Browsing by Subject "inflammation"
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Item Anti-inflammatory and Cytotoxic Activities of Mango (Mangifera indica L. var Keitt) Polyphenols in Cancer and Non-cancer Breast Fibroblasts in Vitro(2013-08-12) Arbizu Berrocal, ShirleyBreast cancer is the leading cause of cancer death among women worldwide and polyphenols are under investigation as an alternative to conventional treatment approaches of breast cancer. The anti-inflammatory and anti-proliferative activities of polyphenols have been demonstrated in many studies, yet cellular targets and the underlying cellular mechanisms remain unclear. The overall goal of this study was to investigate the anti-inflammatory and cytotoxic properties of polyphenol compounds extracted from the mango variety Keitt in MCF-12A breast non-cancer and MDA-MB231 breast cancer cells by assessing the modulation of signaling pathways involved in inflammation and carcinogenesis. Mango polyphenols were identified by HPLC-MS analysis. The generation of reactive oxygen species was performed using fluorescence intensity in the DCFH-DA assay. Gene expression was analyzed by qRT-PCR, and protein expression was conducted by Western Blotting and Multiplex Bead assay analysis. Bioactive compounds identified in the mango pulp by HPLC-MS included a great variety of polyphenols such as gallic acid, galloyl glucosides with different degree of polymerization and other polyphenols. The anti-inflammatory activities of mango polyphenols were evaluated in MCF-12A non cancer breast fibroblasts. An inflammatory microenvironment for MCF-12A breast cells was induced with tumor necrosis factor alpha (TNF-?). The generation of reactive oxygen species was suppressed significantly compared to cells induced with TNF-?, where there was no significant difference between the concentrations of mango polyphenol extract. Results showed a significant down-regulation of mRNA and protein expression of inflammatory genes involved in the PI3K/AKT pathway and related downstream targets such as NF-?B and mTOR involved in biological processes including cell growth, proliferation and survival. Moreover, mango polyphenols had a significant impact on the miRNA-126-PI3K/AKT axis which plays an important role in inflammation and carcinogenesis, suggesting a potential anti-inflammatory underlying mechanism. The cytotoxic effects of mango polyphenols were investigated in MDA-MB231 breast cancer cells. Mango polyphenols decreased the production of reactive oxygen species; however no significant differences were found between the tested concentrations of mango polyphenols. The gene expression of proapoptotic factors involved in the intrinsic mitochondrial pathway such as cytochrome C and caspase-3 were significantly regulated after mango polyphenol treatment. In addition, the suppression of the PI3K/AKT/mTOR pathway and downstream effectors such as HIF-1? and VEGF as well as the disruption of the miRNA-21-PTEN/AKT axis were identified as potential underlying mechanism of the cytotoxic properties of mango polyphenols. Overall, findings from this study show that mango polyphenols counteract inflammatory and cancerous cell signaling processes; therefore the potential of mango polyphenols in the prevention of breast-cancer focusing on the PI3K/AKT/mTOR-axis should be further investigated.Item Anti-inflammatory Protein TSG-6 Promotes Early Gingival Wound Healing: An In Vivo Study(2014-04-14) Beltran, Stacy RenayHuman multipotent mesenchymal stromal cells (hMSCs) produce TNF-?-stimulated gene/ protein 6 (TSG-6). TSG-6, a hyluronan (HA)-binding protein, has been associated with the negative regulation of inflammatory tissue destruction. TSG-6 modulates proinflammatory cytokine cascades and enhances tissue repair. The aim of this study was to test the effects of recombinant human (rh) TSG-6 on the healing of an induced gingival wound within the first 2 days post-surgery. Following gingival resection in one hundred twenty Sprague-Dawley rats (~400 g), 2 ?g recombinant human TSG-6 (rhTSG-6) in 5 ?L of Phosphate Buffered Saline (PBS) or the same volume of PBS solution was injected into gingival tissue approximating the surgical wound. Control animals did not receive injections. Examination of animals occurred at 1-2 hrs, 6-8 hrs, 24 hrs, and 48 hrs post-surgery (n= 10 per group). Photographs were taken for a double blind clinical assessment at each time period. Tissue biopsy samples (4mm) and blood were collected at 1-2 hrs, 6-8 hrs, 24 and 48 hrs following surgery. Specimens were analyzed via histological analysis and enzyme-linked immunosorbent assays (ELISA) for quantification and comparison of inflammatory markers IL-1?, IL-6, TNF-? and myeloperoxidase (MPO) per treatment group. Weights were recorded for all animals pre- and post-surgery. Animals injected with TSG-6 had significantly less severe inflammation based on clinical assessment scores at 6-8 (p=0.01228), 24 (p=0.01675), and 48 hours (p=0.0186). Sham and control animals had more weight loss at 24 and 48 hours. Based on histological analysis, sham and control animals had more pronounced cellular infiltrate. Animals injected with TSG-6 had significantly less myeloperoxidase (MPO) (p=0.027) at 24 hours and IL-1? (p=0.027) at 24 & 48 hours. IL-6 showed marginal significant difference at 6-8 hours. There was no significant difference for TNF-? at any time point. TSG-6 reduced post-operative gingival inflammation by modulating the inflammatory cascade; reducing levels of proinflammatory cytokines and cellular infiltrate. Gingival injection of TSG-6 may offer significant promise as an anti-inflammatory agent for patients requiring gingival surgery.Item Bax-mediated coordination of cognate organelle cell death signaling cascades determines cell death phenotype after trauma in the neonatal rat cortex(2007-08-07) Martin Gill; Regino Perez-Polo; Jose Barral; John Papaconstantinou; Donna Ferriero; David RassinBax translocation to the mitochondria has been well-characterized to induce apoptotic cell death in multiple injury paradigms. However, pro-cell death actions for Bax outside of the mitochondria remain understudied. Bax’s pro-cell death role at other non-mitochondrial locales in response to oxidative stress and energy depletion injury paradigms were investigated in in vitro and in vivo neonatal cortical models.\r\n\r\nIn vivo, hypoxia-ischemia (HI) induces a distinct subcellular time course for Bax in the neonatal cortex, localizing first to the nucleus, then to mitochondria and, finally, to the ER with Bax localization coordinating with the activation of each organelle’s cognate cell death cascade, suggesting a new role for Bax in coordinating the cell death signaling at multiple subcellular organelles.\r\n\r\nIn vitro, using necrotic-like and apoptotic stimuli, we observed both treatments induced early nuclear Bax localization, the apoptotic stimulus increasing mitochondrial Bax localization and caspase-mediated cleavage of á-fodrin, and the necrotic-like stimulus increasing ER Bax localization and calpain-mediated cleavage of á-fodrin. Based on these findings, we concluded apoptotic and necrotic-like stimuli promote differential Bax localization and subsequent differential activation of cognate cell death signaling cascades to induce expression of their characteristic phenotypic cell morphologies.\r\n\r\nFinally, we provide novel mechanistic in vitro and in vivo evidence for Bax coordination of multiple cognate organelle cell death signaling cascades. In vitro, we found 1h pretreatment with immunosuppressive and neuroprotective FK506 inhibited high and low dose rotenone-mediated Bax relocalization and cell death signaling, in toto. In vivo, using 100% O2 as an intervention, we showed 100% O2 increases T2-weighted MRI lesion volumes, via increased inflammatory and necrotic signaling, with no amelioration of cortical apoptotic signaling when compared to HI alone. Furthermore, 100% O2 increased ER calpain activation and increased ER Bax protein levels, suggesting that 100% O2 increases HI-induced Bax-mediated activation of ER cell death signaling to increase inflammation and injury by increasing necrotic-like cell death. Taken together, these findings are the first to show Bax-mediated coordination of multi-organelle cell death signaling, demonstrate a link between ER Bax, ER cell death signaling and necrotic-like cell death, and provide evidence for a general trauma-induced Bax relocalization mechanism.\r\nItem Cellular and Molecular Mechanisms of Corneal Inflammation and Wound Healing(2012-04-19) Gagen, Jenny; Burns, Alan R.; McDermott, Alison M.; Miller, William L.; Rumbaut, Rolando E.Purpose: Accidental or surgical-induced trauma (e.g., refractive surgery) to the corneal epithelium results in keratocyte death immediately below the wound. Subsequent recovery of keratocytes during healing often remains incomplete, even after many years. Keratocytes produce essential proteins that help maintain corneal stability and preserve corneal clarity, and incomplete keratocyte repopulation after injury can result in loss of normal corneal structure (e.g., keratectasia) and impaired vision. To date, very little is known about the mechanisms regulating keratocyte repopulation. In the mouse, central corneal epithelial abrasion not only evokes keratocyte death, but also neutrophil (PMN), platelet, and γδ T cell recruitment out of the limbal vessels and into the stroma. This inflammatory cell recruitment is necessary for efficient epithelial cell and epithelial nerve recovery. CD18, ICAM-1, and P-selectin are adhesion molecules linked to the regulated recruitment of these inflammatory cells. The purpose of this Dissertation is to determine if the molecular mechanisms regulating inflammatory cell recruitment into the injured cornea are necessary for keratocyte repopulation following central epithelial abrasion. Methods: A 2 mm diameter central epithelial region was mechanically debrided from corneas of male wild type C57Bl/6 (WT), CD18 mutant (hypomorphic), ICAM-1-/-, TCRδ-/- (deficient in γδ T cells) and P-selectin-/-mice. Injured and uninjured corneas were prepared for transmission electron microscopy or immunofluorescence microscopy. PMN-keratocyte interactions, total PMN infiltration (per mm2), total and extravascular (EV) platelet accumulation (per mm2), and keratocyte repopulation were morphometrically analyzed using stereological methods. In vitro analyses, using cultured mouse keratocytes and extravasated PMNs, were performed to investigate PMN motility on keratocytes. Results: Previously, it was determined that PMN CD18 mediated close surface contacts between the PMN and keratocyte. The current studies show keratocyte ICAM-1, a ligand for CD18, also mediates PMN-keratocyte surface contact, and contacts were significantly reduced in ICAM-1-/- mice compared to WT (21.5 ± 3.0% versus 39.9 ± 3.5%, respectively), consistent with the idea that ICAM-1 binds to PMN CD18 to mediate the cell-cell close surface contact. Antibody blockade of PMN CD18 or keratocyte ICAM-1 markedly reduced PMN motility on keratocytes, in vitro (by 33% and 47.5%, respectively), suggesting CD18 and ICAM-1 play a functional role in promoting PMN migration on keratocytes. PMN and platelet recruitment were greatest in ICAM-1-/- mice and, 4 days after corneal abrasion, anterior central (AC) keratocyte numbers returned to baseline, demonstrating ICAM-1 negatively regulates PMN infiltration and platelet accumulation. AC keratocyte repopulation in WT and CD18 mutant mice was significantly lower than their respective baseline counts (by 28% and 56%, respectively). There were no differences in PMN infiltration between WT and CD18 mutant mice but platelet accumulation was blunted in CD18 mutant mice, suggesting platelets, not PMNs, participate in keratocyte recovery. Previous studies show platelet P-selectin and γδ T cells are required for efficient epithelial healing. AC keratocyte repopulation in P-selectin-/- and TCRδ-/- mice only recovered to 31% and 23% of their baselines, respectively. However, infusion of WT platelets into P-selectin-/- mice “rescued” keratocyte repopulation, bringing it back to P-selectin baseline values. Additionally, AC keratocyte repopulation in platelet-depleted WT mice recovered to only 29% of WT baseline. Conclusion: Collectively, these data confirm platelet recruitment is necessary for efficient keratocyte recovery. Interestingly, EV platelet recruitment in TCRδ-/- mice was greater than WT (by 62%) although keratocyte repopulation was low; however, EV platelets in TCRδ-/- mice showed less evidence of shape change, suggesting they were less“activated” in the absence of γδ T cells. The evidence provided in this Dissertation demonstrates a role for adhesion molecules and inflammatory cells in mediating PMN-keratocyte interactions, facilitating PMN migration, regulating inflammatory cell recruitment, and promoting keratocyte repopulation following corneal epithelial abrasion. Collectively, the data suggest this inflammatory cascade is necessary for keratocyte recovery during wound healing.Item Effect of Conjugated Linoleic Acid or Oleic Acid Addition on Fatty Acid Composition Profiles of Poultry Meat(2011-08-08) Shin, Dae KeunTwo different studies were conducted to reduce the overall amount of omega-6 fatty acids in broiler chickens. The first experiment was performed to determine the effects of dietary conjugated linoleic acid (CLA) and omega-3 fatty acid combination on the omega-6 fatty acid accumulation in broiler chicken breast and thigh meat. Eight broilers from each treatment were processed at 4 and 6 weeks of age, respectively. Regarding the diets containing five different fat sources, broiler chickens fed CLA and fish oil diet had a lower C20:4 (arachidonic acid, AA, n-6) deposition but showed a higher n-3/n-6 ratio in breast and thigh meat than those fed a flaxseed oil diet and CLA and flaxseed oil diet (P < 0.05). The C20:4 and n-3/n-6 ratio of breast and thigh samples from fish oil diet was similar to those of the conjugated linoleic acid and fish oil combination diet (P > 0.05). However, the addition of CLA and fish oil to the diet resulted in a increase of polyunsaturated fatty acid (PUFA) concentration in broiler chicken breast and thigh meat when compared to that of fish oil diet (P<0.05). The second experiment was conducted based on six different combination of n-3 and n-9 fatty acids. One bird per pen was processed, and each bird was weighed, and blood, liver, breast and thigh samples from the bird were collected. Although the generation of prostaglandin E2 (PGE2) was not affected due to combination of n-3 and n-9 fatty acids in our diets, the deposition of n-6 fatty acids including C18:2 and C20:4 was decreased in broiler chicken breast and/or thigh muscles as n-3 fatty acids were supplied to broiler chickens for 9 weeks. Eicosapentaenoic acid (C20:5, EPA, n-3) addition to poultry diet (FEO) did not reduce the deposition of C18:2 and/or C20:4 as much as C22:6 (FDO) did. When C20:5 and C22:6 were blended to poultry diet (FHO) and fed to broiler chickens for 9 weeks, synergistic effects were observed. Reduction of C20:4 was obtained when FHO diet was fed to broiler chickens, and it may be induced due to decreased expression of delta-6 desaturase mRNA.Item Effect of Omega 3 Polyunsaturated Fatty Acids (PUFAs) on Markers of Inflammation in Young Horses in Training(2011-02-22) Lucia, Jessica LaurenSixteen horses (2 to 4 yr; 357 to 439 kg BW) were utilized in a randomized complete block design for a 140 d trial to determine effect of omega 3 PUFAs (n-3) supplementation on markers of inflammation in young horses in training. Horses were fed treatments consisting of a control diet (n = 8) fed at 1% BW (as fed) or a treatment diet (n = 8) of concentrate fed at 0.75% BW (as fed) and 350 g of a marine n-3 supplement formulated to provide 15 g of eicosapentaenoic acid (EPA) and 20 g of docosahexaenoic acid (DHA). Body weight and body condition scores (BCS) were obtained biweekly and concentrate adjusted accordingly. Horses were exercised 5 d/wk by students in an equine training course. Type of activity and duration was monitored, along with heart rate to quantify workload. Exercise protocol was divided into 2 phases: phase I (d 0 to110) consisted of ground work and early training under saddle, and phase II (d 111 to 140) consisted of advance maneuvers and moderate workload. Synovial fluid was obtained from right radial carpal joint by arthrocentesis every 28 d and was analyzed for white blood cell count (WBC), total protein (TP), and specific gravity (SG). Serum concentrations of carboxypeptide type II collagen (CPII) and chondroitin sulfate 846 (CS-846) were analyzed by ELISA kits. Dietary treatment did not affect synovial WBC, TP, or SG. Also, concentrations of WBC and TP also did not differ over time. SG increased over time (P < 0.001) as horses moved from phase I to phase II of the trial. Dietary treatment did not influence concentrations of CPII or CS-846. CS-846 tended to increase over time (P = 0.09) and CPII concentrations also increased (P < 0.001) in response to changes in exercise. Furthermore, all horses gained BW and BCS throughout the trial (P < 0.001), but values were not influenced by treatment. This data indicates further studies are needed to determine the efficacy of n-3 supplementation as a preventative measure against development of osteoarthritis.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 Evaluation of bone biochemical markers and inflammatory markers in yearlings fed varying ratios of omega-6 and omega-3 polyunsaturated fatty acids(2009-05-15) Ross, Trinette NoelDiets formulated to contain varying ratios of omega 6 to omega 3 fatty acids were fed to exercising yearlings to evaluate bone activity and inflammatory response. Nine Quarter Horse yearlings were arranged within a triplicated 3 X 3 Latin Square experimental design and fed one of three diets. Exercise protocol was designed to stimulate sub-clinical inflammation and normal bone response. Body weight and physical growth measurements were not different between groups (P > 0.05), and feed intake was similar between groups (P > 0.05). Horses consuming soybean oil (SBO) diet had lower fatty acid profiles (% by weight) of C16:0 and C16:1 (P < 0.05) when compared to horses consuming either corn oil (CO) or menhaden/corn oil (MCO) diets. Though numerically different, percentage changes in C16:0 and C16:1 were not different between diets (P < 0.05). Horses consuming MCO had significantly higher measurements of C20:4, C20:5 and C22:6 over the 28 day period when compared to horses consuming SBO or CO. Percent change in mean concentrations of C20:5 were significantly different between the MCO group and the SBO group (P < 0.05) with no observed difference between MCO and CO treatment groups. Overall mean carboxyterminal telopeptide of type I collagen (ICTP) concentrations did not differ between diets (P > 0.05) nor was there a significant change from baseline values when compared to day 28 of the period. Mean Osteocalcin (OC) concentrations did not differ between treatments (P > 0.05). Numerically, OC levels were lower after 14 days, with subsequent increases occurring from day 14 to day 28; however, there was no significant day effect (P > 0.05). Mean measurements of PGE2 and fibrinogen, the two inflammation markers evaluated, did not differ among groups (P > 0.05). However, when fibrinogen data were normalized, horses consuming SBO had a significantly lower change in baseline values of fibrinogen compared to horses fed CO or MCO diets (P< 0.05). In general, horses fed SBO exhibited reduced levels of the inflammatory marker fibrinogen (P< 0.05). No other variable evaluated was influenced by the supplementation of varying ratios of polyunsaturated fatty acids into the equine diet.Item Genome-wide Transcriptome Analysis of Laminar Tissue During the Early Stages of Experimentally Induced Equine Laminitis(2012-02-14) Wang, JixinEquine laminitis is a debilitating disease that causes extreme sufferring in afflicted horses and often results in a lifetime of chronic pain. The exact sequence of pathophysiological events culminating in laminitis has not yet been characterized, and this is reflected in the lack of any consistently effective therapeutic strategy. For these reasons, we used a newly developed 21,000 element equine-specific whole-genome oligoarray to perform transcriptomic analysis on laminar tissue from horses with experimentally induced models of laminitis: carbohydrate overload (CHO), hyperinsulinaemia (HI), and oligofructose (OF). Samples were collected during the developmental (DEV) and Obel grade 1 (OG1) stages of laminitis for the CHO model. For the HI model, samples were collected at the Obel grade 2 (OG2) stage. For the OF model, samples were collected at the 12 h and 24 h time points. Appropriate control samples were obtained for all models. This is the first genome-wide transcriptome analysis of laminar tissue using an equine 21,000 70-mer long oligoarray approach in CHO, HI and OF induced laminitis. Overall, we identified the differential expression of genes encoding S100 calcium binding proteins, extracellular matrix proteins, glycoproteins, transporters, olfactory receptors, genes involved in signal transduction, body?s homeostasis, apoptosis, and immune response. Between CHO and OF models of laminitis, there were more shared genes. We discovered several common differentially expressed genes (i.e., ADAMTS1, CYCS and CXCL14) among all three models that are likely important to the pathogenesis of equine laminitis. We also discovered what appear to be central roles of apoptosis, inflammatory response, and intracellular ion homeostasis molecular processes in CHO and OF models of laminitis. Pathway analysis detected the NOD-like receptor signaling pathway, which is involved in recognition of intracellular bacteria in both the CHO and OF models of laminitis. Genetic network analysis indicated convergent pathway core molecules present in equine acute laminitis: p38 MAPK and NF-?B. Most importantly, our results of overexpression of anti-microbial genes (i.e., DEFB4, PI3, and CXCL14) suggest the central involvement of these genes in the progression of early equine laminitis and will allow refinement of current hypotheses of disease pathogenesis.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 Involvement of PFKFB3/iPFK2 in the Effects of Leucine and n-3 PUFA in Adipocytes(2012-02-14) Halim, VeraStudies had shown that leucine supplementation increases insulin sensitivity and it has been studied that n-3 PUFA may have an anti-inflammatory effect in adipocytes. However, the extent to which dietary sources such as leucine and/or n-3 PUFA act through PFKFB3/iPFK2 to suppress adipocyte inflammatory response has not been studied; PFKFB3/iPFK2 is a regulator that links adipocyte metabolism and inflammatory responses. In this study, the involvement of PFKFB3/iPFK2 in the effects of insulin sensitizing and anti-inflammatory effect of leucine and/or n-3 PUFA are explored using cultured 3T3-L1 adipocytes including wild-type cells, PFKFB3-control cells (iPFK2-Ctrl) and PFKFB3-knockdown cells (iPFK2-KD). In iPFK2-Ctrl cells, leucine supplementation appears to have insulin-sensitizing effects through improving p-Akt/Akt insulin signaling, but have no effect on adiponectin expression, and appear to have limited anti-inflammatory effects. n-3 PUFA supplementation appears to have limited effects on both insulin sensitizing and anti-inflammatory effects in iPFK2-Ctrl. In contrast, n-3 PUFA exhibit pro-inflammatory expression in iPFK2-KD. The results of this study support the hypothesis that PFKFB3/iPFK2 is critically involved in insulin-sensitizing effects of leucine. This role of PFKFB3/iPFK2, however, appears to be independent of anti-inflammatory responses. Given this, it is likely that PFKFB3/iPFK2 only account, in part, for the beneficial effects of leucine. n-3 PUFA stimulate PFKFB3/iPFK2 activity in wild-type adipocytes. However, PUFA do not exhibit anti-inflammatory and insulin-sensitizing effects in controls. In contrast, n3-PUFA exhibit proinflammatory effects in iPFK2-KD cells. Taken together, PFKFB3/iPFK2 is involved, at least in part, in the effects of insulin sensitization of leucine and appears to protect adipocytes from inflammatory responses, which could be exacerbated by n-3 PUFA when PFKFB3/iPFK2 is disrupted.Item Mechanisms of imporvement following functional inhibition of nitrosative stress after brun and smoke-induced acute lung injury(2008-07-28) Aimalohi Esechie; Daniel L. Traber; Simon A. Lewis; Hiroshi Saito; Hal K. Hawkins; George C. Kramer; Csaba SzaboSevere trauma, caused by flame burn and smoke (B + S) inhalation induces acute lung injury (ALI) and results in the loss of pulmonary function. A cascade of molecular and cellular events initiates the formation of reactive oxygen/nitrogen species (ROS/RNS) that in turn drives an inflammatory response and consequently cell death through hyper-activation of poly (ADP-ribose) polymerase (PARP-1). The purpose of this study was to investigate and counteract pulmonary dysfunction associated with nitrosative stress generated after B + S inhalation injury in an ovine and murine model of ALI. \r\nIn our time course experiment, sheep were sacrificed at 4, 8, 12, 18 and 24 hours post B + S injury. From 4 through 24 hours, there was a progressive increase in airway obstruction and lung edema formation. Furthermore, injury was associated with increased ROS/RNS generation, pro-inflammatory cytokine expression and neutrophil accumulation. Additionally, PARP-1 enzymatic activity increased in parallel with Hoechst 3324 nuclear staining in sheep lung sections.\r\nTreatment after ALI with a hydrogen sulfide (H2S) donor compound, a peroxynitrite scavenger, was tested to determine the effect on mortality, pulmonary shunt fraction and gas exchange. The H2S donor increased animal survival. Additionally the rapid decline in PaO2/FiO2, reduced the pulmonary shunt fraction and elevated airway pressures were improved. Likewise, the lung histological assessment demonstrated marked increase in aerated areas in lung sections.\r\nBurn and smoke injury generates reactive oxygen species and nitrogen species and influences inflammatory cytokine expression. Pro- and anti-inflammatory cytokine protein levels were measured in the lung parenchyma as well as 3-nitrotyrosine and protein carbonyl formation (indices of RNS and ROS generation, respectively). In our murine study, treatment with the H2S donor significantly reduced the pro- inflammatory cytokine level and increased the anti-inflammatory cytokine concentration in the lung. Additionally, ROS and RNS generation were significantly lowered. \r\nThese results demonstrate the effectiveness of inhibiting nitrosative stress after burn and smoke injury using a H2S donor and a possible mechanism for improved outcomes may be the altered the expression pattern of pro- and anti-inflammatory cytokines and ROS generation which may contribute to dysfunctional outcomes after B+ S inhalation injury.\r\n\r\nItem Mechanisms of improvement following functional inhibition of neutrophil infiltration after spinal cord injury(2006-12-06) Michael Wayne Carter; Claire E. Hulsebosch; Judith F. Aronson; Donald S. Prough; David J. McAdoo; Alan R. LightSpinal Cord Injury (SCI) is a traumatic event that results in loss of function below the level of injury, and other dysfunctions including pain syndromes, both above and below the level of the lesion. A cascade of biochemical and cellular events leads to secondary events after SCI, exacerbating the injury, and contributes to loss of tissue and increased dysfunctions. While thought to be beneficial, inflammation induced by trauma in the spinal cord contributes to secondary injury early in SCI. Thus, in this project a mammalian model is used to investigate a targeted anti-inflammatory treatment for SCI, and compare it with the current standard therapy of high dose methylprednisolone (MPSS).\r\nTo ensure that the new generation of devices used in experimental SCI can reliably produce an injury that parallels the outcomes seen in the clinical setting, we characterized a rodent model of SCI by adjusting several mechanical parameters of the device. Injuries with higher force or increased duration of compression increased sensitivity to mechanical stimuli and produced loss of locomotion and loss of bladder function, syndromes seen clinically after SCI.\r\nTreatment after SCI with recombinant neutrophil inhibitory factor (rNIF) was tested to determine if inhibition of neutrophils, a primary inflammatory cell, in the first 24 hours after injury would improve outcome measures. Treatment with rNIF reduced neutrophil infiltration after injury by greater than 50% and resulted in decreased sensitivity to mechanical stimuli and improved bladder function. Additionally, the amount of white and grey matter lost secondary to SCI was reduced.\r\nSince neutrophils release proteinases, generate reactive oxygen species, phagocytize cells and influence inflammatory cytokine expression, pro- and anti-inflammatory cytokine protein levels were measured at specific time points after SCI, in both the spinal parenchyma and blood serum. Treatment with rNIF had a significant effect on cytokine expression after injury.\r\nThese results demonstrate the effectiveness of inhibiting secondary injury after SCI using rNIF and one mechanism for improved outcomes may be the altered the expression pattern of pro- and anti-inflammatory cytokines which may contribute to dysfunctional outcomes after SCI.\r\nItem Mechanisms of increased microvascular permeability during acute Rickettsiosis(2007-11-02) Michael Edward Woods; Juan P. Olano; J. Stephen Dumler; Doug S. DeWitt; David H. WalkerRickettsial diseases represent some of the most severe bacterial infections in man including Rocky Mountain spotted fever and epidemic typhus. Rickettsiae primarily target the microvascular endothelium leading to increased microvascular permeability, the mechanisms of which are completely unknown. We sought to determine the impact of host responses to infection on increasing microvascular permeability both in vitro and in vivo. Our work has revealed a role for TNF-á, IL-1â, and IFN-ã as mediators of anti-rickettsial immunity that contribute to increased microvascular permeability in a dose-dependent manner by modulating the function of interendothelial adherens junctions. The permeability-inducing effects of these cytokines appear to occur independently of nitric oxide production since inhibition of iNOS does not prevent cytokine-mediated increases in permeability. Additionally we have shown that iNOS expression in vivo is associated with sites of rickettsial invasion, which also correlates with the leakage of endogenous serum protein. The lack of significantly higher levels of serum cytokines suggests this is primarily a localized response confined to areas of leukocyte infiltration. Likewise we have demonstrated a role for innate endothelial cell responses in modulating adherens junctions following rickettsial invasion. Human endothelial cells infected with rickettsiae produced significantly higher levels of VEGF and IL-6, two cytokines which can have a profound impact on adherens junction stability. This was associated with increased kinase activity in the form of protein kinase C, Src, and focal adhesion kinase. Inhibition of Src during R. rickettsii infection led to a decreased rate of endothelial permeability however this did not prevent rickettsiae-mediated cell death. Finally, we have identified several novel pathways modulated after rickettsial infection that were not previously thought to be important to rickettsial pathogenesis. Future work will be aimed at determining the relative contribution of these pathways to the endothelial dysfunction accrued during rickettsial infection. The work generated here provides a solid foundation for future endeavors aimed at alleviating the vascular dysfunction experienced during severe rickettsial infection.Item A Missing link between lipid metabolism, inflammation and apoptosis: Phospholipase A2-activating protein (PLAA)(2009-03-02) Fan Zhang; Ashok K. Chopra; Thomas G. Wood; Sheila E. Crowe; Johnny W. Peterson; Istvan BoldoghPhospholipase A2-Activating Protein (PLAA) is a novel signaling molecule that regulates the production of arachidonic acid (AA), prostaglandin E2 (PGE2) and TNF-¦Á. Literature suggests that PLAA could be involved in inflammatory responses and apoptosis. However, the in situ function of PLAA is elusive. To elucidate PLAA¡¯s role in TNF-¦Á-induced inflammatory responses and cisplatin-induced apoptosis, we manipulated the expression of the plaa gene at cellular level using overexpression and siRNA approaches. We generated HeLa (Tet-off) cells overexpressing plaa (plaa high) and control (plaa low) cells. We compared plaa high and plaa low cells for transcriptional profiling and their responses to TNF-¦Á stimulation. Overexpression of the plaa gene induced the expression of the proinflammatory cytokine IL-32 and reduced the expression of annexin A4 (a PLA2 inhibitor) and clusterin. We demonstrated that extracellular clusterin limited the production of PGE2. We showed that upon TNF-¦Á stimulation, plaa high cells revealed enhanced PLA2 activation, COX-2 expression and PGE2 production. Furthermore, we found that in response to TNF-¦Á, plaa high cells had significantly enhanced activation of NF-¦ÊB and production of IL-6, compared to the TNF-¦Á-stimulated plaa low cells. To understand regulation of plaa gene expression, we used a luciferase reporter system in normal HeLa cells and identified one stimulatory element, with Sp1 transcription factor-binding site, and one inhibitory element, in exon 1 of the plaa gene. To determine the role of PLAA in apoptosis, we compared the apoptotic responses to cisplatin in plaa high and plaa low cells. Cisplatin-stimulated plaa high cells contained significantly higher levels of DNA fragmentation, caspase activities, PLA2 enzyme activity and mitochondrial damage than did the cisplatin-stimulated plaa low cells. siRNA against PLAA (siRNA-PLAA) reverted the above mentioned trend and promoted cell viability. Further, cisplatin-stimulated plaa high cells produced less cytoprotecive clusterin and more pro-apoptotic IL-32 than did the cisplatin- stimulated plaa low cells. siRNA-PLAA promoted clusterin production and inhibited IL-32 expression from both plaa high and plaa low cells. Finally, our proteomic analysis revealed that cisplatin-stimulated plaa high cells contained higher levels of phosphorylated JNK/c-Jun and FasL than did cisplatin-stimulated plaa low cells.Item Molecular pathogenesis of Salmonella enterica serotype Typhimurium-induced inflammatory responses(2009-05-15) Figueiredo, Josely FerreiraWe demonstrated that infection of HeLa cells, which are non-responsive to flagellin, with wild type Salmonella enterica serotype Typhimurium (S. typhimurium) activated chemokine expression at higher level than S. typhimurium lacking sipAsopABDE2, indicating that the corresponding effector proteins (SipA, SopA, SopB, SopD and SopE2) are required to induce chemokines independent of flagellin. The S. typhimurium sipAsopABDE2 mutant complemented with sipA activated IL-8 expression at significantly higher level than a S. typhimurium sipAsopABDE2 mutant. However, extracellular addition of recombinant SipA failed to induce IL-8. Phosphorylation analyses demonstrated that S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant) induced phosphorylation of CREB1, JUN and p38MAPK, which are proteins involved in IL-8 expression. The contribution of effector proteins to S. typhimurium-induced intracellular Ca2+ mobilization and its role in IL-8 expression and bacterial internalization were also investigated. Our results demonstrated that wild type S. typhimurium significantly increased the amplitude of intracellular Ca2+ beginning 30 sec after infection. However, further analyses of intracellular Ca2+ changes in HeLa cells infected with S. typhimurium mutants indicated no correlation between increased intracellular Ca2+ and IL-8 expression or bacterial internalization. To analyze specific cell populations targeted by wild type S. typhimurium or S. typhimurium carrying a chromosomal copy of sipA (sopABDE2 mutant), laser capture microdissection was performed. Our data indicated that in wild type S. typhimurium-infected bovine Peyer?s patches, high levels of IL-8 were expressed in enterocytes of crypts, whereas Gro-? was expressed in enterocytes of both crypts and absorptive villi. A strain carrying a chromosomal copy of sipA colonized the same cell population as wild type, but induced IL8 and Gro-? in enterocytes of both crypts and absorptive villi. In conclusion, we demonstrated that in vitro S. typhimurium effector proteins induce chemokine expression independent of Ca2+ changes through phosphorylation of proteins related to IL-8 pathway. In vivo, we found higher levels of IL-8 expression in enterocytes of crypts than enterocytes of absorptive villi, although both cell populations contributed to Gro-? expression. These data extend the knowledge of the molecular mechanism by which S. typhimurium induces inflammatory genes by identifying pathogen and host molecules involved in inflammation.Item n-3 Polyunsaturated Fatty Acids Suppress Mitochondrial Translocation to the Immunological Synapse and Modulate Calcium Signaling in T Cells(2011-02-22) Yog, RajeshwariT helper (Th) cell activation is necessary for the adaptive immune response. Formation of an immunological synapse (IS) between Th cells and antigen-presenting cells is the first step in Th cell activation. In vitro studies indicate that formation of the IS induces cytoskeleton-dependent mitochondrial redistribution to the immediate vicinity of the IS. This redistribution of mitochondria to the IS in T cells is necessary to maintain Ca2 influx across the plasma membrane and Ca2 -dependent Th cell activation. Earlier studies have demonstrated that n-3 polyunsaturated fatty acids (PUFA) suppress the localization and activation of signaling proteins at the IS. Therefore, we hypothesized that n-3 PUFA suppress CD4 T cell mitochondrial translocation during the early stages of IS formation and down-modulate Ca2 dependent Th cell activation. CD4 cells derived from fat-1 mice, a transgenic model that synthesizes n-3 PUFA from n-6 PUFA, were co-cultured with anti-CD3-expressing hybridoma cells (145-2C11) for 15 min at 37 degrees C, and mitochondrial translocation to the IS was assessed by confocal microscopy. fat-1 mice exhibited a significantly (P< 0.05) reduced percentage of CD4 T cells with mitochondria which translocated to the IS; fat-1 (30 percent) versus wild type control (82 percent). With respect to an effect on the mitochondrial-to-cytosolic Ca2 ratio, wild type cells showed significant increases at the IS (71 percent) and total cell (60 percent) within 30 min of IS formation. In contrast, fat-1 CD4 T cells remained at basal levels following the IS formation. A similar blunting of the mitochondrial-to-cytosolic Ca2 ratio was observed in wild type cells co-incubated with inhibitors of the mitochondrial uniporter, RU360 or calcium release-activated Ca2 (CRAC) channels, BTP2. Together, these observations provide evidence that n-3 PUFA modulate Th cell activation by limiting mitochondrial translocation to the IS and reducing Ca2 entry.Item Neuropathic pain and the inhibition of learning within the spinal cord(Texas A&M University, 2004-09-30) Ferguson, Adam RichardPrior work from our laboratory has shown that the spinal cord is capable of supporting a simple form of instrumental (response-outcome) learning. In a typical experiment, animals are given a spinal transection at the second thoracic vertebra, and tested 24 h after surgery. If animals are given shock when their leg is in a resting position (controllable shock), they quickly learn to maintain the leg in a flexed position, thereby minimizing shock exposure. Animals exposed to shock that is independent of leg position (uncontrollable shock) fail to learn. This learning deficit can be induced by as little as 6 minutes of shock to either limb or to the tail, and lasts for up to 48 h. The aim of this dissertation was to explore whether the deficit shares behavioral features and pharmacological mechanisms similar to those involved in the induction of neuropathic pain. Work within the pain literature has identified a spinal hyperexcitability that is induced by intense stimulation of pain fibers. This phenomenon, known as central sensitization, is characterized by an increase in tactile reactivity (allodynia) that can be induced by shock or peripheral inflammation. Pharmacological findings have revealed that central sensitization depends on the activation of the N-methyl-D-aspartate (NMDA) and group I metabotropic glutamate receptors (mGluRs). Experiment 1 showed that uncontrollable shock induces a tactile allodynia similar to that observed in central sensitization. Experiment 2 showed that peripheral inflammation caused by a subcutaneous injection of formalin generates a dose-dependent deficit. Experiment 3 indicated that the formalin-induced deficit was observed 24 h after delivery of the stimulus. Experiments 4-8 revealed that the NMDA and group I mGluRs are involved in the deficit. The NMDA receptor was found to be necessary (Experiment 4), but only sufficient to induce a deficit at neurotoxic doses (Experiment 5). Both of the group I mGluRs (subtypes, mGluR1 and mGluR5) were found to be necessary (Experiments 6 & 7). A general group I mGluR agonist summated with a subthreshold intensity of shock to produce a robust deficit (Experiment 8), suggesting shock and mGluR activation produce a deficit through a common mechanism.Item Psychological Well-Being and Spinal Cord Injury Recovery: A Two-Way Street?(2014-08-26) Maldonado, SiouiSpinal cord injury (SCI) leads to increased anxiety and depression in as many as 60% of patients. Yet despite extensive clinical research focused on understanding the variables influencing psychological well-being following SCI, risk factors that decrease psychological well-being remain unclear. We hypothesized that excitation of the immune system, inherent to SCI, may contribute to the decrease in well-being. We used a battery of established behavioral tests to assess depression and anxiety in contused rats and (1) characterized psychological well-being as a function of SCI severity, (2) examined peripheral (serum) and central (hippocampi and spinal cord) inflammation in relation to psychological well-being post SCI, and (3) explored whether social enrichment, as a modulator of psychological well-being, could improve overall recovery post SCI, by housing contused animals either alone, or with an injured or an intact cagemate. Following SCI, the contused subjects showed one of three profiles: depression-like, depression- and anxiety-like, or no signs of decreased psychological well-being. Subjects exhibiting a purely depression-like profile showed higher levels of pro-inflammatory cytokines peripherally, whereas subjects exhibiting a depression- and anxiety-like profile showed higher levels of pro-inflammatory cytokines centrally (hippocampi and spinal cord). These changes in inflammation were not associated with injury severity; suggesting that the association between inflammation and the expression of behaviors characteristic of decreased psychological well-being was not confounded by differential impairments in motor ability. Social enrichment, in the form of group housing, did not improve psychological well-being post SCI. Depression- and anxiety-like signs were found in all group housing conditions. Unexpectedly, we found that the intact animals housed with contused subjects showed depression- and anxiety-like signs similar to those of contused subjects, indicating that their psychological well-being was affected by the presence of an injured cagemate. This is reminiscent of the caregiver effect in humans, specifically the manifestation of symptoms of depression in individuals who care for patients suffering with a chronic illness, such as SCI. These experiments demonstrate that the depression and anxiety patients experience following spinal cord injury is not due solely to psychosocial factors, but may also, in part, result from increased immune activation following the injury.Item Reflectance and Fluorescence Confocal Microscope for Imaging of the Mouse Colon(2012-02-14) Saldua, Meagan AlyssaMany Americans are afflicted with inflammation of the colon. They are also at a higher risk of developing colon cancer. Confocal microscopy of bulk epithelial tissue has the potential to provide information on tissue structural properties that may be lost in the fixation and slicing procedures required for histopathology. Optical sectioning provides images in three dimensions capturing the organizational structure of cells and colon crypts throughout the entire colon. I have constructed a custom built fluorescence and reflectance confocal microscope for imaging molecular and morphological changes associated with development of inflammation in a mouse model. A confocal microscope is a point scanning system that removes out of focus light by placing a pinhole aperture in the conjugate image plane located in front of the detector. We have two sources, 488 nm and 811 nm, for fluorescence and reflectance imaging, respectively. A polygon scanning mirror and a galvanometer scanning mirror allow for a variable scan rate between 8 and 15 fps. The lateral resolution of the system is approximately 3 ?m with an axial resolution of 6 ?m and 4 ?m for reflectance and fluorescence mode, respectively. As colon tissue becomes inflamed, there is a distinct change in the structure and architecture of the tissue. The colon crypts are no longer uniform in size or distribution throughout the tissue. Having a large field of view of 1mm2 allows for many colon crypts to be visualized within a single frame. Histology was performed on the same tissue imaged for the inflammatory study confirming the constructed confocal microscope?s ability to characterize inflamed tissue and the potential use for guided biopsy. Mosaicing, or image tiling, is an imaging technique that stitches single frames together to produce a much larger field of view. An extended frame with 1 mm x 2 cm field of view is achieved within seconds. This extended frame would allow mosaicing of the entire mouse colon much faster than conventional methods without loss of resolution. The acquired confocal images of colon tissue demonstrate the microscope?s ability to resolve cell nuclei lining the colon crypts within a relatively large field of view.