IL-4-transfected tumor cell vaccines activate tumor-infiltrating dendritic cells and promote type-1 immunity

We previously demonstrated that IL-4 gene-transfected glioma cell vaccines induce effective therapeutic immunity in preclinical glioma models, and have initiated phase I trials of these vaccines in patients with malignant gliomas. To gain additional mechanistic insight into the efficacy of this approach, we have treated mice bearing the MCA205 (H-2(b)) or CMS-4 (H-2(d)) sarcomas. IL-12/23 p40(-/-) and IFN-gamma(-/-) mice, which were able to reject the initial inoculation of IL-4 expressing tumors, failed to mount a sustained systemic response against parental (nontransfected) tumor cells. Paracrine production of IL-4 in vaccine sites promoted the accumulation and maturation of IL-12p70-secreting tumor-infiltrating dendritic cells (TIDCs). Adoptive transfer of TIDCs isolated from vaccinated wild-type, but not IL-12/23 p40(-/-), mice were capable of promoting tumor-specific CTL responses in syngeneic recipient animals. Interestingly, both STAT4(-/-) and STAT6(-/-) mice failed to reject IL-4-transfected tumors in concert with the reduced capacity of TIDCs to produce IL-12p70 and to promote specific antitumor CTL reactivity. These results suggest that vaccines consisting of tumor cells engineered to produce the type 2 cytokine, IL-4, critically depend on type 1 immunity for their observed therapeutic efficacy.     

A Nitrate Reductase Inactivator Protein from Spinach. Purification, Molecular Weight and Subunit Composition

A nitrate reductase-inactivator protein has been purified 16,000-foldfrom spinach leaves by pH 5 treatment, chromatography on SE53,Con A-Sepharose, and chromatofocusing. The yield was 12%, thespecific activity was 115 units mg^–1. Polyacrylamide gelelectrophoresis of the final purified inactivator fraction yielded2 major protein bands and both bands exhibited nitrate reductase-inactivatoractivity. Analysis of this inactivator protein by gel filtrationand SDS-gel electrophoresis revealed protein stainable materialonly in a molecular weight range of 110,000–115,000. SDSgel electrophoresis under reducing conditions yielded 2 proteinbands corresponding to molecular weights of 51,000 and 53,000.The proteolytic mapping for the two separated subunits appearedsimilar and possibly identical. (Received October 28, 1991; Accepted February 24, 1992)     

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.     

Translesion synthesis past equine estrogen-derived 2'-deoxycytidine DNA adducts by human DNA polymerases eta and kappa

Estrogen replacement therapy (ERT), composed of equilenin, is associated with increased risk of breast, ovarian, and endometrial cancers. Several diastereoisomers of unique dC and dA DNA adducts were derived from 4-hydroxyequilenin (4-OHEN), a metabolite of equilenin, and have been detected in women receiving ERT. To explore the miscoding property of 4-OHEN-dC adduct, site-specifically modified oligodeoxynucleotides (Pk-1, Pk-2, Pk-3, and Pk-4) containing a single diastereoisomer of 4-OHEN-dC were prepared by a postsynthetic method. Among them, major 4-OHEN-dC-modified oligodeoxynucleotides (Pk-3 and Pk-4) were used to prepare the templates for primer extension reactions catalyzed by DNA polymerase (pol) alpha, pol eta, and pol kappa. Primer extension was retarded one base prior to the lesion and opposite the lesion; stronger blockage was observed with pol alpha, while with human pol eta or pol kappa, a fraction of the primers was extended past the lesion. Steady-state kinetic studies showed that both pol kappa and pol eta inserted dCMP and dAMP opposite the 4-OHEN-dC and extended past the lesion. Never or less-frequently, dGMP, the correct base, was inserted opposite the lesion. The relative bypass frequency past the 4-OHEN-dC lesion with pol eta was at least 3 orders of magnitude higher than that for pol kappa, as observed for primer extension reactions. The bypass frequency past the dA.4-OHEN-dC adduct in Pk-4 was 2 orders of magnitude more efficient than that past the adduct in Pk-3. Thus, 4-OHEN-dC is a highly miscoding lesion capable of generating C --> T transitions and C --> G transversions. The miscoding frequency and specificity of 4-OHEN-dC were strikingly influenced by the adduct stereochemistry and DNA polymerase used.     

Capsazepine is a novel activator of the delta subunit of the human epithelial Na+ channel

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCdelta, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCdeltabetagamma (hENaCdeltabetagamma) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were markedly enhanced by the application of capsazepine (> or =1 microM), and the capsazepine-induced current was mostly abolished by the addition of 100 microM amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 microM. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCdelta homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCalpha and caused a slight decrease on the hENaCalphabetagamma current. In conclusion, capsazepine acts on ENaCdelta and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCdelta.     

Insulin-degrading enzyme antagonizes insulin-dependent tissue growth and Aβ-induced neurotoxicity in Drosophila

Insulin-degrading enzyme (IDE) is implicated in the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimer’s disease (AD). Here we provide genetic evidence that Drosophila Ide (dIde) antagonizes the insulin signaling pathway and human Aβ-induced neurotoxicity in Drosophila. In this study, we also generated a dIde knockout mutant (dIde^KO) by gene targeting, and found that loss of IDE increases the content of the major insect blood sugar, trehalose, thus suggesting a conserved role of IDE in sugar metabolism. Using dIde^KO as a model, further investigations into the biological functions of IDE and its role in the pathogenesis of DM2 and AD can be made.     

Hydrodynamic-based delivery of an interleukin-22-Ig fusion gene ameliorates experimental autoimmune myocarditis in rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.