2011 Abstracts

Inhibition of IL-33 via Caspase-1 blockade in C-10 Mouse Type II Epithelial Lung Cells
Bevin Alison LyBarger, Teri Alyn Girtsman, and Andrij Holian

IL-33 is a recently characterized IL-1 family cytokine that is expressed in inflammatory diseases, including severe asthma and inflammatory bowl disease. Currently, it is unclear how IL-33 is converted to its active form. The current literature has conflicting data associating caspase-1 with both activation of pro IL-33, or cleavage of mature IL-33 to an inactive form. The Berkeley Pit in Butte, Montana, is an acid mine waste reservoir rich in toxic metals and is part of the largest EPA Superfund site in North America. Three unique compounds isolated from microbes collected from the Berkeley Pit (DNA-63, DNA-65 and 4-41-D), previously evaluated in cell free assays, demonstrated potent inhibitory activity to Caspase-1. Engineered nanomaterial (ENM) such as TiO2 and multi-walled carbon nanotubes (MWCNT) have been previously shown to activate the other IL-1 family cytokines, IL-1b and IL-18.  In this study, we investigated the ability of the Berkeley Pit isolates to inhibit IL-33 production in C10 mouse lung type II epithelial cells exposed to ENM. C10 cells were exposed to ENM in the presence of the isolates for 24 hours. Cell viability, and IL-33 secretion into the supernatant were evaluated by MTS cell viability assay and IL-33 ELISA, respectively. We found that ENM induced IL-33 in C10 cells and that this effect was reversed in the presence of the positive control, Caspase-1 inhibitor Ac-YVAD-CHO.  Preliminary results demonstrated that DNA-63, DNA-65 and 4-41-D inhibited IL-33 in C10 cells exposed to MWCNT. Collectively, these data suggest Berkeley Pit isolates as potential anti-inflammatory therapeutics.

The Reduction of SPARC Expression to Reduce Collagen Deposition in Pulmonary Fibrosis
Elizabeth Patty and Elizabeth Putnam

Pulmonary fibrosis affects thousands of people and their families each year. On average about 40,000 people die from pulmonary fibrosis each year.  A symptom of pulmonary fibrosis is collagen deposition, which fills the air space in the lungs, eliminating crucial surface area necessary for the gas exchange of carbon dioxide for oxygen thus hindering respiration. Our lab has determined that collagen expression in the lung may be modulated by a protein called SPARC (Secreted Protein Acidic and Rich in Cysteine). We hypothesize that reducing SPARC expression through siRNA knockdown will reduce the collagen deposition in pulmonary fibrosis. To test this hypothesis, multiple primary lung fibroblast cell strains have been established for use in in-vitro experiments. These cell strains are exposed to asbestos followed by Lentivirus/ shRNA treatment. Western blotting and histochemistry are then used to analyze the resulting changes. In parallel, an asbestos-induced mouse lung model of fibrosis is being used. Asbestos was intratracheally instilled into the mice. After two months of exposure, the lentivirus containing SPARC-specific shRNA sequence will then be intratracheally instilled and the lungs will be harvested 30 days later. These lungs will be analyzed by histochemistry and protein analysis.  These experiments are still in progress. The research project will continue past the 10 weeks of the STEER program and will be completed by Cicily Bull Calf and Elizabeth Putnam.  In the future we hope to see a decrease in asbestos-induced collagen expression after SPARC knockdown via lentivirus/ shRNA.

Targeted Drug Therapy: Novel Anti-Cancer Agents
Gabriella Torti

Telomerase is an enzyme that protects the integrity of coding DNA by adding DNA sequence repeats to non-coding telomeres at the terminal ends of chromosomes.  With time, normal cells down-regulate the expression of telomerase and undergo apoptosis when the telomeres become too short; however, tumor cells re-express high telomerase activity that allows the proliferation of the cancer cells. Telomeric single strands are guanine rich and capable of forming G-quartets that then stack in various arrangements forming G-quadruplexes (G4 DNA).  Recently, there has been increased interest in telomeric G4 DNA as it has been shown that molecules that bind telomeric G4 DNA inhibit telomerase’s ability to elongate the telomere and therefore may possess anti-cancer activity. We hypothesize that a novel series of anthracenyl isoxazole amides (AIMs) that are selective for telomeric G4 DNA will exhibit significant activity against brain tumors.  Two AIMs, MG-70 and MG-71, were studied for their cytotoxicity, mechanism of cell death and their ability to localize in the nuclei of SNB-19 glioblastoma cells. MG-70 and MG-71 had IC₅₀ values of 3.1 +/- 0.5 and 4.8 +/- 0.3 µM, respectively, an indication that they have good potency against this brain cancer cell line. We used the natural fluorescence of the AIMs along with nuclear stains and laser scanning cytometry to determine if the compounds were able to access the DNA in the nucleus by crossing the nuclear membrane. Both MG-70 and MG-71 were found to colocalize in the nuclei of SNB-19 cells suggesting that cytotoxicity may be due to an AIM-DNA interaction.  Work is underway to determine if this interaction occurs selectively with telomeric G4 DNA.

To determine the mechanism of cell death, an annexin V assay was performed using flow cytometry to assess the results.  The cells were drug treated for 2 and 4 hours at a 1uM concentration, and an increase in late apoptosis for MG-70 and MG-71 was observed for the four-hour treatment.  We expect that with drug treatment at 6, 12, and 24 hours this trend will continue and become statistically significant.

Asthma, Wood Smoke, and Antioxidants
Jeff Zwicker

Over 22 million Americans are affected by asthma, and exposure to wood smoke particulate matter has been correlated to an increase in the number of asthma attacks and hospitalizations. It has been shown that dietary factors such as antioxidants play a role in the defense against oxidative damage, especially lipid peroxidation. It has also been suggested that diet could have positive effects on the incidence and severity of asthma. Variation in dietary antioxidant intake is one possible factor to explain why some asthmatic children are more vulnerable to wood smoke than others. In this study we investigate the relationship between the antioxidant vitamin E and 8-isoprostane as a marker of oxidative stress in samples acquired from asthmatic children living in homes with woodstoves. We hypothesized that individuals with higher levels of vitamin E will show lower levels of oxidative stress, indicating that dietary antioxidants could have a protective effect in asthmatic children, particularly those exposed to wood smoke. To explore this hypothesis, urine samples, taken from sixty-six asthmatic children living with woodstoves, were tested for two vitamin E metabolites (a- and g- CEHC) and 8-isoprostane as an indicator of the subject’s asthma severity. Enzyme deconjugation, solvent extraction, and GC-MS analysis were used to measure the levels of a- and g- CEHC. Solid phase extraction and ELISA assays were used to analyze for 8-isoprostane. The resulting data was statistically analyzed, accounting for such factors as age and sex, and the relationships between the antioxidant metabolites and the biomarker 8-sioprostane are discussed.

Neuroprotective capabilities of Endocannabinoid receptor ligands in SH-SY5Y cell line
Kade Grende, Jennene Lyda, Monica Sanchezcontreras, Fernando Cardozo-Pelaez

Oxidative stress results from an increase in the amount of reactive oxygen species (ROS) produced and/or a decrease in a biological systems ability to detoxify these. Reactive oxygen species (ROS) are formed by normal cellular respiration and enzymatic metabolism, as well as insults from exogenous agents. Unquenched ROS may cause accumulation of oxidative damage to proteins, DNA, and lipids. Oxidative stress has been implicated in several neurodegenerative diseases such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). Endocannabinoids and their relative receptors have become a major focus of recent research due to their reported protective role in cellular and animal models of the neurodegenerative diseases described above. We, therefore, hypothesized that endocannabinoid agonists by binding to the cannabinoid receptor type 1 (CB1R) could impact endogenous antioxidants and therefore elicit a neuroprotective effect. This hypothesis was tested by exposing SH-SY5Y cells, which are a neuroblastoma cell line used as a model for many neurodegenerative disease, to endocannabinoid receptor ligands at concentrations of 100nM and 500nM for 24 hours versus control groups. Following exposure, endogenous antioxidants, reduced glutathione (GSH) and super-oxide dismutase (SOD) activity were measured. Although inconclusive at this point, this study explores a potential role for endocannabinoids in increasing antioxidant defenses and offering potential neuroprotection for therapeutic use. Along with the above project this summer research opportunity included genotyping mice by PCR, participation in stereotactic brain surgery on mice, as well as brain extraction followed by assays measuring endogenous antioxidants in these brains.

Acute Pulmonary Toxicity of Multi-walled Carbon Nanotubes Following Instillation
Kristina Finsaas, Ben Seaver, and Celine Beamer

Nanomaterials are becoming a staple of modern society for their use in electronics, medicine, cosmetics, food packaging, etc. and are known to have inflammatory properties—particularly after respiratory exposure. In this study, multi-walled carbon nanotubes (MWCNT) were instilled into the lungs of C57Bl/6 (wild-type), IL-1R deficient (IL-1R-/-) mice, and IL-18R deficient (IL-1R-/-) mice. Pulmonary alterations were evaluated at 24 hrs post-instillation. Biomarkers of pulmonary damage and inflammation were significantly increased post-exposure in wild-type and IL-18R-/- mice, but not IL-1R-/- mice. These findings suggest that respiratory exposure to MWCNTs can induce acute pulmonary inflammation and may exacerbate illness in individuals with existing respiratory diseases.

The initial step in the characterization of the woodsmoke chamber was to perform a detailed evaluation of the performance of the pneumatic valves delivering dilution air. This characterization is still in process.

The M2a Macrophage:  A Key Component and Marker in Particle-Induced Pathology
McKenzie Momany, Gini Porter, and Dr. Christopher Migliaccio

Pulmonary macrophages constitute the first line of defense in exposure to inhaled particulates, and play a key role in the pathology resulting from such exposures.  In the silicosis model, crystalline silica exposure results in pulmonary inflammation, inflammasome activation, fibrosis via a Th2-type immune response (IL-4, IL13), and the presence of the Th2-associated macrophage, M2a.  On the other hand, the health implications of engineered nanomaterials are not understood, but similar pathologies to the silica model result, including inflammasome activation and pulmonary fibrosis, albeit in an apparently accelerated timeframe.  We hypothesized that the presence of IL-33, an inflammasome mediator, following particle exposure is the tipping point in the Th1/Th2 balance and this is characterized by the rise in the M2a early in the disease process.  To test this hypothesis, Balb/c mice were instilled trans-orally with silica, multi-walled carbon nanotubes (FA04 and FA21), or controls.  Samples collected at multiple time points for analyses included whole lung tissue, lavage fluid, and pulmonary macrophages, where Ym1 was used as a M2a subset marker.  In vitro evaluation of our hypothesis included macrophage and epithelial cell cultures, where particles were assessed for the ability to induce IL-33 production from alveolar type II cells (C10).  While both silica and FA21 induced IL-33 production in C10 cells, FA04 did not, and this appears specific to type II cells.  This observation correlated with the relative levels of inflammasome activation as well as changes in interstitial macrophage populations for each material.  Tissue staining and mRNA analyses are currently ongoing.