INF-γ encoding plasmid administration triggers bone loss and disrupts bone marrow microenvironment

IFN-γ is a pleotropic cytokine produced in the bone microenvironment. Although IFN-γ is known to play a critical role on bone remodeling, its function is not fully elucidated. Consistently, outcomes on the effects of IFN-γ recombinant protein on bone loss are contradictory among reports. In our work we explored, for the first time, the role of IFN-γ encoding plasmid (pIFN-γ) in a mouse model of osteopenia induced by ovariectomy and in the sham-operated counterpart to estimate its effects in skeletal homeostasis. Ovariectomy produced a dramatic decrease of bone mineral density (BMD). pINF-γ injected mice showed a pathologic bone and bone marrow phenotype; the disrupted cortical and trabecular bone microarchitecture was accompanied by an increased release of pro-inflammatory cytokine by bone marrow cells. Moreover, mesenchymal stem cells’ (MSCs) commitment to osteoblast was found impaired, as evidenced by the decline of osterix-positive (Osx+) cells within the mid-diaphyseal area of femurs. For instance, a reduction and redistribution of CXCL12 cells have been found, in accordance with bone marrow morphological alterations. As similar effects were observed both in sham-operated and in ovariectomized mice, our studies proved that an increased IFN-γ synthesis in bone marrow might be sufficient to induce inflammatory and catabolic responses even in the absence of pathologic predisposing substrates. In addition, the obtained data might raise questions about pIFN-γ’s safety when it is used as vaccine adjuvant

Genetic immunization against neu /erbB2 transgenic breast cancer

erbB2/neu, an overexpressed oncogene product, has been proposed as a human cancer vaccine target. In the present study, transgenic (rat neuNT oncogene) FVB/neu mice, developing metastasizable mammary carcinoma, were immunized with a plasmid DNA encoding are not tolerant to the self antigen and sequences. We report that transgenic tumour-bearing mice: like some breast cancer patients erbB2 + X, develop anti-neu autoimmune responses, which can be boosted and skewed to a Thl phenotype by DNA immunization. Intramuscular injections of neuNT plasmid. drastically reduced (or even prevented in a small number of treated mice) the outgrowth of mammary neoplasms as well as their metastatic penetrance. Furthermore, DNA immunization caused haemorrhagic necrosis of established cancer nests, leaving a greatly reduced portion of the tumour burden for the host to cope with. The antitumour activities we obtained, in this very challenging model for cancer immunotherapy, lay the foundation for DNA-based immunization to control erbB2/neu-overexpressing neoplasms

neu/HER2 cDNA Vaccination and pregnancy lost.

no abstract availabl

Plasmids encoding protein aggregation domains act as molecular adjuvants for DNA vaccines

BACKGROUND: DNA vaccines provide high tolerability and safety but commonly suffer from suboptimal immunogenicity. We previously reported that a plasmid vector (pATRex), encoding the DNA sequence for the von Willebrand I/A domain of the tumor endothelial marker-8 (TEM8) when given in combination with plasmid-encoded tumor antigens acted as a powerful molecular adjuvant enhancing immunity against breast and melanoma tumors. AIMS: In the present study we addressed two unsolved issues; would the adjuvant action of pATRex extend to a DNA vaccine against infectious disease and, if so, what is the mechanistic basis for pATRex adjuvant action? RESULTS: Here we show in a murine malaria vaccine model that co-administration of pATRex potentiates antibody production elicited by an intramuscular injection of plasmid encoding Plasmodium yoelii merozoite surface protein 4/5 (PyMSP4/5). pATRex enhanced the B-cell response and induced increased IgG1 production consistent with TH2 polarization of the DNA vaccine response. To explore the mechanism of adjuvant action, cells were transfected in vitro with pATRex and this resulted in formation of insoluble intracellular aggregates and apoptotic cell death. Using a structural modeling approach we identified a short peptide sequence (α3-β4) within ATRex responsible for protein aggregation and confirmed that transfection of cells with plasmid encoding this self-assembling peptide similarly triggered intracellular aggregates, caspase activation and cell death. CONCLUSION: Plasmids encoding aggregation-promoting domains induce formation of insoluble intracellular aggregates that trigger caspase activation and apoptotic cell death leading to activation of the innate immune system thereby acting as genetic adjuvants

Immunorecognition of ring skeleton of taxanes by chicken egg yolk antibodies

Anti-10 deacetylbaccatin III (DAB) antibodies (IgY) were elicited in hens immunized with a succinyl-DAB/BSA conjugate and extracted from egg yolk. As shown by indirect competitive inhibition enzyme immunoassay (CIEIA), the addition of free-DAB competitively inhibited the binding of affinity purified anti-DAB IgY to DAB/BSA solid phase conjugated antigen. The assay enabled the detection of DAB in concentrations as low as 7.5ng/ml (13.7 nM DAB), whereas anti-DAB IgY did not react with taxol even at a concentration a thousand times higher. The structural requirements of the diterpenoid nucleus for binding to IgY were considered on the basis of the levels of cross-reaction found with 10 authentic taxanes. The results indicate that anti-DAB IgY represents the first high affinity antibody produced capable of recognizing the ring skeleton of taxol precursors

Co-Expression of Flt-3 Ligand Gene Ablates Tumor Immunity Elicited by HER-2/neu DNA Vaccine in Transgenic Mice

Fms-like tyrosine-kinase 3 ligand (Flt-3L), is a powerful hematopoyetic growth factor, known to modulate the immune response against delivered antigens by acting either as an adjuvant or tolerogenic stimulus. In this study we evaluated the use of murine Flt-3 ligand plasmid (pFl) in combination with a DNA vaccine encoding rat-p185 oncoprotein extra cellular domain (pECD) in the prevention of mammary carcinogenesis in rat-neu HER-2 mutated (neuT) transgenic mice. We demonstrate that intramuscular (i.m.) co-immunization of pFl inhibits the production of anti-HER-2 antibody elicited by pECD vaccine, resulting in the development of spontaneous carcinomas in all co-immunized mice. The inhibitory effect on antibody production by mFlt3 gene appeared to be: dose-dependent, linked to the injection site and timing, and transient in nature. Additionally, we show that co-administration of pFI and pECD plasmids was unable to trigger cytotoxic T-cell immune response in neuT mice. On the other hand, we found that the combination of pFl with pECD had no impact on the ability of pECD to reject HER-2+ transplantable tumors in parental mice. In summary our results demonstrate that, depending on tumor model, co-administration of pFl gene can produce untoward effects to immune response, and thus its application as a vaccine adjuvant should be carefully evaluated