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.

Indiana University-Purdue University Indianapolis (IUPUI) / Flavocytochrome b558, the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91phox (NOX2) and a smaller subunit, p22phox. Localization of flavocytochrome b to the phagosome is essential for microbial killing, yet the subcellular distribution of flavocytochrome b in macrophages and how it is incorporated into macrophage phagosomes is not well characterized. In neutrophils, flavocytochrome b localizes primarily to specific granules that are rapidly mobilized to the phagosome upon stimulation. In contrast to neutrophils, macrophages do not contain specific granules, and trafficking of membrane proteins to the phagosome is more dynamic, involving fission and fusion events with endosomal compartments. We hypothesized that in macrophages, flavocytochrome b localizes to both plasma membrane and endosomal compartments that deliver flavocytochrome b to the phagosome. We generated fluorescently tagged versions of both p22phox and gp91phox, and rigorously verified their functionality in Chinese Hamster Ovary cells. Localization of flavocytochrome b was then examined in both RAW 264.7 murine macrophages and primary murine bone marrow derived macrophages (BMDM) in the presence and absence of interferon gamma (IFNg). We found that in “resting” macrophages, flavocytochrome b localizes primarily to the Rab11-positive endosome recycling compartment that recycles to the plasma membrane. In addition, phagocytosis assays showed flavocytochrome b is incorporated into the phagocytic cup and colocalized with Rab11 at the base of the cup, suggesting Rab11-positive endosomes may be involved in trafficking of flavocytochrome b between intracellular membranes and forming or nascent phagosomes. However, in IFNg activated macrophages, flavocytochrome b was localized predominantly in the plasma membrane, with little present in endosomal compartments. This shift in flavocytochrome b distribution occurred following sustained exposure to IFNg and correlated with increased flavocytochrome b protein expression and increased extracellular production of superoxide. Taken together, our results suggest the IFNg-induced redistribution of flavocytochrome b may be important for enhancing the production of superoxide at the cell surface and may be a potential new mechanism by which IFNg enhances antimicrobial activity in macrophages.

No description available.

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.

WHO has reported that antibiotic resistance is the third major cause of human death all over the globe. Recent study, has focused on the development of new antibacterial resistance drugs. Herein, we tried to synthesis a series of polymers that can mimic the HDPs. HDPs can target the bacterial cell membrane and they have less chances to develop bacterial resistance. We synthesized the amphiphilic polycarbonates that are highly selective to Gram-positive bacteria, including multidrug resistant pathogens. The membrane disruption activity of these polymers was proved by fluorescence and TEM studies and the drug resistance study showed that the polymers don’t develop bacterial resistance. In order to further design the molecules that can target a broad spectrum of bacteria, we have designed a series of lipidated dendrimers that can target the Gram-positive and Gram-negative bacteria. These dendrimers mimic the HDPs and target the bacterial cell membrane. Dendrimers are reported to inhibit the formation of bacterial biofilm which makes them promising for their future development of antibiotic agents. Apart from the synthesis of polymers and dendrimers as antibacterial agents, we have designed a series of small molecular antibacterial agents that are based on the acylated reduced amide scaffold and small dimeric cyclic guanidine derivatives. These molecules display good potency against a panel of multidrug-resistant Gram-positive and Gram-negative bacterial strains. Meanwhile, they also effectively inhibit the biofilm formation. Mechanistic studies suggest that these compounds kill bacteria by compromising bacterial membranes, a mechanism analogous to that of host-defense peptides (HDPs). Lastly, we also demonstrate that these molecules have excellent in vivo activity against MRSA in a rat model. This class of compounds could lead to an appealing class of antibiotic agents combating drug-resistant bacterial strains.

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.

A new paradigm for the treatment of infectious disease is through the modulation of innate immune responses. In this capacity, host defense peptides (HDPs) and synthetic Toll-like receptor 9 (TLR9) ligands have the greatest demonstrated potentials. The work presented here considers mechanisms for the improvement of these treatments through optimization, or in the case of HDPs the minimization, of the interactions of these ligands with sensory receptors.<p>Toll-like Receptor 9 activates the innate immune system in response to microbial DNA or immune-modulating oligodeoxynucleotides. While cell stimulation experiments demonstrate the preferential activating ability of CpG-containing nucleic acids, direct binding investigations have reached contradictory conclusions regarding the sequence-specificity of TLR9 ligand binding. To address this discrepancy the characterization of human TLR9 ligand binding properties is reported. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity of the receptor is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion. <p>Host defense peptides are among the leading candidates to combat antibiotic resistant bacterial strains. Recently, HDPs have been demonstrated to function as ligands for the bacterial sensory kinase PhoQ resulting in the induction of virulence and adaptive responses. Thus, concerns have been raised regarding therapeutic applications of HDPs. Here a methodology is described that permits discrimination and quantification of the distinct, but related, peptide behaviors of direct antimicrobial activity and PhoQ ligand potential. Utilizing peptide derivatives of the model HDP Bac2A it is demonstrated that antimicrobial efficiency is significantly, and inversely, related to PhoQ ligand efficacy. This provides a rational basis for HDP selection with greater therapeutic potential and minimized potential for initiation of bacterial resistance.

mPIP is a mouse homologue of human PIP/GCDFP-15 which is an established marker of both malignant and benign pathological conditions of the mammary gland. mPIP gene expression has been identified in both lacrimal and salivary glands of healthy mice and the mPIP protein has been detected in saliva. The mPIP protein has been found to bind oral bacteria, showing the highest affinity for streptococci, suggesting a potential function of mPIP in the non-immune host defense in the mouse oral cavity. Since the exact functions of mPIP are still unknown, we examined the roles of mPIP through both in vitro and in vivo studies, specifically to address the possible role of this protein in non-immune host response through modulating the oral flora. The in vitro studies were primarily focused on elucidation of the consequences of interaction between mPIP and oral bacteria, in particular to examine whether mPIP plays a role in bacterial aggregation. The in vivo studies addressed the roles of mPIP through the analysis of an mPIP knockout mouse model generated in our laboratory. Following confirmation of the null mutation, the delineating the phenotype of this model was pursued through morphopathological analysis as well as examination of the impact of the lack of mPIP on the mouse oral flora. The null mutation in the mPIP knockout mice was confirmed by both the gene and protein analysis. Histological analysis revealed lymphocytic proliferation in both the submaxillary and prostate glands of the mPIP knockout mice. In addition, both quantitative and composition differences in the oral flora of mPIP knockout mice were identified when compared with wild-type controls. Specifically, a higher proportion of the oral bacteria of mPIP knockout mice were found to belong to genus Streptococcus and certain genera were found to be absent from the oral cavity of these mice. The effect of knockout mouse saliva, which lacks mPIP, on the aggregation of oral bacteria was compared to wild-type mouse saliva. Our data suggests that mPIP contributes to saliva-induced bacterial aggregation. While oral flora has multiple functions, including protection against infection, mPIP might play a role in the non-innate host defense through modulating the resident oral flora in the mouse. The identification of lymphocytic proliferation in submaxillary and prostate glands of mPIP knockout mice suggests that mPIP might also interfere with lymphocyte activity, playing a possible immunomodulatory role. / May 2008