Effective neovascularization is crucial for recovery after cardiovascular events.
Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that ...target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation.
Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation.
The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.
Spontaneous tumors in dog have been demonstrated to share many features with their human counterparts, including relevant molecular targets, histological appearance, genetics, biological behavior and ...response to conventional treatments. Mammary tumors in dog therefore provide an attractive alternative to more classical mouse models, such as transgenics or xenografts, where the tumour is artificially induced. To assess the extent to which dog tumors represent clinically significant human phenotypes, we performed the first genome-wide comparative analysis of transcriptional changes occurring in mammary tumors of the two species, with particular focus on the molecular pathways involved.
We analyzed human and dog gene expression data derived from both tumor and normal mammary samples. By analyzing the expression levels of about ten thousand dog/human orthologous genes we observed a significant overlap of genes deregulated in the mammary tumor samples, as compared to their normal counterparts. Pathway analysis of gene expression data revealed a great degree of similarity in the perturbation of many cancer-related pathways, including the 'PI3K/AKT', 'KRAS', 'PTEN', 'WNT-beta catenin' and 'MAPK cascade'. Moreover, we show that the transcriptional relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries.
Our data confirm and further strengthen the value of the canine mammary cancer model and open up new perspectives for the evaluation of novel cancer therapeutics and the development of prognostic and diagnostic biomarkers to be used in clinical studies.
Background/Aims Hepatitis C virus leads to chronic hepatitis in the majority of infected individuals. The mechanism of viral persistence is not completely understood. Hepatitis C virus core protein ...is produced within hepatocytes and is secreted during HCV infection. Our study characterizes the effects of core protein on T cell priming in mice. Methods We used a system of antigen-specific in vitro priming of CD4+ and CD8+ T cells by myeloid dendritic cells, hepatoma cells or primary hepatocytes. Core protein was either added to the cultures or expressed by antigen-presenting cells. Results Antigen-presenting cells treated with core protein showed reduced surface expression of major histocompatibility molecules. Myeloid dendritic cells showed also reduced expression of costimulatory molecules. CD4+ and CD8+ T cells primed by these cells showed defects in activation, proliferation, and cytokine production. Importantly, CD4+ and also CD8+ T cells primed in the presence of core protein showed an increase in interleukin-10 production resembling the phenotype of regulatory T cells. Conclusions Hepatitis C virus core protein inhibits priming of antigen-specific CD4+ and CD8+ T cell responses by downregulation of major histocompatibility molecules and costimulatory molecules on antigen-presenting cells and induces development of IL-10-producing T cells.
Oligonucleotides containing an immune-stimulatory motif and an immune-regulatory motif act as antagonists of Toll-like receptor (TLR)7 and TLR9. In the present study, we designed and synthesized ...oligonucleotide-based antagonists of TLR7, 8 and 9 containing a 7-deaza-dG or arabino-G modification in the immune-stimulatory motif and 2'-O-methylribonucleotides as the immune-regulatory motif. We evaluated the biological properties of these novel synthetic oligoribonucleotides as antagonists of TLRs 7, 8 and 9 in murine and human cell-based assays and in vivo in mice and non-human primates. In HEK293, mouse and human cell-based assays, the antagonist compounds inhibited signaling pathways and production of a broad range of cytokines, including tumour necrosis factor alpha (TNF-α), interleukin (IL)-12, IL-6, interferon (IFN)-α, IL-1β and interferon gamma-induced protein (IP)-10, mediated by TLR7, 8 and 9. In vivo in mice, the antagonist compounds inhibited TLR7- and TLR9-mediated cytokine induction in a dose- and time-dependent fashion. Peripheral blood mononuclear cells (PBMCs) obtained from antagonist compound-treated monkeys secreted lower levels of TLR7-, 8- and 9-mediated cytokines than did PBMCs taken before antagonist administration. The antagonist compounds described herein provide novel agents for the potential treatment of autoimmune and inflammatory diseases.
Genetic vaccines are emerging as a powerful modality to induce T-cell responses to target tumor associated antigens (TAA). Viral or plasmid DNA or RNA vectors harbor an expression cassette encoding ...the antigen of choice delivered in vivo by vaccination. In this context, immunizations with minigenes containing selected, highly antigenic, T-cell epitopes of TAAs may have several advantages relative to full-length proteins. The objective of this study was to identify an optimal scaffold for minigene construction. We generated a number of minigenes containing epitopes from the carcinoembryonic antigen (CEA) model TAA and utilized muscle DNA electro-gene-transfer (DNA-EGT) to vaccinate HLA-A*0201 (HHD) and CEA/HHD double transgenic mice. The components utilized to construct the minigenes included CD8
+
T cell epitopes and (or) anchor modified analogs that were selected on the basis of their predicted binding to HLA-*A0201, their uniqueness in the human proteome, and the likelihood of cancer cell natural processing and presentation via MHC-I. Other candidate components comparatively tested included: helper CD4
+
T-cell epitopes, flanking regions for optimal epitope processing (including both proteasome-dependent and furin-dependent polypeptide processing mechanisms), and immunoenhancing moieties. Through a series of comparative studies and iterations we have identified an optimal minigene scaffold comprising the following elements: human tissue plasminogen activator (TPA) signal peptide, T-cell epitopes connected by furin sensitive linkers, and the E. Coli enterotoxin B subunit. The selected epitope modified minigenes (EMM) delivered by DNA-EGT were able to break immune tolerance in CEA/HHD mice and induce a strong immune response against all epitopes tested, independently of their relative positions within the scaffold. Furthermore, the optimized EMMs delivered via DNA-EGT were more immunogenic and exerted more powerful antitumor effects in a B16-CEA/HHD metastatic melanoma model than a DNA vector encoding the full-length protein or a mixture of the same peptides injected subcutaneously. Our data may shed light on the optimal design of a universal vehicle for epitope-targeted, genetic cancer vaccines.
Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, ...including IL-1β. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases.
Oligodeoxynucleotides containing unmethylated CpG dinucleotides induce innate and adaptive immunity through Toll-like receptor 9 (TLR9). In the present study, we have examined the ability of a novel ...agonist of TLR9, called immunomodulatory oligonucleotide (IMO), to enhance effects of a HER-2/neu plasmid DNA electroporation/adenovirus (DNA-EP/Ad) vaccine.
BALB/NeuT mice were treated with DNA-EP vaccine alone, IMO alone, or the combination of two agents starting at week 13, when all mice showed mammary neoplasia. Tumor growth and survival were documented. Antibody and CD8+ T-cell responses were determined. Peptide microarray analysis of sera was carried out to identify immunoreactive epitopes. Additionally, microCT and microPET imaging was carried out in an advanced-stage tumor model starting treatment at week 17 in BALB/NeuT mice.
The combination of DNA-EP and IMO resulted in significant tumor regression or delay to tumor progression. 2-Deoxy-2-18Ffluoro-D-glucose microPET and microCT imaging of mice showed reduced tumor size in the DNA-EP/IMO combination treatment group. Mice treated with the combination produced greater antibody titers with IgG2a isotype switch and antibody-dependent cellular cytotoxicity activity than did mice treated with DNA-EP vaccine. An immunogenic B-cell linear epitope, r70, within the HER-2 dimerization domain was identified through microarray analysis. Heterologous DNA-EP/Ad vaccination combined with IMO increased mice survival.
The combination of HER-2/neu genetic vaccine and novel agonist of TLR9 had potent antitumor activity associated with antibody isotype switch and antibody-dependent cellular cytotoxicity activities. These results support possible clinical trials of the combination of DNA-EP/Ad-based cancer vaccines and IMO.
Matrix metalloproteinases (MMP) are zinc-dependent endopeptidases that mediate numerous physiologic and pathologic processes, including matrix degradation, tissue remodeling, inflammation, and tumor ...metastasis. To develop a vaccine targeting stromal antigens expressed by cancer-associated fibroblasts, we focused on MMP11 (or stromelysin 3). MMP11 expression correlates with aggressive profile and invasiveness of different types of carcinoma.
To show the efficacy of a vaccine targeting MMP11, we constructed a series of plasmid DNA vectors expressing murine MMP11. Mice were vaccinated by i.m. injection followed by in vivo DNA electroporation. A chemically induced, MMP11-overexpressing colon cancer model was established and characterized. Antibody and T-cell responses were determined, and immunoreactive epitopes were characterized. To analyze the possible use of MMP11 as tumor-associated antigen in cancer patients, HLA-A2.1 transgenic mice (HHD) were used to identify reactive epitopes as tools to assess immunogenicity in humans.
Using microarray, we confirmed the overexpression of MMP11 mRNA in a large panel of human tumor samples. MMP11 vaccine induced cell mediated and antibody immune response and exerted significant antitumoral protection in mice with colon cancer in prophylactic and therapeutic settings. HHD transgenic mice were vaccinated with a plasmid encoding human MMP11, and a HLA-A2.1--restricted epitope (hMMP(237)) was identified. hMMP(237) was shown to be immunogenic in human peripheral blood mononuclear cells (PBMC) by in vitro priming.
Our study describes the identification of MMP11 as a novel broadly expressed tumor associated antigen as target candidate for cancer immunotherapy.
Abstract
Systemic Lupus Erythematosus is an autoimmune disease characterized by production of autoantibodies against nucleic acid-associated antigens. Endogenous DNA and RNA associated with these ...antigens stimulate inflammatory responses through Toll-like receptors (TLRs) and exacerbate lupus disease pathology. We have evaluated an antagonist of TLR7, 8 and 9 as a therapeutic agent in lupus-prone NZBW/F1 mice. NZBW/F1 mice treated with the antagonist had lower serum levels of autoantibodies targeting DNA, RNP, Smith antigen, SSA and SSB than did untreated mice. Reduction in blood urea nitrogen and proteinuria and improvements in kidney histopathology were observed in antagonist-treated mice. The antagonist treatment also reduced serum IL-12 and IL-1β and increased IL-10 levels. Levels of mRNA for IL-6, iNOS and IL-1β were lower in the kidneys and spleen of antagonist-treated mice than in those of untreated mice. Levels of mRNA for IP-10, TNFRSF9 and FASL were lower and IL-4 mRNA were higher in spleens of antagonist-treated mice than in spleens of untreated mice. mRNA for the inflammasome component NLRP3 was lower and mRNA for the antioxidant enzymes, catalase and glutathione peroxidase 1 was higher in the kidneys of antagonist-treated mice than in those of untreated mice. These results show that the antagonist of TLR7, 8 and 9 effectively inhibits inflammatory pathways involved in the development of lupus in NZBW/F1 mice and constitutes a potential therapeutic approach for the treatment of lupus and other autoimmune diseases.
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