28 and 36 weeks after administering the vinyl carbamate. Other mice were administered the drugs for 2 weeks before killing at Week 20. At Week 20, the rank order for prevention of lung tumors was the combined treatment > budesonide > R115777. At later killings, R115777 was no longer effective, whereas budesonide and the combinations continued to prevent tumors, albeit at a reduced efficacy. DNA hypomethylation in lung tumors was prevented by treatment with R115777, budesonide and the combinations. When administered starting at Week 18 to tumor-bearing mice, the drugs reversed DNA hypomethylation in the tumors. In summary, combined treatment with budesonide and R115777 produced the following results: (i) it was more efficacious in preventing lung tumors than the individual drugs; and (ii) it prevented and reversed DNA hypomethylation in lung tumors. These results support the combined use of budesonide and R115777 in prevention of lung tumors and suggest that reversal of DNA hypomethylation in lung tumors would be useful as a surrogate end-point biomarker for prevention.

Prevention of mouse lung tumors and modulation of DNA methylation by combined treatment with budesonide and R115777 (ZarnestraMT).

Budesonide (an anti-inflammatory glucocorticoid), R115777 (a farnesyl transferase inhibitor, Zarnestra, Tipifarnib) or combinations of them were evaluated for prevention of lung tumors and for modulation of DNA methylation in tumors. Lung tumors were induced by vinyl carbamate in female Strain A mice. One week later, mice received 60 or 100 mg/kg R115777 by oral gavage and 5 days/week, 0.8 or 1.6 mg/kg of budesonide in their diet, or their combined treatment until killed at 20, 28 and 36 weeks after administering the vinyl carbamate. Other mice were administered the drugs for 2 weeks before killing at 20 weeks. At Week 20, the rank order for prevention of lung tumors was the combined treatment>budesonide>R115777. At later killings, R115777 was no longer effective, whereas budesonide and the combinations continued to prevent tumors, albeit at a reduced efficacy. DNA hypomethylation in lung tumors was prevented by treatment with R115777, budesonide and the combinations. When administered starting at Week 18 to tumor-bearing mice, the drugs reversed DNA hypomethylation in the tumors. In summary, combined treatment with budesonide and R115777 produced the following results: (i) it was more efficacious in preventing lung tumors than the individual drugs; and (ii) it prevented and reversed DNA hypomethylation in lung tumors. These results support the combined use of budesonide and R115777 in prevention of lung tumors and suggest that reversal of DNA hypomethylation in lung tumors would be useful as a surrogate endpoint biomarker for prevention.

Prevention of mouse lung tumors by targretin.

Targretin has indicated chemotherapeutic activity against nonsmall-cell lung cancer and chemoprevention in rat mammary gland. Therefore, targretin was evaluated for the prevention of 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol (NNK) and vinyl carbamate-induced lung tumors in female strain A mice. Three experiments were performed: (i) a dose-response study with vinyl carbamate-induced tumors; (ii) a limited treatment study also with vinyl carbamate and (iii) prevention of NNK-induced tumors. In the dose-response study, 0, 10, 30, 100 and 300 mg/kg targretin were administered after vinyl carbamate. Dose levels of 30 mg/kg and greater significantly decreased tumor multiplicity by >19%. However, the efficacy of 30 and 300 mg/kg was not significantly different demonstrating a shallow dose-response relationship. In the limited treatment study, 200 mg/kg targretin was administered to the mice from 4-13, 4-19, 4-25 and 23-25 weeks after vinyl carbamate. Administering targretin from weeks 4-19 and 4-25 decreased the multiplicity of tumors from 35.3 +/- 1.43 to 29.1 +/- 1.51 and 25.0 +/- 0.93, respectively, and along with administering it from weeks 23-25 decreased tumor size. In the third study, when targretin (100 and 300 mg/kg) was administered for 3 weeks after NNK followed by a 20 weeks holding period, tumor multiplicity was reduced from 10.6 +/- 1.13 to 6.38 +/- 0.75 and 4.60 +/- 0.70, respectively. Hence, targretin demonstrated both preventive and therapeutic activity with respect to mouse lung tumors supporting its further development as a preventive and therapeutic agent for lung cancer.