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Abstract

Objective—To investigate the effects of insulin-like growth factor-II (IGF-II) on DNA and glycosaminoglycan (GAG) synthesis and the expression of matrix-related genes in equine articular cartilage explants and chondrocytes, respectively, with and without interleukin 1-β (IL1-β).

Sample Population—Articular cartilage from 12 adult horses.

Procedure—Articular cartilage was incubated in standard media with and without equine IL1-β (10 ng/mL) containing various concentrations of IGF-II for 72 hours. Synthesis of DNA and GAG was determined by incorporation of thymidine labeled with radioactive hydrogen (3H) and sulfate labeled with radioactive sulfur (35S), respectively. Total GAG content of the explants and spent media was determined by use of the 1,9-dimethylmethylene blue assay. Northern blots of RNA from cultured equine articular cartilage chondrocytes were hybridized with cDNA of major matrix molecules.

Results—Insulin-like growth factor-II stimulated DNA and GAG synthesis at concentrations of 25 and 50 ng/mL, respectively. In cartilage explants conditioned with IL1-β, IGF-II stimulated DNA and GAG synthesis at concentrations of 500 and 50 ng/mL, respectively. Insulin-like growth factor-II had no effect on total GAG content as determined by the 1,9-dimethylmethylene blue assay. No specific effects on steady-state levels of messenger RNAs were observed.

Conclusions and Clinical Relevance—Insulin-like growth factor-II stimulated DNA and GAG synthesis in equine adult cartilage and may have potential application in vivo. (Am J Vet Res 2004;65:238–244)

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in American Journal of Veterinary Research

DiI-Ac-LDL 1,1′-dioctadecyl-3,3,3′,3′ tetramethylindocarbocyanine perchlorate–labeled acetylated low-density lipoprotein IL Interleukin XTT 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide Footnotes a. EGM-2 with

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in American Journal of Veterinary Research

SUMMARY

In each of 4 horses, sterile synovitis was induced by intra-articular injection of 3 μg of Escherichia coli endotoxin (lipopolysaccharide, LPS) into one antebrachiocarpal joint; an equal volume (2 ml) of phosphate-buffered saline solution (PBSS) was injected into the opposite, control carpus. Blood and 1.5 ml of synovial fluid were obtained at postinjection hours (pih) 0, 2, 4, 8, 12, 18, 42, 66, and 144. Synovial fluid sample collection was accomplished by use of an indwelling, intra-articular catheter through pih 12, and by arthrocentesis subsequently. Joint fluid samples were analyzed for cell counts, protein concentration, cytologic variables, and tumor necrosis factor (tnf), interleukin 6 (IL-6), and prostaglandin E2 (PGE2) values. Tumor necrosis factor and 1L-6 activities and WBC count were also measured in blood. To monitor local inflammation, skin temperature of each carpus was imaged, using a thermographic scanner prior to each sample collection time.

Horses had minimal systemic effects. Mean (± SEM) rectal temperature increased significantly to 39 02 ± 0.15 C only at pih 18 after intra-articular injection of LPS. One horse had signs of mild depression from pih 7 to 18, but its vital signs did not change appreciably. Each horse had mild signs of discomfort in the LPS-injected limb from pih 1 to 3 until pih 8 to 10. Mean peak surface temperature of the LPS-injected carpi was significantly higher than that of control carpi from pih 8 to 144 (P < 0.05).

Mean synovial fluid WBC count in the LPS-injected and control carpi increased after injection and peaked by pih 8 (193,125 ± 8,528 cells/μl, LPS-treated; 16,425 ± 8,336 cells/μl, controls). Mean values for LPS-treated (principal) joints were significantly greater than values for control joints from pih 2 until after pih 66 (P < 0.05). Mean synovial fluid protein concentration increased in the principal and control joints, with values for the principal joints remaining significantly greater than values for control joints from pih 4 to 144 (P < 0.05). Mean TNF activity in synovial fluid was maximal at pih 2 (10,573 ± 5,924 U/ml). Interleukin-6 activity increased more gradually and peaked at pih 8 (1.78 ± 0.71 × 106 U/ml). Tumor necrosis factor activity did not increase above the minimal detectable value of 6 U/ml in the control joints. Mean PGE2 concentration in the principal joints peaked by pih 2 (3.6 ± 0.37 ng/ml) and remained significantly (P < 0.05) greater than the value for control joints from pih 2 through 66. These results indicate that a humane model of synovitis was created by intra-articular injection of LPS and can be used to establish the basic responses of synovial TNF, IL-6, and PGE2 in horses with early inflammatory joint disease.

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in American Journal of Veterinary Research

Abstract

Objective—To determine whether glucosamine and chondroitin sulfate (CS) at concentrations approximating those achieved in plasma by oral administration would influence gene expression of selected mediators of osteoarthritis in cytokine-stimulated equine articular chondrocytes.

Sample Population—Samples of grossly normal articular cartilage obtained from the metacarpophalangeal joint of 13 horses.

Procedure—Equine chondrocytes in pellet culture were stimulated with a subsaturating dose of recombinant equine interleukin (reIL)-1β. Effects of prior incubation with glucosamine (2.5 to 10.0 µg/mL) and CS (5.0 to 50.0 µg/mL) on gene expression of matrix metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase 1 and 2; inducible nitric oxide synthase (iNOS); cyclooxygenase (COX)-2; nuclear factor κB; and c-Jun- N-terminal kinase (JNK) were assessed by use of a quantitative real-time polymerase chain reaction assay.

Results—Glucosamine at a concentration of 10 µg/mL significantly reduced reIL-1β–induced mRNA expression of MMP-13, aggrecanase 1, and JNK. Reductions in cytokine-induced expression were also observed for iNOS and COX-2. Chondroitin sulfate had no effect on gene expression at the concentrations tested.

Conclusions and Clinical Relevance—Concentrations of glucosamine similar to those achieved in plasma after oral administration in horses exerted pretranslational regulation of some mediators of osteoarthritis, an effect that may contribute to the cartilage- sparing properties of this aminomonosaccharide. Analysis of results of this study indicated that the influence of CS on pretranslational regulation of these selected genes is limited or lacking. (Am J Vet Res 2005;66:1861–1869)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of glucosamine (GLN) and chondroitin sulfate (CS), at concentrations attainable in vivo, on expression of genes encoding proteolytic enzymes, enzyme inhibitors, and macromolecules of articular cartilage in interleukin-1(IL- 1)–challenged bovine cartilage explants.

Sample Population—Articular cartilage explants harvested from 9 steers.

Procedures—Cartilage explants were exposed to media containing 10% fetal bovine serum (FBS) only, IL- 1 (50 ng/mL), IL-1 with GLN (5 µg/mL), IL-1 with CS (20 µg/mL), or IL-1 with GLN and CS for 24 and 48 hours. Cartilage was frozen, and RNA was extracted. Gene expression of matrix metalloproteinases (MMPs)-2, -3, -9, -13, and -14; aggrecanases (Aggs)-1 and -2; tissue inhibitors of metalloproteinases (TIMPs)-1, -2, and -3; and type II collagen and aggrecan were assessed with quantitative real-time polymerase chain reaction.

Results—Upregulated MMP-3, MMP-13, and Agg-1 transcripts at 24 hours were repressed by the GLN and CS combination by at least approximately 6-fold. Glucosamine was effective in suppressing IL-1–induced mRNA expression of MMP-13, Agg-1, and Agg-2, whereas CS was effective in decreasing IL-1–induced MMP-13 transcript at 24 hours. At 48 hours, GLN and CS added separately and in combination significantly abrogated Agg-1 and Agg-2 gene induction. The combination also decreased IL-1–stimulated MMP-13 transcript.

Conclusions and Clinical Relevance—GLN and CS, at concentrations that are within the range measured in synovial fluid and blood after oral administration, may regulate expression of matrix degrading enzymes and their inhibitors at the transcriptional level, providing a plausible mechanism for their purported chondroprotective properties. (Am J Vet Res 2005;66:1870–1876)

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in American Journal of Veterinary Research

Abstract

Objective—To determine the effects of interleukin- 1β (IL-1β) and tumor necrosis factor-α (TNF-α) on expression and regulation of several matrix-related genes by equine articular chondrocytes.

Sample Population—Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses.

Procedure—Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1β or TNF-α. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis.

Results—IL-1β and TNF-α increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected.

Conclusions and Clinical Relevance—Treatment of cultured equine chondrocytes with IL-1β or TNF-α resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1β and TNF-α play a role in the degradation of articular cartilage in arthritis. (Am J Vet Res 2000;61: 624–630)

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in American Journal of Veterinary Research

Abstract

Objective—To develop an in vitro model of the bovine alveolar-capillary interface and to evaluate the roles of interleukin-8 (IL-8) and platelet-activating factor (PAF) in neutrophil-mediated endothelial injury induced by infection with Mannheimia haemolytica.

Sample Population—Cultured bovine pulmonary microvascular endothelial cells, freshly isolated bovine neutrophils, and monocyte-derived bovine macrophages.

Procedure—A coculture system was developed in which endothelial cells were grown to confluence in tissue culture inserts, neutrophils were added to the inserts, and macrophages were added to tissue culture wells. Mannheimia haemolytica-derived lipopolysaccharide (LPS) or supernatant was added to activate macrophages, and inhibitors of PAF or IL-8 were added to the insert. Endothelial cell cytotoxicity and permeability (ie, albumin leakage) and neutrophil activation (ie, adhesion, degranulation [lactoferrin expression], and superoxide production) were assessed.

Results—The addition of M haemolytica-derived LPS to bovine macrophages in the coculture system resulted in significant increases in endothelial cell cytotoxicity and permeability and neutrophil degranulation and adhesion. Inhibition of IL-8 reduced endothelial cell permeability and neutrophil degranulation induced by exposure to M haemolytica-derived supernatant, whereas inhibition of PAF decreased superoxide release by neutrophils.

Conclusions and Clinical Relevance—In vitro activation of bovine macrophages by M haemolyticaderived LPS resulted in neutrophil activation and neutrophil- mediated endothelial damage. Neutrophilmediated endothelial injury and neutrophil degranulation were, at least in part, mediated by IL-8, whereas PAF promoted superoxide release by neutrophils in this in vitro system designed to mimic the in vivo events that occur during the early stages of bovine pneumonic pasteurellosis. (Am J Vet Res 2002; 63:394–401)

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in American Journal of Veterinary Research

Abstract

Objective—To examine the effects of DNA dose, site of vaccination, and coadministration of a cytokine DNA adjuvant on efficacy of H1-subtype swine influenza virus hemagglutinin (HA) DNA vaccination of pigs.

Animals—24 eight-week-old mixed-breed pigs.

Procedure—2 doses of DNA were administered 27 days apart by use of a particle-mediated delivery system (gene gun). Different doses of HA DNA and different sites of DNA administration (skin, tongue) were studied, as was coadministration of porcine interleukin-6 (pIL-6) DNA as an adjuvant. Concentrations of virus-specific serum and nasal mucosal antibodies were measured throughout the experiment, and protective immunity was assessed after intranasal challenge with homologous H1N1 swine influenza virus.

Results—Increasing the dose of HA DNA, but not coadministration of pIL-6 DNA, significantly enhanced virus-specific serum antibody responses. Pigs that received DNA on the ventral surface of the tongue stopped shedding virus 1 day sooner than pigs vaccinated in the skin of the ventral portion of the abdomen, but none of the vaccinated pigs developed detectable virus-specific antibodies in nasal secretions prior to challenge, nor were they protected from challenge exposure. Vaccinated pigs developed high virus-specific antibody concentrations after exposure to the challenge virus.

Conclusions and Clinical Relevance—Co-administration of pIL-6 DNA did not significantly enhance immune responses to HA DNA vaccination or protection from challenge exposure. However, HA DNA vaccination of pigs, with or without coadministration of pIL-6 DNA, induced strong priming of the humoral immune system. (Am J Vet Res 2002; 63:653–659)

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in American Journal of Veterinary Research

Abstract

Objective—To determine whether concentrations of proinflammatory cytokines, acute-phase proteins, and cortisol differ at parturition among 3 categories of sows (noninoculated, clinically affected and nonaffected following intramammary inoculation with Escherichia coli).

Animals—16 sows.

Procedure—Sows were allocated to inoculated (n = 12) or noninoculated (4) groups. Inoculated sows received intramammary administration of E coli (serotype O127) during the 24-hour period preceding parturition. Blood samples were collected from noninoculated and inoculated sows for 3 consecutive days within 3 to 11 days before farrowing and inoculation. Samples were also collected 0, 24, 48, 72, and 96 hours after farrowing and inoculation. Inoculated sows were further categorized as affected (4 sows) or nonaffected (8 sows) based on clinical signs of disease. Serum tumor necrosis factor (TNF)-α, plasma interleukin (IL)-6, and serum amyloid A (SAA) concentrations were measured by use of ELISA; serum haptoglobin concentration was assayed by use of a hemoglobin- binding method; and plasma cortisol concentration was determined by use of radioimmunoassay.

Results—Plasma or serum concentrations of TNF-α, IL-6, and SAA of both categories of inoculated sows were significantly increased by 24 hours after intramammary inoculation of E coli, compared with concentrations in noninoculated sows. Concentrations of serum TNF-α and plasma IL-6 were significantly higher in inoculated sows that developed clinical mastitis than in nonaffected inoculated sows.

Conclusions and Clinical Relevance—Concentrations of TNF-α and IL-6 are promising markers for the identification of periparturient sows with subclinical coliform mastitis. Identification of such sows should help improve the health and survival of piglets. (Am J Vet Res 2004;65:1434–1439)

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in American Journal of Veterinary Research

Abstract

Objective—To determine functional characteristics of monocytes obtained from cows with subclinical infection with Mycobacterium avium subsp paratuberculosis (MAP) that may have predisposed those cows to becoming infected with MAP.

Sample Population—Monocytes obtained from 5 uninfected cows and 5 cows subclinically infected with MAP in a herd with a high prevalence of paratuberculosis (ie, Johne's disease).

Procedures—Monocytes from uninfected and subclinically infected cows were incubated with MAP for 2, 6, 24, 72, or 96 hours. Variables measured included expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-12, transforming growth factor-β, and suppressor of cytokine signaling-3 (SOCS-3); apoptosis of monocytes; acidification of phagosomes; and killing of MAP.

Results—Monocytes from infected cows had greater expression of IL-10 and SOCS-3 at 2 hours of coincubation with MAP and lower expression of TNF-α and IL-12 when results for all incubation times were combined. Monocytes from infected cows had a greater capacity to acidify phagosomes. No differences were observed in the rate of apoptosis or capacity of monocytes to kill MAP organisms.

Conclusions and Clinical Relevance—Monocytes obtained from cows with subclinical infection with MAP had upregulated expression of IL-10 and SOCS-3 within the first 2 hours after exposure to MAP organisms. Although this did not inhibit acidification of phagosomes, apoptosis of monocytes, or attenuation of the capacity to kill MAP organisms, it may have attenuated the capacity of mononuclear phagocytes to initiate inflammatory and adaptive immune responses. (Am J Vet Res 2005;66:1114–1120)

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in American Journal of Veterinary Research