Evaluating the effect of prodrug metabolism on the bystander effect in cancer gene therapy

Suicide gene therapy for cancer offers a selective approach to eradicating tumor cells by using non-toxic prodrugs in conjunction with prodrug-activating enzymes expressed at tumor sites. The efficacy of suicide gene therapy is often limited by the lack of a suitable gene delivery system and by the low activity of suicide enzymes towards the prodrug. These limitations may be overcome by using mutant and/or fusion enzymes that display increased activity towards a prodrug or by using enzymes that elicit a strong bystander effect (BE). The BE refers to the capability of transfected cells to induce death in neighboring untransfected cells, typically through intercellular transfer of cytotoxic prodrug metabolites. The Escherichia coli or bacterial cytosine deaminase (bCD) converts the prodrug 5-fluorocytosine (5FC) to the chemotherapeutic agent 5-fluorouracil (5FU). When coupled with E. coli uracil phosphoribosyltransferase (bUPRT), the resultant fusion enzyme, bCD/UPRT, induces greater cytotoxicity compared to bCD alone. Several bCD mutants generated in our laboratory also increase cytotoxicity relative to bCD. One mutant, bCD1525, was coupled with bUPRT in an attempt to augment cell death. It was hypothesized that expression of this fusion enzyme, bCD1525/UPRT, would enhance 5FC metabolism, thus generating higher concentrations of cytotoxic metabolites. We predicted that altered metabolite concentrations would shift the BE mechanisms away from 5FU diffusion (the mechanism typically reported for the 5FC/bCD system). We anticipated different mechanisms, namely transfer of apoptotic vesicles and metabolite movement through gap junctions, to play a more central role in the BE of cells expressing fusion enzymes. To examine 5FC metabolism, we developed two novel high-performance liquid chromatography (HPLC) methods, one to quantify 5FC anabolic products produced by suicide enzymes, the other to quantify a 5FC catabolic product generated by endogenous mammalian enzymes. Additionally, we examined the contribution of apoptotic vesicles to the BE and assessed the ability of our cancer cell lines to transfer compounds through gap junctions. Taken together, these data provide a more complete understanding of the relationship between prodrug metabolism, cytotoxicity, and the BE. This knowledge will aid in future endeavors to improve enzymes for more effective suicide gene therapy

Assessing B Cell Differentiation Following Asbestos Fiber Exposure

B cells are the main pathogenesis drivers in autoimmune diseases. Subtypes of B cells, known as B regulatory (Breg) or B10 cells contain a suppressive function, which down regulates the immune response. This is primarily conducted by IL-10, which is an inhibitory cytokine that is produced by the B10 subset. Recent evidence indicates that exposure to different asbestos fiber cause the activation of the immune system, however there is a difference in the response created by each fiber type. The Libby amphibole (LA) fiber leads to autoimmune response, whereas Chrysotile (Chry) fiber exposure does not. We hypothesize that this difference occurs because Chry exposure increases the production of B10 cell to keep the immune system suppressed. To test this, we will collect mouse splenocytes and expose the cells to the different asbestos fibers for 48 or 96 hours. As a control, the splenocytes will be exposed to the proteins, BAFF or IL-21. These proteins have been shown to stimulate proliferation of B10 cells. Next, we will use antibodies against cell surface markers and flow cytometry in order to identify B cell subtypes. Thus, we can investigate B cell differentiation, with the hypothesis that LA will decrease Breg differentiation, whereas Chry will increase Breg differentiation. This investigation will increase our understanding of how different asbestos fibers induce Breg production

Assessing the Free Radical Scavenging Ability and Antioxidant Efficiency of a Synthetic Flaxseed Lignan in an Asbestos Exposure Model

Asbestos exposure is known to cause pleural fibrosis, asbestosis, and other respiratory conditions, as well as systemic autoimmune disease (SAID). In order to help treat/prevent such illnesses, we are investigating the therapeutic potential of LGM2605, a synthetic derivative of the flaxseed lignan secoisolariciresinol diglucoside (SDG). This drug is an antioxidant and free radical scavenger which may block early immune responses (i.e. inflammation) observed with asbestos exposure. Studies have shown that mesothelial cells act as key regulators of inflammation through activation of the NLRP-3 inflammasome and the production of pro-inflammatory cytokines. Thus, we predicted that LGM2605 may help reduce these activities. We performed several DCFDA ROS assays comparing the production of reactive oxygen species (ROS) in mesothelial cells exposed to various asbestos fiber types. We found that Libby Amphibole (LA) fibers yield the highest level of ROS activity, suggesting higher levels of inflammasome activation and pathogenicity. Next, we performed additional ROS assays to determine the effects of LGM2605 treatment. We found that LGM2605 reduces ROS production in mesothelial cells exposed to asbestos fibers, which we suspect may also reduce inflammasome activation. Subsequent investigation will determine the effects of LGM2605 treatment in mice that have been exposed to LA through intraperitoneal injection

Investigating the Correlation Between Immune Responses and Collagen Deposition in Lungs of Amphibole Asbestos-Exposed Mice Through the Use of Multiphoton Microscopy and Paraffin Tissue Embedding

Asbestos are naturally occurring fibers found in rock outcroppings but also used in many commercial processes; therefore, exposures are both environmental and occupational. When inhaled, amphibole asbestos can lead to various health issues, such as cancers, autoimmune diseases, and pleural fibrosis (excess collagen deposition). Previous research in our lab has shown that anti-mesothelial cell autoantibodies (MCAAs) are produced following amphibole asbestos exposure and may be the driving force of collagen formation during fibrosis. In the present work, we are confirming the functional relationship between the presence of autoantibodies and presence of collagen deposition. We are using multiphoton microscopy to image lung samples from mice that have been exposed to different forms of amphibole asbestos (Libby amphibole or Arizona amphibole) and have shown MCAA production. We hypothesize that mice with MCAA will show higher collagen levels in the pleural tissue This research will allow for a better understanding of the role MCAA has in fibrosis development. This information could lead to fibrosis treatment strategies aimed at blocking MCAA function

Macrophage Cytotoxicity and Global DNA Methylation Following Carbon Fiber Dust Exposure

Carbon fiber composite materials are used in a wide variety of industries including automobile, golf, and archery applications. Modification of carbon materials by cutting or machining can generate particulate matter which represents a potential occupational health risk. While the pulmonary health effects of carbon nanotubes (1-10nm) and carbon nanofibers (100-1000nm) have been well studied, the toxicity of carbon fibers (1-10um) has not been well characterized. Here, we evaluate the biophysical properties and cytotoxic potential of carbon fibers generated during arrow cutting. An initial determination of carbon fiber size and shape was accomplished using FESEM. The fibers had mean width of 7.21um (SD=0.93) and mean length of 33.99um (SD=8.95). Then, we demonstrated dose-dependent cellular toxicity in mouse RAW264.7 macrophages using a CellTiter-Blue assay and contextualized this with data generated from asbestos exposure (a similarly sized and shaped toxicant). Further, we explored a potential mechanism that might explain this cytotoxicity using bisulfite pyrosequencing to measure DNA methylation in repetitive elements. Carbon fiber exposed mouse macrophages did not demonstrate differential DNA methylation at LINE1 or IAP repetitive elements compared to untreated controls. Future directions for this work include examination of other epigenetic regions of interest for DNA methylation differences, interrogation of cell signaling cascades that could result in cell death, and additional in vitro exposures using human-derived cells or primary cultured cells

Assessing the Effects of a Synthetic Flaxseed Lignan on the Gut Microbiome

In healthy individuals, the gut microbiome serves many functions: it aids in digestion, confers protection against pathogenic organisms, synthesizes essential vitamins and minerals, and plays an important role in the immune system. Dysbiosis of the gut microbiome can cause dysregulation of these functions and has been linked to systemic autoimmune disease (SAID) in both humans and animal models. Dietary intervention has been shown to be an effective means of improving gut health and ameliorating inflammatory responses associated with SAID by beneficially altering the diversity of gut microbes. Previous studies have demonstrated that dietary flaxseed administration may attenuate gut dysbiosis and reduce acute inflammation. Therefore, we hypothesized that oral administration of LGM2605, a synthetic flaxseed derivative, would beneficially alter the gut microbiome of mice. To investigate this, mice were treated with LGM2605 via oral gavage over a 10-day period. Mouse cecums were then extracted, and 16S rRNA gene sequencing was performed in order to identify microbial genera of interest. The abundance and diversity of gut microbes were compared between treatment groups using Microbiome Analyst and R. Alterations in the composition of the gut microbiome as a result of LGM2605 treatment were observed. Analyses of specific changes in gut microbial communities are ongoing

Plasminogen Binding and Activation at the Mesothelial Cell Surface Promotes Invasion through a Collagen Matrix

Plasminogen (Plg) activation to the serine protease plasmin (Pla) plays a key role in regulating wound healing and fibrotic responses, particularly when bound to cell surface receptors. Our previous work suggested that mesothelial cells bind Plg at the cell surface, though no Plg receptors were described for these cells. Since mesothelial cells contribute to injury responses, including cellular differentiation to a mesenchymal-like phenotype and extracellular matrix remodeling, we hypothesized that Plg binding would promote these responses. Here, we confirm that Plg binds to both pleural and peritoneal mesothelial cells via the lysine-binding domain present in Plg, and we demonstrate the presence of three Plg receptors on the mesothelial cell surface: α-Enolase, Annexin A2, and Plg-RKT. We further show that bound-Plg is activated to Pla on the cell surface and that activation is blocked by an inhibitor of urokinase plasminogen activator or by the presence of animal-derived FBS. Lastly, we demonstrate that Plg promotes mesothelial cell invasion through a type I collagen matrix but does not promote cellular differentiation or proliferation. These data demonstrate for the first time that mesothelial cells bind and activate Plg at the cell surface and that active Pla is involved in mesothelial cell invasion without cell differentiation

Autoimmunity and Asbestos Exposure

Despite a body of evidence supporting an association between asbestos exposure and autoantibodies indicative of systemic autoimmunity, such as antinuclear antibodies (ANA), a strong epidemiological link has never been made to specific autoimmune diseases. This is in contrast with another silicate dust, crystalline silica, for which there is considerable evidence linking exposure to diseases such as systemic lupus erythematosus, systemic sclerosis, and rheumatoid arthritis. Instead, the asbestos literature is heavily focused on cancer, including mesothelioma and pulmonary carcinoma. Possible contributing factors to the absence of a stronger epidemiological association between asbestos and autoimmune disease include (a) a lack of statistical power due to relatively small or diffuse exposure cohorts, (b) exposure misclassification, (c) latency of clinical disease, (d) mild or subclinical entities that remain undetected or masked by other pathologies, or (e) effects that are specific to certain fiber types, so that analyses on mixed exposures do not reach statistical significance. This review summarizes epidemiological, animal model, and in vitro data related to asbestos exposures and autoimmunity. These combined data help build toward a better understanding of the fiber-associated factors contributing to immune dysfunction that may raise the risk of autoimmunity and the possible contribution to asbestos-related pulmonary disease

Late Inflammation Induced by Asbestiform Fibers in Mice Is Ameliorated by a Small Molecule Synthetic Lignan

Exposure to Libby amphibole (LA) asbestos-like fibers is associated with increased risk of asbestosis, mesothelioma, pulmonary disease, and systemic autoimmune disease. LGM2605 is a small molecule antioxidant and free radical scavenger, with anti-inflammatory effects in various disease models. The current study aimed to determine whether the protective effects of LGM2605 persist during the late inflammatory phase post-LA exposure. Male and female C57BL/6 mice were administered daily LGM2605 (100 mg/kg) via gel cups for 3 days before and 14 days after a 200 µg LA given via intraperitoneal (i.p.) injection. Control mice were given unsupplemented gel cups and an equivalent dose of i.p. saline. On day 14 post-LA treatment, peritoneal lavage was assessed for immune cell influx, cytokine concentrations, oxidative stress biomarkers, and immunoglobulins. During the late inflammatory phase post-LA exposure, we noted an alteration in trafficking of both innate and adaptive immune cells, increased pro-inflammatory cytokine concentrations, induction of immunoglobulin isotype switching, and increased oxidized guanine species. LGM2605 countered these changes similarly among male and female mice, ameliorating late inflammation and altering immune responses in late post-LA exposure. These data support possible efficacy of LGM2605 in the prolonged treatment of LA-associated disease and other inflammatory conditions