Elucidation of the cell signaling pathways mediating innate immunity and host-pathogen interactions

The ability to generate a robust immune response is integral to organismal homeostasis. Cells of the innate immune system are considered the first responders of immunity, and are therefore responsible for sensing both pathogens and endogenous danger signals and initiating a protective inflammatory response. To appropriately sense pathogens and danger signals, cells have developed intricate mechanisms for transducing signals from the extracellular environment into the cell. The integration of these signals is complex, resulting from crosstalk between many signaling pathways, but is critical to generating a coordinated biological response. In additional to the specialized mechanisms of innate immune cells to respond to antigens, these cells (like most) have evolved a complex set of adaptive mechanisms that maintain homeostasis during cell stress. Activation of innate immunity via pathogen invasion or the presence of danger signals can be considered an especially intense form of cell stress, thereby implicating these homeostatic pathways as components of the innate immune response. The work presented in this thesis relates to the molecular mechanisms by which cells of the innate immune system integrate signals from the microenvironment to produce a coordinated biological response. The aim was to elucidate the mechanisms by which innate macrophages transduce extracellular signals to activate important effector pathways, and to describe crosstalk between cell signaling pathways that mediate adaptive responses to cell stress. Finally, we looked to extend our understanding to pathophysiological settings, and investigated the mechanisms by which pathogens that cause cell stress generate an aberrant inflammatory response. In doing so, we described novel components of these signaling pathways, which may be exploited in designing novel therapeutics. In paper I, Gαi2 was identified as a critical signaling molecule in macrophage phenotype determination, functioning to transduce signals from the microenvironment to fine tune macrophage propensity towards an M1 inflammatory or M2 anti-inflammatory phenotype. In paper II, the immune receptor CD38 was shown activate the master transcriptional regulation of the autophagic/lysosome machinery, TFEB. We further identified the large kinase LRRK2 as essential in signal transduction downstream of CD38. In paper III, we described adaptive crosstalk between TFEB, an essential component of the cell stress response, and the typically proliferative WNT signaling pathway. Finally, in paper IV we describe how the SARS-Coronavirus open reading frame-3a causes multimodal necrotic death by activating multiple cell stress and innate immune pathways, resulting in aberrant inflammation. In summary, the work presented in this thesis extends our current understanding of the molecular mechanisms mediating the integration of signals in innate immune cells. We have identified several novel signaling mechanisms, which could lay the foundation for the development of targeted therapeutics

Studies on the removal of Lissamine Green B (LGB) from soil in comparison with contemporary approaches

Studies on the removal of Lissamine Green B (LGB) from soil in comparison with contemporary approaches. N. NABAR and D.F. MARTIN. Vast amounts of chemical dyes (around 106 tons) are made annually worldwide. Dye effluents can make their way into runoff and wastewater, eventually settling in the soil. Previous extraction methods have had limited success in removal from soil, and a recent approach involved the use of Fenton’s reagent with electrochemistry (Rosales et al., J. Environ Sci. Hlth. 2009, 44, 1104-1111). We examined the efficacy of dye removal using a kaolin-clay model for soil and Lissamine Green B (LGB) dye. The removal of LGB from a LGB/kaolin mixture was recorded after a 24- and 48- hour extraction with hot water using a Soxhlet apparatus. Previously, aqueous Lissamine B removal was achieved using column chromatography with commercially available Octolig®, a polyethyldiamine molecule attached to a high surface area silica gel (Chang et al., Tech. Innov, 2010, 12 71-1277). The results indicated complete removal of LGB from kaolin using 24-hour Soxhlet extractions. Removal of dyes using hot water and/or chromatography with Octolig® could provide a potential safe, large-scale solution to treating soils contaminated with dyes or other organics with suitable functional groups

Correction: Cell Therapy: A Safe and Efficacious Therapeutic Treatment for Alzheimer's Disease in APP+PS1 Mice.

[This corrects the article DOI: 10.1371/journal.pone.0049468.]

Cell Therapy: A Safe and Efficacious Therapeutic Treatment for Alzheimer’s Disease in APP+PS1 Mice

Previously, our lab was the first to report the use of antigen-sensitized dendritic cells as a vaccine against Alzheimer\u27s disease (AD). In preparation of this vaccine, we sensitized the isolated dendritic cells ex vivo with Aβ peptide, and administered these sensitized dendritic cells as a therapeutic agent. This form of cell therapy has had success in preventing and/or slowing the rate of cognitive decline when administered prior to the appearance of Aβ plaques in PDAPP mice, but has not been tested in 2 × Tg models. Herein, we test the efficacy and safety of this vaccine in halting and reversing Alzheimer\u27s pathology in 9-month-old APP + PS1 mice. The results showed that administration of this vaccine elicits a long-lasting antibody titer, which correlated well with a reduction of Aβ burden upon histological analysis. Cognitive function in transgenic responders to the vaccine was rescued to levels similar to those found in non-transgenic mice, indicating that the vaccine is capable of providing therapeutic benefit in APP+PS1 mice when administered after the onset of AD pathology. The vaccine also shows indications of circumventing past safety problems observed in AD immunotherapy, as Th1 pro-inflammatory cytokines were not elevated after long-term vaccine administration. Moreover, microhemorrhaging and T-cell infiltration into the brain are not observed in any of the treated subjects. All in all, this vaccine has many advantages over contemporary vaccines against Alzheimer\u27s disease, and may lead to a viable treatment for the disease in the future

Behavioral results.

<p>A) Latency. The Tg PDFM with high antibody titer group performed moderately better than the Tg control group, though not reaching statistical significance. The low-responders performed significantly worse than the Tg Control (<i>P</i><0.05). B) Error. The Tg PDFM with High Antibody Titer group performed slightly better than the Tg Control Group, though not significant. The low-responders performed significantly worse than the Tg control (<i>P</i><0.05).</p

Antibody response of individual mice.

<p>A) Antibody responses on all Tg and select Non-Tg mice are shown. B) The mean and 75^th percentile value was used to determine level of antibody response. Mice 8 and 14 did not have a robust, sustained antibody response and were deemed low responders.</p

Ig Isotyping.

<p>A) Total Ig isotyping. Tg PDFM group showed an increase in IgG1/IgG2a ratio after treatment (<i>P</i><0.05) B) Aβ Specific isotyping. There was no significant difference in Aβ specific isotyping during the duration of the experiment. Note: Ig isotyping was only done for the Tg PDFM group. Aβ specific Ig isotyping was not able to be done at Day 0, as no Aβ antibody was present.</p

Calculated T-cell binding affinities for different Aβ start sites.

<p>Calculated T-cell binding affinities for different Aβ start sites.</p

Calculated binding affinity for mutated Aβ peptides.

<p>Calculated binding affinity for mutated Aβ peptides.</p