The definition of Natural Killer (NK) cells has undergone dramatic modification with the advance of immunological research tools. NK cells can no longer be classified only as indiscriminate killer innate immune cells. NK cells are now known to form functional relationships with accessory cells to shape and promote the adaptive immune response. Recently, antigen specific and cytokine induced memory NK cells have been demonstrated to exhibit the characteristic phases of an adaptive immune cell, including extended persistence and robust function in response to reexposure. New findings in basic NK immunobiology indicate that the role of NK cells is underappreciated and these cells may potentially be manipulated for the development of anti-viral therapies.

Aims: The ability of lipopolysaccharide (LPS) pre-treatment to induce cardioprotection following ischaemia/reperfusion (I/R) has been well documented; however, the mechanisms have not been fully elucidated. LPS is a Toll-like receptor 4 (TLR4) ligand. Recent evidence indicates that there is cross-talk between the TLR and phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathways. We hypothesized that activation of PI3K/Akt signalling plays a critical role in LPS-induced cardioprotection. Methods and results: To evaluate this hypothesis, we pre-treated mice with LPS 24 h before the hearts were subjected to ischaemia (45 min) and reperfusion (4 h). We examined activation of the PI3K/Akt/GSK-3β signalling pathway. The effect of PI3K/Akt inhibition on LPS-induced cardioprotection was also evaluated. LPS pre-treatment significantly reduced infarct size (71.25%) compared with the untreated group (9.3 ± 1.58 vs. 32.3 ± 2.92%, P < 0.01). Cardiac myocyte apoptosis and caspase-3 activity in LPS-pre-treated mice were significantly reduced following I/R. LPS pre-treatment significantly increased the levels of phospho-Akt, phospho-GSK-3β, and heat shock protein 27 in the myocardium. Pharmacological inhibition of PI3K by LY294002 or genetic modulation employing kinase-defective Akt transgenic mice abolished the cardioprotection induced by LPS. Conclusion: These results indicate that LPS-induced cardioprotection in I/R injury is mediated through a PI3K/Akt-dependent mechanism.

Recognition of microbial components by germ-line encoded pattern recognition receptors (PRR) initiates immune responses to infectious agents. We and others have proposed that pairs or sets of PRR mediate host immunity. One such pair comprises the fungal β-glucan receptor, Dectin-1, which collaborates through an undefined mechanism with Toll-like receptor 2 (TLR2) to induce optimal cytokine responses in macrophages. We show here that Dectin-1 signaling through the spleen tyrosine kinase (Syk) pathway is required for this collaboration, which can also occur with TLR4, 5, 7 and 9. Deficiency of either Syk or the TLR adaptor MyD88 abolished collaborative responses, which include TNF,MIP-1α andMIP-2 production, and which are comparable to the previously described synergy between TLR2 and TLR4. Collaboration of the Syk and TLR/MyD88 pathways results in sustained degradation of the inhibitor of kB (IkB), enhancing NFkB nuclear translocation. These findings establish the first example of Syk-and MyD88-coupled PRR collaboration, further supporting the concept that paired receptors collaborate to control infectious agents.

This thesis addresses the exponential growth of experimental data and the resulting computational complexity seen in two major scientific applications, which account for significant cycles consumed on today’s supercomputers. The first application concerns computation of the adaptive optics system in next-generation ground-based telescopes, which will expand our knowledge of the universe but confronts the astronomy community with daunting real-time computation requirements. The second application deals with emerging frequency-domain redatuming methods, e.g., Marchenko redatuming, which are game-changers in exploration geophysics. They are valuable to oil and gas applications and will soon be to geothermal exploration and carbon capture storage. However, they are impractical at industrial scale due to prohibitive computational complexity and memory footprint. We tackle the aforementioned challenges by designing high-performance algebraic and stochastic algorithms, which exploit the data sparsity structure of the matrix operator. We show that popular randomized algorithms from machine learning can also solve large covariance matrix problems that capture the correlations of wavefront sensors detecting the atmospheric turbulence for ground-based telescopes. Algebraic compression based on low-rank approximations that retains the most significant portion of the spectrum of the operator provides numerical solutions at the accuracy level required by the application. In addition, selective use of lower precisions can further reduce the data volume by trading off application accuracy for memory footprint. Reducing memory footprint has ancillary implications for reduced energy expenditure and reduced execution time because moving a word is more expensive than computing with it on today’s architectures. We exploit the data sparsity of matrices representative of these two scientific applications and propose four algorithms to accelerate the corresponding computational workload. In soft real-time control of an adaptive optics system, we design a stochastic Levenberg-Marquardt method and high-performance solver for Discrete-time Algebraic Riccati Equations. We create a tile low-rank matrix-vector multiplication algorithm used in both hard real-time control of ground-based telescopes and seismic redatuming. Finally, we leverage multiple precisions to further improve the performance of seismic redatuming applications We implement our algorithms on essentially all families of currently relevant HPC architectures and customized AI accelerators and demonstrate significant performance improvement and validated numerical solutions.

Although the relationship between the effects of cold and infection has been studied for a long time, the mechanisms contributing to this phenomenon have not yet been discovered. While short-term exposure to cold triggers a stress response and the production of proinflammatory cytokines, long-term cold exposure induces adaptation and anti- inflammatory shift. The role of cold adaptation in the development and the process of the organism's response to infection remains an unresolved issue. In this diploma thesis, we focused on determining the effect of cold stress or cold adaptation on the immune system of rats which was stimulated by ligands of Toll-like receptors (TLRs). The obtained results showed the importance of γδT lymphocytes and their cytokine production in the process of cold adaptation. To determine how cold adaptation affects the response to infectious agents, we studied changes in the proportion of different immune cell populations in rats treated with the TLR2/6 ligand Pam2CSK4. We determined significant changes in the activation of myeloid cells and B lymphocytes, but also in the population of γδT lymphocytes. Our obtained results suggested the importance of γδT lymphocytes and the protective effect of cold adaptation. Key words: immune system, cold stress, cold adaptation, infection,...

Toll-like receptor (TLR) ligands and phytochemical feed additives (PFAs) were evaluated in this study to determine the effects of immune stimulation on gut immunity in the zebrafish, Danio rerio, and the channel catfish, Ictalurus punctatus. Rag1-/- (MT) zebrafish were used to study how the TLR ligands β-glucan and resiquimod (R848) affect the innate immune system in the gut of MT zebrafish. Enhanced expression of marker genes (NITR9, NCCRP-1 and MPEG-1) indicated stimulation of Natural Killer (NK) cells, non-specific cytotoxic cells (NCCs) and macrophages. After challenge with Edwardsiella ictaluri, MT zebrafish stimulated with β-glucan demonstrated higher survival and the presence of more macrophages/monocytes in the gut than control MT zebrafish. A PFA test diet containing a blend of prebiotic fiber, oregano, thyme, cinnamon essential oils, and Yucca schidigera (ONE Current™, OC) was fed to channel catfish for 3 months in ponds to determine the effect on channel catfish fingerling growth. Fish were fed in ponds and a tank bacterial challenge followed to test the efficacy of the product. Catfish fed OC demonstrated greater weight gain and feed conversion ratios, higher survival after challenge with E. ictaluri, greater phagocytosis or binding by macrophages and cytotoxic cells. Catfish fed OC also demonstrated greater gut surface area after 2 months feeding OC. To elucidate the effect(s) of each of the compounds in the OC diet on gut immune responses and to determine if PFAs can decrease bacterial colonization and replication within gut tissues, WT and MT zebrafish were fed diets containing different compounds included in OC. Quantification of live bacteria from gut and kidney tissue was determined after challenge with E. ictaluri. Expression levels of immune response genes were evaluated after ingestion of PFAs. Actifibe, Essential oil 25 ppm (EO 25) and Actifibe + EO demonstrated the lowest infection and colonization rate, upregulation of immune response genes, and significantly higher survival when challenged with E. ictaluri. This study demonstrates the potential for application of TLR ligand and feed administered PFAs to improve fish health. Our findings provide a more comprehensive understanding of host gut/pathogen interactions as well as suggestions for novel disease control measures.

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No description available.

Salmonella enterica serovar Typhimurium is an important cause of gastroenteritis in the United States and the developing world. Biofilm growth is an significant mechanism, which S. Typhimurium utilizes to contaminate food products and survive in the environment. Biofilms are also an important part of the infectious process for many pathogenic bacteria. As part of the biofilm, S. Typhimurium produces an extracellular matrix consisting of cellulose, extracellular DNA, and most importantly, the amyloid protein curli. Similar to amyloids associated with human diseases, curli is recognized by the innate immune system through Toll-Like Receptors (TLRs). Here, we studied the immune receptors recognizing curli as well as interactions between eDNA and curli during biofilm development in order to glean a better understanding of these complex bacterial communities and the immune response to them. Recently, our lab demonstrated that curli fibers are recognized by the TLR2/TLR1 complex. CD14 has been shown to be a common adaptor protein for TLR2/TLR1 complex in response to one of its ligands, tri-acylated lipopeptide, Pam3CSK4. In order to study the role of CD14 in the immune receptor complex recognizing curli, we utilized HeLa 57A cells, a human cervical cancer cell line that has a stably transfected luciferase reporter for Nf-κB activation. When these cells were transiently transfected with TLR2 and TLR1 together or with the addition of membrane-bound CD14, NfκB activation was enhanced by the presence of CD14 in response to purified curli, GST-tagged curli subunit (GST-CsgA), and the control lipopeptide Pam3CSK4. Soluble CD14 also increased NfκB activation in response to purified curli. Bone marrow derived macrophages (BMDM) from wild type (C57BL/6) mice produced more IL-6 and nitric oxide in response to stimulation with purified curli, GST-CsgA, and Pam3CSK4, than BMDMs deficient in CD14. Binding assays demonstrated direct binding of curli to all members of this hypothesized trimolecular complex, TLR2, TLR1, and CD14. Utilizing synthetic peptides corresponding to the fourth and fifth repeat of the CsgA monomer, CsgA R4-5, and its modified version, CsgA R4-5N122A deficient in forming amyloid fibers, we also showed that binding to CD14, and CD14 enhancement of IL-6 production required the fibrillar amyloid structure of curli. To study interactions between curli and eDNA in biofilms and the resulting immune response generated to composites formed by these ECM components, we analyzed biofilms of GFP expressing S. Typhimurium using confocal laser scanning microscopy (CLSM). Staining for amyloids with Congo Red revealed the presence of curli in the biofilms and staining with propidium iodide demonstrated the presence of extracellular DNA in the biofilms. Co-staining with TOTO-1, a nucleic acid stain, and Congo Red showed co-localization of the fluorescent signal for these molecules within the biofilms. DNase I treatment of the biofilms produced no significant change in biofilm thickness by confocal microscopy signifying that the biofilm, possibly eDNA, was resistant to DNase treatment. This was further confirmed by the presence of DNA in purified curli fibers, which were treated twice with DNase and RNase. Polymerization assays showed acceleration of amyloid polymerization in the presence of DNA from both bacteria and salmon sperm. CLSM of bone marrow derived dendritic cells demonstrated that DCs are able to sample antigens from biofilms. BMDCs also produced robust quantities of proinflammatory cytokines in response to wild type, msbB, and ΔfliCfljB S. Typhimurium biofilms and purified amyloid/DNA composites as measured by ELISA. Using BMDCs deficient in TLR2 and TLR9, we found that this cytokine production was partially dependent on TLR2, but did not require TLR9. Together, these findings significantly broaden our understanding of S. Typhimurium biofilms and the immune response to ECM components present in its biofilms. We now understand that a trimolecular complex of TLR2/TLR1/CD14 is required for full response to curli by innate immune cells. We also discerned that interactions between biofilm components aid biofilm development and create composites that are highly immunogenic. This new information enhances the need to explore the interaction between composite ligands and the immune system rather than only studying ligands individually. / Microbiology and Immunology

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. In this dissertation, the single N. vectensis TLR (Nv-TLR) is characterized. It is demonstrated that Nv-TLR can activate canonical NF-κB signaling in human cells, the intracellular TIR domain of Nv-TLR can interact with human TLR adapter proteins MAL and MYD88, and the TIR domain of Nv-TLR is required for NF-κB activation. It is shown that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, Nv-TLR is shown to be expressed in a subset of cnidocytes and many of these Nv-TLR-positive cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR, many innate immune pathway homologs, and can engulf V. coralliilyticus. Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. The characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a novel model for studying the molecular basis of cnidarian disease and immunity.