A method has been developed to track infectious diseases by using data mining of active Twitter accounts and its efficacy was demonstrated during the West African Ebola outbreak of 2014. Using a meme based n-gram semantic usage model to search the Twitter database for indications of illness, flight and death from the spread of Ebola in Africa, principally from Guinea, Sierra Leone and Liberia. Memes of interest relate disease to location and severity and are coupled to the density of Tweets and re-Tweets. The meme spreads through the community of social users in a fashion similar to nonlinear wave propagation- like a shock wave, visualized as a spike in Tweet activity. The spreading was modeled as a system isomorphic to a modified SIR (Susceptible, Infected, Removed disease model) system of three coupled nonlinear differential equations using Twitter variables. The nonlinear terms in this model lead to feedback mechanisms that result in unusual behavior that does not always reduce the spread of the disease. The resulting geographic Tweet densities are coupled to geographic maps of the region. These maps have specific threat levels that are ported to a mobile application (app) and can be used by travelers to assess the relative safety of the region they will be in.

Vaccines and anthelmintics induce an inflammatory response in equids. Since they are commonly given concurrently, it is practical to study any interaction between them. This study evaluated whether IVM and PYR would modulate the acute phase inflammatory response, the systemic gene expression of pro-inflammatory cytokines, and vaccine-specific titers induced by WNV, EHV, and KLH vaccines. Naturally-infected, yearling ponies were sorted by gender, then fecal epgs. They were randomly assigned to three treatment groups: IVM, PYR, and control. All ponies received vaccinations intramuscularly on days 0 and 29. Whole blood, serum, and plasma samples were collected 1, 3, and 14 days post-vaccination. Samples were analyzed for inflammatory markers, cytokine, mRNA expression, and vaccine-specific IgG titers by ELISA. The acute-phase inflammatory marker data showed no statistical significance; they did show an increase in SAA, haptoglobin, and fibrinogen, and a decrease in iron after vaccination. The mRNA data showed that anthelmintics had a significant effect on interleukin mRNA levels, but not on TNF-α or IFN-γ levels. The ELISA assays showed no biologically significant reduction in IgG as compared to the control group. We conclude that deworming does not affect vaccine IgG titers; therefore, ceasing vaccinating and deworming concurrently is not necessary.

The study involved Indigenous communities of Saskatchewan and Alberta which are adversely impacted by industrial activities in their traditional territory. The overall goal of this study is: the assessment of social media in risk studies among Indigenous communities of western Canada. The methods used were: interviews, focus group discussion (FGD), and net-mapping. Results showed that the majority of youths communicated around risk using new social media (NSM) in event of risk outbreak, while Indigenous Elders, communicated face-to-face and via cell-phone. Results also showed that youths use Traditional Knowledge learnt from the Elders to understand Chronic wasting disease (CWD) risk, interpret, communicate and mobilize around mitigation. The study concluded that the use of NSM is becoming increasingly important for scoping information around wildlife decline and emergency in these Indigenous communities.

OBJECTIVES: Measles and rubella are nationally notifiable, vaccine-preventable diseases targeted for elimination by the Pan American Health Organization (PAHO). To support national and international elimination efforts, surveillance optimization is important to ensure rapid case detection, document endemic transmission interruption, identify susceptible populations and inform immunization strategies. While current national surveillance captures confirmed-case data, its performance cannot be assessed using PAHO-recommended surveillance indicators as suspect-case investigation data are required for their estimation. In Canada, the investigation of clinically-suspect measles-like illness (MLI) is highly dependent on laboratory evidence, providing an opportunity to use laboratory data to estimate MLI investigation rates. The Measles and Rubella Surveillance (MARS) pilot project was developed to address existing surveillance challenges with the central hypothesis that (I) ‘it is feasible to develop and implement a real‐time, web‐based measles and rubella surveillance system in the Canadian setting’, and the following sub‐hypotheses: (II) ‘implementation of real‐time surveillance in MARS pilot provinces will result in increased timeliness of national measles and rubella surveillance when compared with established confirmed-case surveillance’, and (III) ‘it is possible to use augmented laboratory data to estimate the performance of national measles and rubella surveillance using adapted PAHO indicators’. METHODS: A MARS application was designed to support centralized real-time measles/ rubella investigation reporting and alerting with integration of non-nominal laboratory and epidemiological data, then developed and piloted using the web-based Canadian Network for Public Health Intelligence platform in British Columbia, Alberta and Newfoundland from June/2011-May/2012. Pre- and post-pilot laboratory surveys were conducted to retrospectively assess national surveillance performance in ‘outbreak’ and ‘non-outbreak’ settings during the 2005‐2011 and pilot years using various surveillance indicators and attributes. Measles IgM serology testing was used as a laboratory-based proxy for MLI investigation to support indicator estimation. RESULTS: Real-time, integrated surveillance was successfully implemented in MARS pilot provinces as modeled within the context of established reporting roles, and surveillance indicators and attributes were estimated using augmented laboratory data. MARS surveillance was more timely than confirmed-case surveillance, and real-time MARS reports exceeded all laboratory-related PAHO targets evaluated: 100% met ‘sample collection’ and ‘receipt’ timelines, and 91.7% met ‘result' timelines (Targets:≥80%); 99.8% of all MLI investigations were discarded (Target:≥95%). A national ‘non-outbreak’ baseline rate of 14 MLI investigations/100 000 population was estimated, whereas MARS pilot sites averaged 22 MLI investigations/100 000 population during the pilot year. While ‘non-outbreak’ investigation rates varied between provinces, all annual provincial and national rates estimated for the 2005‐2011 and MARS pilot years exceeded the PAHO investigation target of ≥2 suspected cases/100 000 population in settings attempting elimination. CONCLUSIONS: The MARS model supported more timely and integrated national measles and rubella surveillance, and enabled indicator‐based performance assessment. Results underscore the importance of laboratory data when evaluating and documenting surveillance performance to support elimination efforts. Consideration should be given to national MARS implementation and its use as a model adaptable to the case-based surveillance of other nationally notifiable diseases.

Lyme disease is one of the most common infectious diseases present in the United States today and it is clear that the changing climate will affect the geographical range of it across the country. Climate change may impact the range of the Lyme vector species, ticks, which will in turn expand the range of human risk. Although I could not successfully map the possible spread of tick populations or Lyme disease incidence as a result of climate change, my research shows a direct connection between infected tick geographic distribution and key climatic variables, such as temperature, humidity, and precipitation. It is expected that as the climate changes, particularly as it warms, the range of suitable habitat for ticks will expand into high latitudes and altitudes. The expansion of tick populations will put previously unaffected human populations at greater risk of Lyme disease. It is essential that further research be done to confirm the possible consequences of climate change on Lyme disease in the United States and to gain a more precise understanding of how and where effects will be seen. Health officials and policymakers must be informed so they can properly educate and prepare people preemptively for potential Lyme disease outbreaks.

T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression. The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods of immune evasion. Myelodysplastic Syndrome (MDS) is a pre-malignant dysplastic disorder of the bone marrow characterized by ineffective hematopoiesis and clonality in the myeloid lineage, where lack of immune response has been implicated in the propensity for progression to acute myeloid leukemia (AML). Leukemia progression is associated with the acquisition of complex genetic abnormalities. Alterations in immune system regulation have been implicated in various stages of the disease process, although the role of the immune system in response to several therapies in MDS has not been fully discovered. Lenalidomide is a small molecule therapeutic preferentially effective in MDS patients with an interstitial chromosome 5q deletion (del(5q)). Improved erythropoiesis has also been reported to occur in 20-30% of low-risk, non-del(5q) patients. Although lenalidomide is a potent immunomodulatory drug that potentiates T-cell and NK-cell responses, the T-cell compartment in MDS is highly deregulated by aberrant repertoire skewing, decreased function and abnormal naïve and memory cell homeostasis. The presence of lymphoid infiltrates in the bone marrow of lenalidomide-responsive patients suggests that T-cells may participate in the hematopoietic response, but it is unclear if lenalidomide is capable of reversing these functional T-cell defects. We therefore assessed immunological changes in low-risk MDS patients before and after 16-weeks of lenalidomide therapy, and assessed the relationship to erythroid response. Although MDS T-cells were anergic prior to treatment, we have shown that T-cells in responders have a significant increase in antigen-induced proliferative response and T helper type-1 (Th1) cytokine production (IL-2, IFN-γ, TNF-α) compared to non-responders. The change in function positively correlated with an increase in naïve T-cells and a decrease in memory cells, indicating that lenalidomide has immunomodulatory activity to reverse anergy in MDS. Although it is known that lenalidomide may increase T-cell activation and proliferation in the absence of co-stimulatory signals, a direct mechanism of action has yet to be elucidated. Since CD28 is one of the most important co-stimulatory molecules deregulated in cancer, we therefore set out to determine if the expression of CD28 was essential for lenalidomide's mechanism in T-cells. We knocked out CD28 expression in healthy donor T-cells, and sorted on inherently deficient, CD28null, T-cells that accumulate in older healthy donors and found that lenalidomide-induced proliferation and function were completely ablated within the CD28null subset. These data indicate the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor are necessary for lenalidomide action. Interestingly, during the natural aging process, repeated exposure to antigens results in the accumulation of CD28null T-cells that are phenotypically distinct and functionally deficient due to excessive proliferative history in vivo. We therefore examined whether CD28 expression on MDS patient T-cells affected responses to lenalidomide, and if this could be used as a predictive biomarker of responsiveness. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM), which are inherently CD28null, and that non-responders had an overall increase in CD4+ and CD8+CD28null T-cells, as well as an increase in CD28null cells within the TEM compartment compared to hematologic responders. We then sought to determine the particular protein target of lenalidomide responsible for increased CD28 receptor signaling in T-cells. Several targets in a variety of cell types have been postulated, although the direct mechanism in T-cells is unclear. Our group has previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases located within the commonly deleted region (CDR) on chromosome 5q in the MDS myeloid clone, Protein Phosphatase 2A (PP2A) and Cdc25c. PP2Ac is known to bind CD28 and is hypothesized to inhibit T-cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit the phosphatase activity of PP2A which leads to increased activation of T-cell proximal signals dependent on CD28 expression. We examined this hypothesis using molecular modeling and virtual screening and found that all of the IMiDs (lenalidomide, pomalidomide, and thalidomide) can theoretically interact with the catalytic pocket of the PP2A heterotrimer, potentially inhibiting PP2Ac activity. In vitro phosphatase activity assays supported these findings as lenalidomide-inhibition of PP2Ac activity was seen in both ad293 and Jurkat cell lines, and in primary T-cells. Mutations of theorized lenalidomide hydrogen-bond sites within the catalytic pocket of PP2A rendered the enzyme catalytically dead, indicating that these are important residues for enzymatic activity, but unfortunately could not be used to determine if lenalidomide activity was disrupted by mutation of those sites. These data together suggest that the ability of lenalidomide to augment immune activation in vivo in MDS patients, and potentially other diseases, is extremely important to patient response. Also, that CD28 expression on T-cells is essential for lenalidomide immune-mediated tumor eradication through CD28 downstream signaling, and potentially through inhibition of PP2A. These results are useful in designing future lenalidomide-combination therapy trials in other hematologic and solid malignancies, and could be used to help stratify patients for future therapeutic decisions in MDS and other malignancies.

With over 35 million people currently infected, the World Health Organization considers HIV a global pandemic. HIV is characterized by a high mutation rate, which allows it to evade the host immune system and develop resistance to drugs. However, this extraordinary adaptive ability may also be the key to HIV’s demise. Through the field of phylodynamics, the evolutionary behavior of the virus is being studied in an attempt to control the epidemic. In this thesis, three papers are presented in which we analyze sequences generated through the Canadian HIV Strain and Drug Resistance Surveillance program. In chapter 2 we validate a classifier which distinguishes between recent and established infections based on the proportion of mixed bases observed in population-based pol sequences. Our results will help identify recent infections and improve incidence calculations. In chapter 3, we investigate immune-induced patterns in HIV that are shared by patients of the same ethnicity. An understanding of the forces shaping HIV evolution is instrumental to the development of a vaccine relevant to the Canadian epidemic. In chapter 4, we present preliminary results of a historical reconstruction of HIV across the provinces of Canada. This analysis will highlight strategies that have succeeded or failed in controlling the epidemic. Furthermore, our work will establish whether non-B subtypes of HIV are an increasing threat to Canadian public health. Overall, this thesis provides the first country-wide evolutionary and phylogenetic analysis of the HIV epidemic.

<p>In this dissertation, I present a general statistical framework for phylodynamic inference that can be used to estimate epidemiological parameters and reconstruct disease dynamics from pathogen genealogies. This framework can be used to fit a broad class of epidemiological models, including nonlinear stochastic models, to genealogies by relating the population dynamics of a pathogen to its genealogy using coalescent theory. By combining Markov chain Monte Carlo and particle filtering methods, efficient Bayesian inference of all parameters and unobserved latent variables is possible even when analytical likelihood expressions are not available under the epidemiological model. Through extensive simulations, I show that this method can be used to reliably estimate epidemiological parameters of interest as well as reconstruct past disease dynamics from genealogies, or jointly from genealogies and other common sources of epidemiological data like time series. I then extend this basic framework to include different types of host population structure, including models with spatial structure, multiple-hosts or vectors, and different stages of infection. The later is demonstrated by using a multistage model of HIV infection to estimate stage-specific transmission rates and incidence from HIV sequence data collected in Detroit, Michigan. Finally, to demonstrate how the approach can be used more generally, I consider the case of dengue virus in southern Vietnam. I show how earlier phylodynamic inference methods fail to reliably reconstruct the dynamics of dengue observed in hospitalization data, but by deriving coalescent models that take into consideration ecological complexities like seasonality, vector dynamics and spatial structure, accurate dynamics can be reconstructed from genealogies. In sum, by extending phylodynamics to include more ecologically realistic and mechanistic models, this framework can provide more accurate estimates and give deeper insight into the processes driving infectious disease dynamics.</p> / Dissertation

Host defense peptides (HDPs) provide innate immune defense against invasive S. aureus infection. Recent studies suggest potential cross-resistance between HDPs and the lipopeptide antibiotic, daptomycin (DAP). Isolates that exhibit DAP non-susceptible phenotypes may have virulence advantages and pose challenges to effective treatment. The current studies were performed to compare the efficacies and mechanisms of action of native and engineered HDPs vs. clinical S. aureus strain pairs which differed in susceptibility to daptomycin in vitro. Ultrasensitive radial diffusion and multi-colored flow cytometry were employed to analyze distinctive susceptibilities and mechanisms of resistance, respectively. Overall efficacies were greater vs. DAP-susceptible (DSSA) vs. DAP non-susceptible (DNSA) S. aureus isolates for some but not all HDPs. Efficacy profiles of certain HDPs were influenced by pH, regardless of whether the particular isolate was DSSA or DNSA phenotype. Mechanistically, DSSA and DNSA isolates differed in responses to specific HDPs regarding cell energetics, membrane permeability, cytoplasm membrane turnover, and cell death protease induction. DSSA and DNSA strain pairs exhibited non-identical mechanisms of resistance to HDPs. At pH 7.5, as expected, HDPs hNP-1 and RP-1 exerted significantly greater efficacy on susceptible control strain ISP479C vs. its resistant counterpart ISP479R. These data suggest different mechanisms of HDP resistance are active in differing DNSA strains. These preliminary results are under further investigation, as are the genetic determinant(s) that may emerge during infection. If substantiated, these findings would imply multiple modes of survival of S. aureus in the face of DAP or HDPs.

Interleukin-10 (IL-10) deficient dendritic cells (DCs) are potent antigen presenting cells during Chlamydia infection. To further understand the mechanism underlying this protective property in IL-10 DCs, we combined two proteomic techniques: 2DIGE and liquid chromatography mass spectrometry. We then performed western blotting on proteins from Chlamydia infected wild type (WT) and IL-10 knock out (KO) DCs. The results showed that alpha enolase (ENO1), a metabolic enzyme involved in the ninth step of glycolysis, was significantly upregulated in Chlamydia infected IL-10KO DCs compared to WT DCs. We further studied the role of ENO1 by silencing ENO1 gene using lentiviral siRNA technology. Flow cytometry, confocal microscopy, cytokine analysis, infectivity analysis and T-cell proliferation analysis were also used to determine DC function. We then analyzed the effect of the ENO1 knockdown on DC metabolism during Chlamydia stimulation. The results showed that DC maturation and activation were decreased in ENO1 knockdown DCs that were stimulated with Chlamydia compared to the Chlamydia stimulated WT DCs. In addition, pyruvate concentration decreased significantly in ENO1 knockdown DCs stimulated with Chlamydia. The mitochondrial structure of Chlamydia stimulated ENO1 knockdown DCs appeared damaged compared to the Chlamydia stimulated WT DCs. The function of ENO1 in the immune response to Chlamydia trachomatis infection is unknown. However, the results from this study indicates that ENO1 may contribute to DC metabolism and mitochondrial homeostasis which may play a role in the maturation and antigen presenting function of DCs.