Neuroinflammation is a common pathology found in patients with Parkinson's disease (PD). PD involves a loss of dopamine (DA) neurons and an increase in activated microglia with subsequent proinflammatory cytokine secretion in the substantia nigra (SN) and striatum. A loss of DA neurons is found in the offspring of animals exposed prenatally to the bacteriotoxin, lipopolysaccharide (LPS) (Ling et al., 2002). Activation of the extracellular regulated kinases, ERK1/2 and ERK5, the downstream targets in the mitogen-activated protein kinase (MAPK) pathway, has been shown to be involved in the dysregulation of the inflammatory process (Cuschieri and Maier, 2005). Consequently, LPS-induced activation of ERK1/2 and ERK5 may cause an increase in production and secretion of proinflammatory cytokines in activated microglia. LPS-mediated activation of ERK1/2 has been shown to be decreased by the phytochemical resveratrol (Zhang et al., 2010). However, the effect of LPS or resveratrol on ERK5 signaling has not been explored. The purpose of this study was to determine (1) the effect of resveratrol on LPS-induced dopaminergic deficits in pups exposed prenatally to LPS, (2) the impact of resveratrol on LPS-induced BV-2 microglial cell activation and (3) the roles of ERK1/2 and ERK5 in resveratrol mediated inhibition of LPS-induced BV-2 cell activation. To test our hypothesis, pregnant rats received an intraperitoneal (i.p.) injection 10,000 EU/kg LPS at gestational day 10.5 (E10.5) and were fed a resveratrol-enriched diet for 20 days (E3 - E22.5). LPS-induced dopaminergic deficits in pups exposed prenatally at postnatal day 21 (P21), but not at P10 or P40. These deficits were exhibited by a loss of striatal 3,4-dihydroxyphenylacetic acid (DOPAC) and DA content and tyrosine hydroxylase (TH) expression in the P21 animals. However, dietary resveratrol supplementation increased TH expression, DA and DOPAC levels in the P21 pups following prenatal exposure to LPS. Thus, these data suggest that resveratrol treatment may restore the homeostasis of the DA neuronal system in vivo. However, contrary to previous reports it was determined in vitro that LPS-mediated BV-2 activation and ERK1/2 phosphorylation was not inhibited by resveratrol pretreatment. Interestingly, at 6 hours the MEK inhibitor U0126 decreased LPS-mediated ERK1/2 activation and TNF-α release. ERK5 was not activated by LPS, but preliminary data suggest that the MEK5 inhibitor BIX02189 inhibited LPS-induced TNF-α release. Therefore, BIX02189 may be inhibiting a distinct pathway in our model. Overall, these studies suggest that the use of dietary resveratrol supplementation may be protective against LPS-induced loss of striatal dopaminergic deficits in a time-dependent manner and inhibition of ERK signaling may reduce LPS-mediated microglial activation. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmacology / MS / Thesis

Published Article / The initial section of this manuscript focus on the ultra-structure of a unique class of heat stable cell-bound lipopolysaccharides (endotoxin) produced by Gram-negative bacteria. Subsequently, this paper summarises literature on the human body's response when challenged with endotoxins present in food and further explores the influence of food manufacturing and storage practices on endotoxin production and release by bacteria commonly isolated from food. Finally, this paper presents a brief description on the methods applied by the food industry to quantify endotoxins.

Nineteen yearling Quarter Horses were utilized in a randomized, complete block design to evaluate systemic cytokine gene expression and circulating leukocyte population in young horses following an intra-articular lipopolysaccharide (LPS) challenge. Horses were administered an injection of 0.25 ng (n = 7) or 0.50 ng (n = 6) of LPS or lactated Ringer?s solution (n = 6; control). Blood was collected via jugular catheter at pre-injection h 0 and at 2, 6, 12, and 24 h following aseptic injection of the left radiocarpal joint. Aseptic arthrocentesis was performed at the same times to sample synovial fluid for a companion study. Total RNA was isolated from leukocytes using a commercially available kit and real-time PCR was used to determine relative gene expression of the cytokines; interleukin (IL)-1beta (beta), IL-6, IL-8, IL-10, and tumor necrosis factor-alpha (TNF-alpha). Determination of total leukocyte subpopulations and differentials was performed by Texas Veterinary Medical Diagnostic Laboratory. Data were analyzed using the PROC MIX procedure of SAS. Gene expression of all cytokines analyzed was unaffected by treatment. However, changes over time were observed in some cytokines. Interleukin-1? was increased above baseline at 6, 12, and 24 h (P = 0.04), IL-6 was decreased slightly at 6 and 12 h and then increased at 24 h (P = 0.002), and TNF-alpha was increased at 6 and 12 h (P = 0.01). Only IL-8 exceeded a 2-fold change in expression (P = 0.01), peaking at 12 h and indicating greater responsiveness to arthrocentesis than was observed in the other cytokines. No treatment effects on the leukocyte population were observed; however, total circulating leukocytes increased over time (P = 0.04), peaking at 6 h post-injection. Similarly, an increase over time was observed in monocytes (P = 0.002) and in platelets (P = 0.01) at 24 h post-injection. The results indicate that regardless of treatment, a mild immune response was elicited, likely due to repeated arthrocentesis. Future experiments should consider the effects of arthrocentesis and potential systemic inflammatory response, even in control animals, when administering intra-articular LPS to young horses.

This project established an in vivo method to identify and manipulate expression of markers of osteoarthritis (OA). Specifically, strategies that predictably induce joint inflammation to evaluate dietary methods of OA prevention in young horses have yet to be accomplished. Therefore, the 3 studies described herein were conducted to determine effectiveness of an intra-articular lipopolysaccharide (LPS) challenge on markers of inflammation and cartilage metabolism in young horses and potential of dietary glucosamine hydrochloride (HCl) to mitigate these alterations. In the first study, horses were challenged with 0.25 ng or 0.50 ng of intra-articular LPS solution or lactated ringer’s solution (control). Injection of LPS increased inflammation based on synovial prostaglandin E2 (PGE2) concentrations. Carboxypeptide of type II collagen (CPII), a maker of type II collagen synthesis, also increased in a dose-dependent manner. However, clinical parameters of health were not influenced and remained within normal ranges. Carpal circumference increased in response to repeated arthrocentesis. Lameness scores increased with LPS injection when compared to controls. This model of joint inflammation (0.5 ng LPS) was used in the second study to evaluate potential chondroprotective effects of oral glucosamine HCl supplementation in yearling horses. Specifically, the oral absorption of glucosamine HCl versus saline was determined by nasogastric dosing and incorporation of dietary glucosamine HCl into plasma and synovial fluid over time. Plasma and synovial fluid concentrations of glucosamine tended to increase over the 98-d period. In the third study, yearlings were challenged with intra-articular LPS to determine the potential of glucosamine HCl to mitigate inflammation when compared to contralateral joints. Injection of LPS increased synovial PGE2 and cartilage biomarkers CPII and collagenase cleavage neopeptide (C2C), a marker of type II collagen degradation. Oral glucosamine HCl decreased PGE2 and C2C concentrations, but increased levels of CPII. Results of these 3 studies provide a clearer understanding of joint inflammation and cartilage turnover in young horses and demonstrated a potential role of oral glucosamine to mitigate these effects and possibly prevent OA in horses.

Members of the Burkholderia cepacia complex (Bcc) are plant and human opportunistic pathogens. Essentially all Bcc isolates demonstrate in vitro broad-spectrum antibiotic resistance. In fact, many clinical isolates are resistant to all currently available antibiotics, rendering therapy ineffective. There is a substantial need to develop new antimicrobial therapies. The potential use of phage-tail-like high molecular weight bacteriocins, or "tailocins", as alternative anti-bacterial agents against Bcc was investigated. A tailocin, designated Bcep0425, produced by B.cenocepacia strain BC0425 was determined to have broad host range activity against members of the Bcc. Targeted mutagenesis of genes involved in the biosynthesis of the bacterial lipopolysaccharide (LPS) was conducted to determine the receptor site and it was determined that L-rhamnose and alpha-glucose associated with the LPS core were the receptors. Genetic analysis and targeted mutagenesis of the tailocin encoding genes was conducted in the host strain, B. cenocepacia BC0425, to determine the genetic organization of the tailocin Bcep0425 gene cluster and to confirm gene functions. We report for the first time genes involved in replication and integration that are associated with a pyocin/tailocin gene cluster. Additionally, a new class (IV) of holin was identified as part of the lysis cassette. Genetic analysis of the tailocin encoding genes revealed a high degree of similarity to defective phages identified in sequenced Burkholderia genomes. Two novel transcriptional regulators, bctN and bctR, along with recA were found to be involved in the induction of Bcep0425. Numerous studies have focused on the characterization of pyocins from Pseudomonas, but there have been no molecular investigations of tailocins from Burkholderia. This constitutes the first molecular characterization of a phage tail-like bacteriocin from Burkholderia.

Current understanding of molecular-level interactions is inadequate to explain the initial moments of bacterial adhesion. Such information is required to develop appropriate models for bacteria-surface interactions and predictions of cell transport in subsurface environments. Bacterial adhesion is influenced by bacterial surfaces, substratum physical-chemical characteristics, and solution chemistry. Extracellular polymeric substances (EPS), surface proteins, and lipopolysaccharides (LPS) mediate cell adhesion and conditioning film formation via direct bonding to a substrate. The goal of this dissertation is to probe molecular-scale interactions of cell surface macromolecules at mineral surfaces under environmentally-relevant conditions. Four primary investigations are presented in this dissertation. The first study uses in situ attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy to reveal that prior to Mn-oxidation via Pseudomonas putida GB-1, cell adhesion to ZnSe is favorable. Subsequent Mn-oxidation results in increased extracellular proteins expression. Conversely, planktonic cell adhesion is inhibited for Mn-oxide coated cells via blocking of surface proteins. The second investigation reveals the formation of inner-sphere complexes between bacteria surface phosphoryl groups and nanohematite (α-Fe₂O₃). Spectra of bacteria (P. aeruginosa PAO1, Shewanella oneidensis MR-1, and Bacillus subtilis) on α- Fe₂O₃ contain peaks indicative of P-OFe inner-sphere bonding. Spectra collected for oxide-adsorbed model P-containing compounds give spectral signatures similar to those P-OFe bonding interactions observed for whole cell and EPS. The behavior of P. aeruginosa serotype 10 LPS in aqueous solutions was investigated in the third study. Ionic strength, pH, and electrolyte composition were varied during collection of ATR-FTIR and dynamic light scattering (DLS) data. Results reveal stable aggregate Na-LPS aggregates, whereas binding of Ca²⁺ to phosphate groups in the lipid A region leads to aggregate reorientation and increased interaction with ZnSe (hydrophobic). DLS data demonstrate decreasing hydrodynamic radius of LPS aggregates with increasing I and decreasing pH. In the fourth investigation, ATR-FTIR was used to probe the solid-solution interface of LPS on surfaces of ZnSe, Ge, α-Fe₂O₃, and α-Al₂O₃ in solutions of varying ionic composition and pH. Na-LPS aggregates remain stable and spectra are biased towards solution phase LPS. Ca-LPS aggregates are disrupted, leading to enhanced interaction with surfaces via hydrophobic (lipid A- ZnSe) and electrostatic (O-antigenhydrophilic surfaces) interactions.

Fps/Fes and Fer are members of a distinct subfamily of cytoplasmic protein tyrosine kinases that have recently been implicated in the regulation of innate immunity. Previous studies showed that mice lacking Fps/Fes are hyper-sensitive to systemic lipopolysaccharide (LPS) challenge. This study identifies physiological, cellular and molecular defects that contribute to the hyper-inflammatory phenotype in Fps/Fes-null mice. We showed that plasma tumour necrosis factor (TNF) - levels were elevated in LPS challenged Fps/Fes-null mice as compared to wild type mice, and cultured Fps/Fes-null peritoneal macrophages treated with LPS showed increased TNF- production. Cultured Fps/Fes-null macrophages also displayed prolonged LPS-induced degradation of IB-, increased phosphorylation of the p65 subunit of NF-B, and defective TLR4 internalization. Next, we showed a role for Fps in the regulation of recruitment of inflammatory leukocytes. Using the cremaster muscle intravital microscopy model, we observed increased leukocyte adherence to venules, and increased rates and degrees of transendothelial migration in Fps/Fes-null mice, compared to wild type. There was also increased neutrophil migration into the peritoneal cavity subsequent to thioglycollate challenge. Using flow cytometry, we observed prolonged expression of the selectin ligand PSGL-1 on peripheral blood neutrophils from Fps/Fes-knockout mice stimulated ex-vivo with LPS. Finally, we examined the role of Fps/Fes in regulating apoptosis in response to inflammation. Upon intra-peritoneal challenge with LPS, Fps/Fes-null mice displayed a decreased depletion of macrophages from the peritoneal cavity. In response to ex-vivo TNF- stimulation, macrophages from Fps/Fes-null mice underwent decreased apoptosis and necrosis as assessed by flow cytometry. Immunoblot analysis revealed that Fps/Fes-null macrophages displayed more TNF--induced degradation of IB-α in Fps/Fes-null cells, with corresponding increases in the phosphorylation of the p65 subunit of NF-B. In addition, stimulation of macrophages with TNF-α up-regulated PARP expression in wild-type macrophages; this up-regulation was not observed in Fps/Fes-null macrophages. Finally we observed a decreased recruitment of macrophages to the peritoneal cavities of Fps/Fes-null mice, with a corresponding increase in neutrophil recruitment, 5 days after thioglycollate challenge. Overall, we show that there is a role for Fps/Fes in regulating inflammation at the physiological, cellular, and molecular levels, and that this might be relevant in inflammatory disease. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-08-27 11:39:23.393

The effects of induced subacute ruminal acidosis (SARA) using grain pellet-based (GPI) and alfalfa pellet-based diet models on systemic immunological parameters were evaluated in nonlactating Holstein cows. The systemic immunological parameters analysed in this study included rectal temperature, blood cell leukogram, expression of lipopolysaccharide (LPS) recognition receptors on leukocytic cells, and plasma and serum proteins. Also, blood biochemistry was analysed. There were no significant differences in rectal temperature, blood cell leukogram, expression of LPS recognition receptors and fibrinogen or haptoglobin concentrations between control and SARA induction treatments. Concentrations of serum amyloid A and lipopolysaccharide-binding protein increased while total protein concentrations decreased in response to GPI SARA compared to control. Blood glucose and urea concentrations increased and decreased, respectively, with GPI SARA treatment. Grain pellet-induced SARA resulted in changes to serum proteins and acute phase proteins but did not affect other systemic immunological parameters suggesting a localized inflammatory response was initiated.

No description available.

The effects of induced subacute ruminal acidosis (SARA) using grain pellet-based (GPI) and alfalfa pellet-based diet models on systemic immunological parameters were evaluated in nonlactating Holstein cows. The systemic immunological parameters analysed in this study included rectal temperature, blood cell leukogram, expression of lipopolysaccharide (LPS) recognition receptors on leukocytic cells, and plasma and serum proteins. Also, blood biochemistry was analysed. There were no significant differences in rectal temperature, blood cell leukogram, expression of LPS recognition receptors and fibrinogen or haptoglobin concentrations between control and SARA induction treatments. Concentrations of serum amyloid A and lipopolysaccharide-binding protein increased while total protein concentrations decreased in response to GPI SARA compared to control. Blood glucose and urea concentrations increased and decreased, respectively, with GPI SARA treatment. Grain pellet-induced SARA resulted in changes to serum proteins and acute phase proteins but did not affect other systemic immunological parameters suggesting a localized inflammatory response was initiated.