Browsing by Subject "lipopolysaccharide"
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Item Characterization of a Broad Host Range Tailocin from Burkholderia(2012-10-19) Duarte, IrisMembers 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.Item Effects of Intra-Articular Lipopolysaccharide Injection on Systemic Cytokine Gene Expression and Leukocyte Population in Young Horses(2012-02-14) Mueller, CarrieNineteen 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.Item Influence of an Intra-articular Lipopolysaccharide Challenge on Markers of Inflammation and Cartilage Metabolism and the Ability of Oral Glucosamine to Mitigate these Alterations in Young Horses(2013-02-04) Lucia, Jessica LaurenThis 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.Item The Relationship Amongst Stress, Temperament, and Immune Function in Brahman Cattle(2011-10-21) Burdick, Nicole CassandraThe studies described herein were designed to determine the influence of temperament on stress hormones and the immune system in response to various stressors. These stressors included transportation, lipopolysaccharide (LPS) challenge, and adrenocorticotropin (ACTH) challenge. In the first transportation study, bulls (8 Calm, 8 Intermediate, and 8 Temperamental) were loaded into a trailer and transported for 9 hr. Rectal temperature (monitored via indwelling recorders) increased within 0.5 hr of transportation, with greater peak rectal temperature in Temperamental than Calm bulls. Pre- and post-transport concentrations of cortisol and epinephrine were not affected by transportation, but were greater in Temperamental than Calm bulls. A second transportation study utilized 2 automatic sampling devices to allow the recording of rectal temperature and collection of blood samples, respectively. Rectal temperature was not affected by transportation or temperament in response to 4-hr of transport. Average heart rate oscillated between 60 and 130 bpm in Temperamental bulls, but remained around 100 bpm in Calm bulls. Transportation did not affect concentrations of epinephrine, although concentrations were greater in Temperamental bulls than Calm bulls. Cortisol concentrations increased in Calm bulls but not in Temperamental bulls in response to transportation. Additionally, there were limited effects of transportation on peripheral blood mononuclear cell proliferation, IgM production, and cytokine gene expression. Specifically, proliferation tended to be greater post-transport. Expression of the glucocorticoid receptor was, and the expression of toll-like receptor 4 tended to be, reduced post-transport, as analyzed by quantitative real-time RT-PCR. In a study utilizing a LPS challenge, basal stress hormone concentrations during the pre-challenge period were greater in Temperamental bulls than Calm bulls. However, in response to the LPS challenge, only the epinephrine response was influenced by temperament. Additionally, Temperamental bulls exhibited a smaller increase in rectal temperature and sickness behavior than Calm bulls. In the last study, change in gene expression in peripheral blood mononuclear cells in response to acute increases in cortisol was assessed. Plasma cortisol and gene expression of cytokines and the glucocorticoid receptor tended to increase in response to placement of jugular cannula. Additionally, administration of ACTH significantly increased plasma concentrations of cortisol and the gene expression of some cytokines (interleukin-4 and interleukin-10). This suggests that acute increases in cortisol may have positive effects on immune function in Brahman calves. Through an increased understanding of the interaction between the stress response and animal temperament, as well as how stress hormones and temperament influence immune function, animal management practices can be modified to reduce negative impacts on growth and productivity.