The ICE-flox Integrative Conjugative Element of Legionella pneumophila.

Legionella pneumophila is an accidental human pathogen that causes the bacterial pneumonia Legionnaire’s Disease. The bacteria are ubiquitous in freshwater environments and are spread by aerosolization of contaminated water from the built environment. Accessory traits carried on mobile genetic elements diversify the L. pneumophila and may contribute to persistence in stressful environments. One class of mobile elements are Integrative Conjugative Elements (ICEs) which encode cargo genes as well as type IV secretion system (T4SS) transfer apparatuses to direct their own transmission among a bacterial population. In this dissertation, I demonstrate that ICE-βox enhances L. pneumophila resistance to oxidative stresses encountered in vitro (such as bleach) and in macrophages. Specifically, this mobile element protects L. pneumophila from the toxic activities of the macrophage phagocyte oxidase. In addition to cargo genes predicted to repair oxidative damage, ICE-βox encodes a paralog of the master L. pneumophila life cycle regulator csrA. Bioinformatic analyses of 34 L. pneumophila ICE-associated T4SS reveals four families based on apparatus composition. Each T4SS family is genetically and phylogenetically linked with a distinct csrA paralog, suggesting functional interactions. Indeed, the ICE-βox csrA paralog csrT can repress ICE-βox traits as well as motility of the host bacterium. Finally, a preliminary epidemiologic survey identified ICE-βox in a majority of built environment L. pneumophila isolates. Accordingly, the hypothesis that chlorine-based disinfectants enrich for ICE-βox and increase L. pneumophila resilience and virulence warrants testing. By understanding the fitness advantages, regulation and prevalence of ICE-βox, disinfection protocols can be designed to eradicate persistent L. pneumophila and reduce its risk to humans.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113493/1/kjflynn_1.pd

Characterization of dysphagia onset in a mouse model of amyotrophic lateral sclerosis (ALS

The primary goal of this study was to characterize dysphagia onset and progression in the low copy number SOD1-G93A (LCN-SOD1) mouse model of ALS. A secondary goal was to determine the effect of serial radiation exposure throughout the lifespan on dysphagia severity. To accomplish this goal, we used our lab's established Videofluoroscopic Swallow Study (VFSS) assay to objectively assess swallow function in 54 mice, divided into serial versus single radiation exposure groups. The serial X-ray exposure group (n=24; 13 LCN-SOD1, 11 nontransgenic control) underwent VFSS testing once a month, starting at 2 months of age until disease end-stage. The single X-ray exposure group (n=30; 15 LCNSOD1, 15 non-transgenic control) underwent VFSS testing only once at disease end-stage. VFSS videos from both groups were analyzed to quantify 8 swallow metrics. Results showed that all swallow metrics were similar within and between genotypes from 2 to 6 months of age, which coincided with the pre-clinical disease stage in LCN-SOD1 mice. At disease end-stage, LCN-SOD1 mice had significantly altered swallow function for 5 of the 8 VFSS metrics under investigation, compared to age-matched controls. Between disease onset and end-stage, LCN-SOD1 mice demonstrated highly variable disease phenotypes and survival durations, which rendered it impossible to characterize the onset and rate of dysphagia progression with the small sample size. However, two main findings emerged from this study. First, dysphagia onset in LCN-SOD1 mice did not occur until after 6 months of age. This finding suggests that treatments for dysphagia in this mouse model of ALS should begin after 6 months of age (i.e., after clinical disease onset) for optimal translational potential to humans with ALS. Our second novel finding was that dysphagia severity at disease end-stage was similar for single versus serial radiation exposure in LCN-SOD1 mice, which provides evidence that our lab can continue to perform longitudinal VFSS studies in this small animal without confounding outcomes relative to dysphagia. However, the majority of mice developed evidence of surface-level radiation toxicity (fur depigmentation and dry eyes) as the number of x-ray exposures increased, even though it did not affect swallow function. Therefore, we are taking proactive measures to reduce radiation exposure during VFSS and thereby prevent skin and eye morbidities for future longitudinal dysphagia investigations.Dr. Teresa E. Lever, Thesis Supervisor.Includes bibliographical references (pages 40-47)

Inducible expression quantitative trait locus analysis of the MUC5AC gene in asthma in urban populations of children

BACKGROUND: Mucus plugging can worsen asthma control, lead to reduced lung function and fatal exacerbations. MUC5AC is the secretory mucin implicated in mucus plugging, and MUC5AC gene expression has been associated with development of airway obstruction and asthma exacerbations in urban children with asthma. However, the genetic determinants of MUC5AC expression are not established. OBJECTIVE: To assess single-nucleotide polymorphisms (SNPs) that influence MUC5AC expression and relate to pulmonary functions in childhood asthma. METHODS: We used RNA-sequencing data from upper airway samples and performed cis-expression quantitative trait loci (eQTL) and allele specific expression (ASE) analyses in two cohorts of predominantly Black and Hispanic urban children, a high asthma-risk birth cohort and an exacerbation-prone asthma cohort. We further investigated inducible MUC5AC eQTLs during incipient asthma exacerbations. We tested significant eQTLs SNPs for associations with lung function measurements and investigated their functional consequences in DNA regulatory databases. RESULTS: We identified two independent groups of SNPs in the MUC5AC gene that were significantly associated with MUC5AC expression. Moreover, these SNPs showed stronger eQTL associations with MUC5AC expression during asthma exacerbations, consistent with inducible expression. SNPs in one group also showed significant association with decreased pulmonary functions. These SNPs included multiple EGR1 transcription factor binding sites suggesting a mechanism of effect. CONCLUSIONS: These findings demonstrate the applicability of organ specific RNA-sequencing data to determine genetic factors contributing to a key disease pathway. Specifically, they suggest important genetic variations that may underlie propensity to mucus plugging in asthma and could be important in targeted asthma phenotyping and disease management strategies

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

On the intrinsic sterility of 3D printing.

3D printers that build objects using extruded thermoplastic are quickly becoming commonplace tools in laboratories. We demonstrate that with appropriate handling, these devices are capable of producing sterile components from a non-sterile feedstock of thermoplastic without any treatment after fabrication. The fabrication process itself results in sterilization of the material. The resulting 3D printed components are suitable for a wide variety of applications, including experiments with bacteria and cell culture

On the intrinsic sterility of 3D printing.

3D printers that build objects using extruded thermoplastic are quickly becoming commonplace tools in laboratories. We demonstrate that with appropriate handling, these devices are capable of producing sterile components from a non-sterile feedstock of thermoplastic without any treatment after fabrication. The fabrication process itself results in sterilization of the material. The resulting 3D printed components are suitable for a wide variety of applications, including experiments with bacteria and cell culture