The Development of Methods for the Production of New Molecular Vaccines and Appropriate RNA Fragments to Counteract Unwanted Genes: A Pilot Study.

The potential of viruses as appropriate vectors for the development of new therapeutic strategies, as well as for the design of molecular (DNA, RNA, and/or protein) vaccines via substitution of nucleotide sequences, has been proven. Among the most appropriate DNA and/or RNA fragments, members belonging to families Parvoviridae (particularly adeno-associated virus, AAV) and Poxviridae have frequently been suggested for this purpose. In previous studies, the vaccine avipoxvirus strains FK (fowl) and Dessau (pigeon) have been proven able to infect mammalian cells (as well as avian cells), and to replicate productively in a small number of them; thus, we may be able to adapt them using incubation, and in these conditions. Additionally, we have previously proved, based on AAV recombinant DNA vectors, that it is possible to transfer appropriate genes of interest via mouse embryonic stem cells (mESCs). In the current study, we develop methods for the application of the same vaccine avipoxviral strains, based on the AAV DNA genome recombinant constructs, to be used for gene transfer in cells, for the transfer of DNA and/or RNA fragments (for the suppression of unwanted viral and/or cellular genes), and for the production of molecular (DNA, RNA, and/or protein) anti-cancer and anti-viral vaccines. To this end, sub-populations of embryonic mammalian cells infected with the two forms of both vaccine avipoxviral strains were frozen in the presence of cryo-protector dimethylsulfoxide (DMSO), subsequently thawed, and re-incubated. In most cases, the titers of the intra-cellular forms of the two strains were higher than those of their extra-cellular forms. These data were explained by the probable existence of the intra-cellular forms as different sub-forms, including those integrated in the cellular genome proviruses at a given stage of the cellular infection, and suggest the possibility of transferring nucleotide (DNA and/or RNA) fragments between cellular and viral genomes; this is due to the influence of activated fusion processes on DMSO, as well as drastic temperature variations.

IL-12 regulates the expansion, phenotype, and function of murine NK cells activated by IL-15 and IL-18.

NK cells, which are composed of phenotypically and functionally heterogeneous subpopulations, play critical roles in immunity against cancer. The mechanism of generation of distinct subsets such as the effector and regulatory subtypes is unclear. Here, we show that this process comprises several steps, including generation of proliferating, highly cytotoxic cells activated by IL-15/IL-18 and differentiation into distinct cell populations induced with IL-12. Freshly prepared murine splenic NK cells expressed IL-15Rs and IL-18Rs and rapidly began to proliferate following stimulation with IL-15/IL-18. The proliferating NK cells highly expressed various activation markers such as B220, CD49b (DX5), lysosome-associated membrane glycoprotein 1 (LAMP-1), DNAX accessory molecule 1, perforin, and granzyme B and showed reduced expression of natural killer cell p46-related protein (NKp46) and IL-18Rα. These cells exerted strong cytotoxicity against YAC-1 cells, but did not secrete cytokines. IL-12 rapidly activated STAT4 in these cells, induced IFN-γ production, and then upregulated p21 and p27, leading to withdrawal from the cell cycle. In parallel, IL-12-stimulated cells gradually reduced cytotoxicity, decreased expression of activation markers, and instead increased expression of Sca-1, CD25, CD49a, and NKp46. Some IL-15/IL-18-induced cells strongly expressed PD-1, whereas NK cells induced with IL-15/IL-18 and IL-12 expressed high levels of T cell immunoglobulin mucin-3, LAG-3, and natural killer group 2 A. Furthermore, these cells spontaneously secreted IL-10 and TGF-ß following prolonged incubation. Thus, IL-12 regulates expansion of NK cells activated with IL-15/IL-18, influences the population size of highly cytotoxic cells, and induces differentiation to unique cells sharing some phenotypes of ILCs.

Upregulation of Natural Killer Cells Proliferation by Cytokine Stimulation.

Natural killer (NK) cells can discriminate between normal and cancer cells and are known to directly recognize and kill malignant cells or induce apoptosis. Thus, activation of NK cells is considered as a promising strategy for cancer treatment. However, clinical application has been somewhat limited because of difficulties in the preparation of sufficient number of highly cytotoxic/activated NK cells in vitro. We used cytokine stimulation to provide a suitable environment (activating receptor-ligand interactions) for the expansion of NK cells. This method potently expanded NK cells, and the final product was composed of highly proliferating NK cells. The expanded NK cells showed significant upregulation of various activation receptors such as CD69 and NKG2D. The latter is a particularly important receptor for triggering NK cell responses toward tumor cells.

Еvaluation of biocompatibility and antioxidant efficiency of chitosan-alginate nanoparticles loaded with quercetin.

The present study deals with development and evaluation of the safety profile of chitosan/alginate nanoparticles as a platform for delivery of a natural antioxidant quercetin. The nanoparticles were prepared by varying the ratios between both biopolymers giving different size and charge of the formulations. The biocompatibility was explored in vitro in cells from different origin: cultivated HepG2 cells, isolated primary rat hepatocytes, isolated murine spleen lymphocytes and macrophages. In vivo toxicological evaluation was performed after repeated 14-day oral administration to rats. The study revealed that chitosan/alginate nanoparticles did not change body weight, the relative weight of rat livers, liver histology, hematology and biochemical parameters. The protective effects of quercetin-loaded nanoparticles were investigated in the models of iron/ascorbic acid (Fe2+/AA) induced lipid peroxidation in microsomes and tert-butyl hydroperoxide oxidative stress in isolated rat hepatocytes. Interesting finding was that the empty chitosan/alginate nanoparticles possessed protective activity themselves. The antioxidant effects of quercetin loaded into the nanoparticles formulated with higher concentration of chitosan were superior compared to quercetin encapsulated in nanoparticles with higher amount of sodium alginate. In conclusion, chitosan/alginate nanoparticles can be considered appropriate carrier for quercetin, combining safety profile and improved protective activity of the encapsulated antioxidant.

Augmentation of Immune Checkpoint Cancer Immunotherapy with IL18.

PURPOSE: Recent clinical trials and animal models demonstrated that immune checkpoint blockade enhanced effector cell responses and tumor rejection; however, further development and improvement of cancer immunotherapy is necessary for more favorable objective responses. In this study, we examined the effect of IL18 on the antitumor effect of immune checkpoint inhibitors. EXPERIMENTAL DESIGN: We examined the effect of IL18 on the peritoneal dissemination of CT-26 cells or tail vein injection metastasis of B16/F10 cells using antiprogrammed death-1 ligand-1 (αPD-L1) and/or anti-CTL-associated antigen-4 (αCTLA-4) mAbs. RESULT: Massive ascites developed after intraperitoneal inoculation of CT-26, resulting in animal death within 30 days. Treatment of mice with αPD-L1 and/or αCTLA-4 significantly prolonged their survival, and a combination of the antibodies and IL18 provided a much greater therapeutic benefit. The combination modality led to the accumulation of precursor of mature natural killer (pre-mNK) cells in the peritoneal cavity together with increased CD8(+) T and decreased CD4(+)CD25(+)Foxp3(+) T cells. Depletion of the pre-mNK cells abrogated the therapeutic effects and increased the number of CD4(+)CD25(+)Foxp3(+) T cells. The combination treatment also suppressed tail vein injection metastasis of B16/F10 cells. CONCLUSIONS: The results demonstrated that IL18 enhanced therapeutic effects of immune checkpoint blockade against peritoneal dissemination of carcinoma or tail vein injection metastasis of melanoma through accumulation of pre-mNK cells, memory-type CD8(+) T cells, and suppression of CD4(+)CD25(+)Foxp3(+) T cells. A combination of immune checkpoint inhibitors with IL18 may give a suggestion to the development of next-generation cancer immunotherapy. Clin Cancer Res; 22(12); 2969-80. ©2016 AACR.

Antagonism of IL-4 signaling by a phosphodiesterase-4 inhibitor, rolipram, in human T cells.

BACKGROUND: Phosphodiesterase (PDE4) inhibitors prevent breakdown of cAMP and affect the increase in cellular levels of cAMP, which is known to regulate immune cell functions. Because IL-4 plays a causal role in the pathogenesis of allergic disorders, we were interested to study the modulatory mechanisms of a PDE4 inhibitor, rolipram, in IL-4-mediated signaling in T cells. METHODS: Human peripheral T cells were stimulated with IL-4 in combination with rolipram, and RT-PCR was performed using primers specific for IL-5. To monitor activation of transcription factors, immunostaining was employed. RESULTS: Rolipram or a cAMP-analogue, 8-Br-cAMP, significantly downregulated IL-4-induced expression of IL-5 mRNA. The rolipram-induced inhibition of IL-5 mRNA was mediated by activation of protein kinase A (PKA), because rolipram-downregulated mRNA expression of IL-5 was restored by PKA inhibitors. Immunostaining revealed that rolipram interfered with IL-4-induced nuclear translocation of activator protein (AP)-1 components. CONCLUSIONS: This is the first demonstration of suppression of IL-4 signaling by PDE4 inhibitors via prevention of nuclear translocation of AP-1.