Antitumor Potential of Extracellular Vesicles Released by Genetically Modified Murine Colon Carcinoma Cells With Overexpression of Interleukin-12 and shRNA for TGF-β1

Recent developments demonstrate that tumor-derived extracellular vesicles (EVs) could become a highly effective tool for delivery of antitumor factors. The main objective of the study was to determine whether EVs secreted by MC38 colon carcinoma cells genetically engineered for overproduction of interleukin (IL-)12 and/or shRNA targeting TGF-β1 are effectively loaded with these molecules and whether the obtained EVs could be an efficient tool for antitumor therapy. Fractions of EVs released by genetically modified MC38 cells [both modified tumor-derived exosomes (mTEx) and modified microvesicles (mTMv)] and those released by unmodified, wild-type MC38 cells were characterized in terms of loading efficacy, using real-time PCR and ELISA, as well as their antitumor potential. In order to examine the therapeutic potential of mTEx, they were applied in the form of sole treatment as well as in combination with dendritic cell (DC)-based vaccines stimulated with mTMv in the therapy of mice with subcutaneously growing MC38 tumors. The results demonstrated that genetic modification of wild-type MC38 tumor cells is an effective method of loading the molecules of interest into extracellular vesicles secreted by the cells (both TEx and TMv). The results also showed that mTEx secreted by cells engineered for overproduction of IL-12 and/or shRNA for TGF-β1 are able to induce tumor growth inhibition as opposed to TEx from unmodified MC38 cells. Additionally, antitumor therapy composed of mTEx (especially those deprived of TGF-β1) and DC-based vaccines allowed for regeneration of antitumor immunity and induction of the systemic Th1 response responsible for the sustained effect of the therapy. In conclusion, tumor-derived exosomes loaded with IL-12 and/or deprived of TGF-β1 could become an efficient adjuvant supporting induction of a specific antitumor response in both immuno- and chemotherapeutic schemes of treatment

Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.

Lipopolysaccharide (LPS, endotoxin, pyrogen) constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol). During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU) in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 10(3) and 10(5) EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 10(3)-10(5) EU/10(9) PFU (plaque forming units) down to an average of 2.8 EU/10(9) PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli) and F8 (P. aeruginosa)

Outline of purification protocol.

<p>Outline of purification protocol.</p

Bacteriophage Tail Proteins as a Tool for Bacterial Pathogen Recognition—A Literature Review

In recent years, a number of bacterial detection methods have been developed to replace time-consuming culture methods. One interesting approach is to mobilize the ability of phage tail proteins to recognize and bind to bacterial hosts. In this paper, the authors provide an overview of the current methodologies in which phage proteins play major roles in detecting pathogenic bacteria. Authors focus on proteins capable of recognizing highly pathogenic strains, such as Acinetobacter baumannii, Campylobacter spp., Yersinia pestis, Pseudomonas aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Enterococcus spp., Salmonella spp., and Shigella. These pathogens may be diagnosed by capture-based detection methods involving the use of phage protein-coated nanoparticles, ELISA (enzyme-linked immunosorbent assay)-based methods, or biosensors. The reviewed studies show that phage proteins are becoming an important diagnostic tool due to the discovery of new phages and the increasing knowledge of understanding the specificity and functions of phage tail proteins

Aggregation/dispersion transitions of T4 phage triggered by environmental ion availability

Abstract Background Bacteriophage survives in at least two extremes of ionic environments: bacterial host (high ionic-cytosol) and that of soil (low ionic-environmental water). The impact of ionic composition in the micro- and macro-environments has not so far been addressed in phage biology. Results Here, we discovered a novel mechanism of aggregation/disaggregation transitions by phage virions. When normal sodium levels in phage media (150 mM) were lowered to 10 mM, advanced imaging by scanning electron microscopy, atomic force microscopy and dynamic light scattering all revealed formation of viral packages, each containing 20–100 virions. When ionic strength was returned from low to high, the aggregated state of phage reversed to a dispersed state, and the change in ionic strength did not substantially affect infectivity of the phage. By providing the direct evidence, that lowering of the sodium ion below the threshold of 20 mM causes rapid aggregation of phage while returning Na+ concentration to the values above this threshold causes dispersion of phage, we identified a biophysical mechanism of phage aggregation. Conclusions Our results implicate operation of group behavior in phage and suggest a new kind of quorum sensing among its virions that is mediated by ions. Loss of ionic strength may act as a trigger in an evolutionary mechanism to improve the survival of bacteriophage by stimulating aggregation of phage when outside a bacterial host. Reversal of phage aggregation is also a promising breakthrough in biotechnological applications, since we demonstrated here the ability to retain viable virion aggregates on standard micro-filters

Boron-Rich Boron Carbide Nanoparticles as a Carrier in Boron Neutron Capture Therapy: Their Influence on Tumor and Immune Phagocytic Cells

The aim of the work was to study the interaction between boron-rich boron carbide nanoparticles and selected tumor and immune phagocytic cells. Experiments were performed to investigate the feasibility of the application of boron carbide nanoparticles as a boron carrier in boron neutron capture therapy. Boron carbide powder was prepared by the direct reaction between boron and soot using the transport of reagents through the gas phase. The powder was ground, and a population of nanoparticles with an average particle size about 80 nm was selected by centrifugation. The aqueous suspension of the nanoparticles was functionalized with human immunoglobulins or FITC-labeled human immunoglobulins and was then added to the MC38 murine colon carcinoma and to the RAW 264.7 cell line of mouse macrophages. Flow cytometry analysis was used to determine interactions between the functionalized boron carbide nanoparticles and respective cells. It was shown that B4C–IgG nanoconjugates may bind to phagocytic cells to be internalized by them, at least partially, whereas such nanoconjugates can only slightly interact with molecules on the cancer cells’ surface

Table_1_Inhibition of MC38 colon cancer growth by multicomponent chemoimmunotherapy with anti-IL-10R antibodies, HES-MTX nanoconjugate, depends on application of IL-12, IL-15 or IL-18 secreting dendritic cell vaccines.docx

BackgroundThe tumor microenvironment (TME) provides a conducive environment for the growth and survival of tumors. Negative factors present in TME, such as IL-10, may limit the effectiveness of cellular vaccines based on dendritic cells, therefore, it is important to control its effect. The influence of IL-10 on immune cells can be abolished e.g., by using antibodies against the receptor for this cytokine - anti-IL-10R. Furthermore, the anticancer activity of cellular vaccines can be enhanced by modifying them to produce proinflammatory cytokines, such as IL-12, IL-15 or IL-18. Additionally, an immunomodulatory dose of methotrexate and hydroxyethyl starch (HES-MTX) nanoconjugate may stimulate effector immune cells and eliminate regulatory T cells, which should enhance the antitumor action of immunotherapy based on DC vaccines. The main aim of our study was to determine whether the HES-MTX administered before immunotherapy with anti-IL-10R antibodies would change the effect of vaccines based on dendritic cells overproducing IL-12, IL-15, or IL-18.MethodsThe activity of modified DCs was checked in two therapeutic protocols - immunotherapy with the addition of anti-IL10R antibodies and chemoimmunotherapy with HES-MTX and anti-IL10R antibodies. The inhibition of tumor growth and the effectiveness of the therapy in inducing a specific antitumor response were determined by analyzing lymphoid and myeloid cell populations in tumor nodules, and the activity of restimulated splenocytes.Results and conclusionsUsing the HES-MTX nanoconjugate before immunotherapy based on multiple administrations of anti-IL-10R antibodies and cellular vaccines capable of overproducing proinflammatory cytokines IL-12, IL-15 or IL-18 created optimal conditions for the effective action of these vaccines in murine colon carcinoma MC38 model. The applied chemoimmunotherapy caused the highest inhibition of tumor growth in the group receiving DC/IL-15/IL-15Rα/TAg + DC/IL-18/TAg at the level of 72.4%. The use of cellular vaccines resulted in cytotoxic activity increase in both immuno- or chemoimmunotherapy. However, the greatest potential was observed both in tumor tissue and splenocytes obtained from mice receiving two- or three-component vaccines in the course of combined application. Thus, the designed treatment schedule may be promising in anticancer therapy.</p

Structural patterns enhancing the antibacterial activity of metallacarborane-based antibiotics

Healthcare systems heavily rely on antibiotics to treat bacterial infections but widespread of the multidrug-resistant bacteria puts this strategy in danger. Novel drugs capable of overcoming current resistances are needed if our ability to treat bacterial infections is to be maintained. Boron clusters offer a valuable possibility to create a new class of antibiotics and expand antibiotic’s chemical space beyond conventional carbon-based molecules. In this work, we identified the two promising structural patterns providing cobalta bis(dicarbollide)(COSAN)-based compounds with potent and selective activity toward Staphylococcus aureus (including clinical strains): introduction of the α-amino acid amide and addition of iodine directly to the metallacarborane cage. Furthermore, we found that proper hydrophilic-lipophilic balance is crucial for the selective activity of the tested compounds toward S. aureus over mammalian cells. The patterns proposed in this paper can be useful in the development of metallacarborane-based antibiotics with potent antibacterial properties and low cytotoxicity