Host Defense Peptides (HDPs) are small, cationic and amphipathic molecules with inherent antimicrobial and immunomodular function. However their effects on blood-derived T cells is unknown and is the focus of this investigation. In this thesis, porcine peripheral blood mononuclear cells (PBMCs) were stimulated with bovine myeloid antimicrobial peptide (BMAP)-27, BMAP-28, Indolicidin (Indol), or HH2 in the presence and absence of Concanavalin A (ConA). It was observed that BMAP-27, BMAP-28, and Indol inhibited ConA-stimulated porcine PBMC proliferation. To ensure that the observed effect on cell proliferation was not simply due to a physical interaction between the peptide and ConA, addition of peptide and ConA was staggered. Porcine CD4+/CD8+ T cells were isolated from blood using magnetic activating cell sorting (MACS) and it was determined that BMAP-27 and BMAP-28 inhibited ConA-stimulated T cell proliferation. They did not promote T cell necrosis, but approximately 40 % of the activated T cells undergoes apoptosis in the presence of BMAP-27 and BMAP-28. The remaining 60 % of the T cells consumed very little ATP and showed an increase in expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), indicating the induction of T cell anergy. The addition of exogenous IL-2 decreased the surface expression of CTLA-4 in ConA- activated CD4+ T cells and induced renewed CD4+/CD8+ T cell proliferation, an indicator that these cells underwent activation-induced anergy. Thus, we submit that BMAP-27 and BMAP-28 may play a role in returning the activated T cell population to a homeostatic state through induction of peripheral tolerance mechanisms.

Host Defense Peptides (HDPs) are small, cationic and amphipathic molecules with inherent antimicrobial and immunomodular function. However their effects on blood-derived T cells is unknown and is the focus of this investigation. In this thesis, porcine peripheral blood mononuclear cells (PBMCs) were stimulated with bovine myeloid antimicrobial peptide (BMAP)-27, BMAP-28, Indolicidin (Indol), or HH2 in the presence and absence of Concanavalin A (ConA). It was observed that BMAP-27, BMAP-28, and Indol inhibited ConA-stimulated porcine PBMC proliferation. To ensure that the observed effect on cell proliferation was not simply due to a physical interaction between the peptide and ConA, addition of peptide and ConA was staggered. Porcine CD4+/CD8+ T cells were isolated from blood using magnetic activating cell sorting (MACS) and it was determined that BMAP-27 and BMAP-28 inhibited ConA-stimulated T cell proliferation. They did not promote T cell necrosis, but approximately 40 % of the activated T cells undergoes apoptosis in the presence of BMAP-27 and BMAP-28. The remaining 60 % of the T cells consumed very little ATP and showed an increase in expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), indicating the induction of T cell anergy. The addition of exogenous IL-2 decreased the surface expression of CTLA-4 in ConA- activated CD4+ T cells and induced renewed CD4+/CD8+ T cell proliferation, an indicator that these cells underwent activation-induced anergy. Thus, we submit that BMAP-27 and BMAP-28 may play a role in returning the activated T cell population to a homeostatic state through induction of peripheral tolerance mechanisms.

Host Defense Peptides (HDPs) are small, cationic and amphipathic molecules with inherent antimicrobial and immunomodular function. However their effects on blood-derived T cells is unknown and is the focus of this investigation. In this thesis, porcine peripheral blood mononuclear cells (PBMCs) were stimulated with bovine myeloid antimicrobial peptide (BMAP)-27, BMAP-28, Indolicidin (Indol), or HH2 in the presence and absence of Concanavalin A (ConA). It was observed that BMAP-27, BMAP-28, and Indol inhibited ConA-stimulated porcine PBMC proliferation. To ensure that the observed effect on cell proliferation was not simply due to a physical interaction between the peptide and ConA, addition of peptide and ConA was staggered. Porcine CD4+/CD8+ T cells were isolated from blood using magnetic activating cell sorting (MACS) and it was determined that BMAP-27 and BMAP-28 inhibited ConA-stimulated T cell proliferation. They did not promote T cell necrosis, but approximately 40 % of the activated T cells undergoes apoptosis in the presence of BMAP-27 and BMAP-28. The remaining 60 % of the T cells consumed very little ATP and showed an increase in expression of cytotoxic T lymphocyte antigen-4 (CTLA-4), indicating the induction of T cell anergy. The addition of exogenous IL-2 decreased the surface expression of CTLA-4 in ConA- activated CD4+ T cells and induced renewed CD4+/CD8+ T cell proliferation, an indicator that these cells underwent activation-induced anergy. Thus, we submit that BMAP-27 and BMAP-28 may play a role in returning the activated T cell population to a homeostatic state through induction of peripheral tolerance mechanisms.

Salmonella and Campylobacter are the leading causes of bacterial foodborne illness in the United States. Commonly found in the gastrointestinal tract of poultry, Salmonella and Campylobacter may show little to no signs of infection in birds. The objective of this dissertation was to evaluate the influence on mRNA abundance of nutrient transporters and host defense peptides (HDPs) during a Salmonella or a Campylobacter challenge in young commercial broilers. Comparisons were made between non-challenged and challenged (106, 107, or 108 colony forming units of Salmonella or Campylobacter) broilers on expression of nutrient transporters and host defense peptides in the duodenum, jejunum, ileum and cecum at various days after inoculation. During a Salmonella challenge, changes in mRNA abundance of nutrient transporters and avian beta-defensins (AvBD) vary by day, tissue and challenge dose. ZnT1 may play an important role during a Salmonella challenge as mRNA abundance of ZnT1 significantly increased (P<0.05) by day 7 in the 108 group compared to the control. Early changes in LEAP2 mRNA abundance were observed in the 106 group than the 107 and 108 groups. However, at a later time point post challenge, a lower abundance of almost all AvBD mRNA (P<0.05) was observed in the lower gastrointestinal tract especially in the 107 and 108 groups compared to the control group, indicating that the pathogen may be influencing intestinal expression of AvBD mRNA. In Campylobacter, analyses revealed that expression of zinc transporter 1 (ZnT1) increased (P<0.05) in the duodenum, ileum and ceca in the 106 group on day 7. An increase (P<0.05) in the expression of avian beta-defensins were observed on day 14 in the ileum and ceca in the 106 group compared to the control group. Pathogens like Salmonella and Campylobacter may have an influence on the mRNA abundance of nutrient transporters and HDPs. Manipulation of these genes may ensure the survivability of these pathogens. Through sequestration of nutrients, the pathogen would have the ability to colonize the host and replicate. However, it must evade the host immune system as well. The processing of infected poultry with these pathogens may lead to foodborne illness in humans. Further research is needed to investigate possible methods to counter the influence these pathogens have on host immunity genes. / Ph. D. / Foodborne pathogens such as Salmonella and Campylobacter are commonly found in the gastrointestinal tract of poultry, causing little to no disease symptoms in poultry. Consumption of uncooked or mishandled meat and eggs from infected poultry could result in foodborne illness in humans. Little is known, however, about the influence of Salmonella and Campylobacter on the intestinal expression of nutrient transporters and immune genes such as host defense peptides in broiler chickens. Nutrient transporters are responsible for the transport of a variety of nutrients across the intestinal lumen to the blood. Host defense peptide are small peptides, which can be effective against invading bacteria, viruses and fungi. Therefore, the effects of Salmonella and Campylobacter at low, medium, and high challenge doses were determined in broiler chickens. In chickens challenged with Salmonella, changes in the expression of nutrient transporters and host defense peptides were dependent on day, intestinal segment and challenge dose. The expression of the zinc transporter increased in chickens challenged with the highest Salmonella dose. In chickens challenged with Campylobacter, changes in expression of nutrient transporters and host defense peptides were also observed. Expression of the zinc transporter increased in chickens challenged with the lowest Campylobacter dose. Expression of host defense peptides increased in chickens challenged with the lowest Campylobacter challenge dose. These results indicate that cellular zinc levels as well as host defense peptides may play an important role in modulating a Salmonella and Campylobacter infection.

The emergent resistance of bacteria against the conventional antibiotics has motivated the search for novel antimicrobial agents. Nature abounds with a number of antimicrobial peptides that are a part of our innate immune system and protect us against a variety of pathogenic bacteria. While they are broad-spectrum in their activity and show less drug-resistance induction, their intrinsic metabolic stability limits their potential therapeutic applications. Herein we describe the development of novel broad-spectrum bioactive antimicrobial peptidomimetics AA-peptides. AApeptides were designed based on chiral PNA backbone. Substitution of nucleobases yields AApeptides that are resistant to proteolysis and capable of mimicking peptides. Two types of AApeptides are discussed in this dissertation "[aacute]-AApeptides" and " ã-AApeptides" The therapeutic potential of these AApeptides were accessed by conducting antibacterial assays against a series of both gram-positive, gram-negative bacteria and fungi. These oligomers were characterized using MALDI-TOF and Circular Dichroism spectroscopy (CD). Their invitro toxicity was evaluated against human erythrocytes .We attempted to study their mechanism of action via membrane depolarization assay. We have successfully identified them as antimicrobial agents, pro-inflammatory immune response suppressing agents and as anti-biofilm agents.

The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads.

The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads.

The emergence of pathogens resistant to available drug therapies is a pressing global health problem. Antimicrobial peptides (AMPs) may potentially form new therapeutics to counter these pathogens. AMPs are key components in the mammalian innate immune system and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. This thesis describes computational methods for the identification of novel natural and synthetic AMPs. A bioinformatic resource was constructed for classification and discovery of gene- coded AMPs, consisting of a database of clustered known AMPs and a set of hidden Markov models (HMMs). One set of 146 clusters was based on the mature peptide sequence, and one set of 40 clusters was based on propeptide sequence. The bovine genome was analyzed using the AMPer resources, and 27 of the 34 known bovine AMPs were identified with high confidence and up to 69 AMPs were predicted to be novel peptides. One novel cathelicidin AMP was experimentally verified as up-regulated in response to infection in bovine intestinal tissue. A chemoinformatic analysis was performed to model the antibacterial activity of short synthetic peptides. Using high-throughput screening data for the activities of over 1400 peptides of diverse sequence, quantitative structure-activity relation (QSAR) models were created using artificial neural networks and physical characteristics of the peptide that included three-dimensional atomic structure. The models were used to predict the activity of a set of approximately 100,000 peptide sequence variants. After ranking the predicted activity, the models were shown to be very accurate. When 200 peptides were synthesized and screened using four levels of expected activity, 94% of the top 50 peptides expected to have the highest level of activity were found to be highly active. Several promising candidates were synthesized with high quality and tested against several multi- antibiotic-resistant pathogens including clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. These peptides were found to be highly active against these pathogens as determined by minimal inhibitory concentration; this serves as independent confirmation of the effectiveness of high-throughput screening and in silico analysis for identifying peptide antibiotic drug leads. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Dendritic cells (DCs) are potent antigen presenting cells (APCs) that link the innate and adaptive immune system by their unique ability to induce and direct immune responses towards various T helper (Th)-type of immune responses such as Th1-, Th2-, Th9-, Th17-, Th22- or T regulatory (TR). The type of Th response generated very much depends on the nature of the antigen encountered and allows for an effective and proficient immune response. For example, Th1 responses are used to clear intracellular pathogens while Th2 responses are needed to clear extracellular pathogens The ability to specifically modulate Th-responses is an area of intense research, as it allows for the development of more effective vaccines and immunotherapeutics. Immunomodulation of DCs is one strategy by which specific Th-type immune responses may be tailored. Current research is focused on identifying agents that have the capacity to immunomodulate DCs such as host defense peptides (HDPs). Apart from their anti-microbial activities, HDPs have a number of immune functions including recruitment and subsequent activation of DCs. The goal of this study was to examine the immunomodulatory effects of HDPs on porcine DC functions. This research was part of a larger multinational research project to develop a novel adjuvant platform for single-immunization vaccines against pertussis in neonates. The pig model was used for this research because of its physiological similarities to humans and the recently developed pertussis infection model in young piglets. A series of experiments was conducted to characterize and describe porcine DC functions. Two subsets of DCs were successfully characterized and tested for their response to stimulation with HDPs. Initial results demonstrated a minimal effect of HDPs on DC functions, therefore we expanded the number of HDPs used to include both synthetic derivatives of HDPs known as innate defense regulators (IDRs) and naturally- occurring HDPs. We examined these effects on peripheral blood mononuclear cells (PBMC) in vitro and found that HDPs induce expression of the chemokine interleukin (IL)-8, which resulted in PBMC recruitment in vitro. We then proceeded to evaluate the HDPs in vivo by intradermally administering them into the flank of pigs. Surprisingly, treatment with the HDPs did not result in recruitment of neutrophils in vivo. We also examined the effects of formulating IDR-1002 as an adjuvant with the academic antigen Keyhole Limpet Hemocyanin (KLH) on the development of KLH-specific immune responses in vaccinated pigs. While there was no difference in antibody titers between vaccinated and control animals, we found that co-formulation with IDR-1002 decreased both antigen-specific and mitogen-induced proliferation in KLH/IDR-1002 vaccinated animals as long as four weeks post-treatment. These results demonstrate that specific IDRs can suppress certain aspects of the pro-inflammatory immune response making them potentially highly versatile tools to modulate and tailor the immune response in disease states characterized by a pro-inflammatory component.

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.