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Summary

Because hepatocyte-stimulating factor/interleukin 6 (il-6) is the principal inducer of acute-phase protein synthesis in the liver, quantification of its activity in blood provides an early and sensitive assessment of the acute-phase response. Circulating il-6 activity was monitored in 4 adult horses for 72 hours after iv administration of endotoxin. In 4 experiments performed at weekly intervals and in randomized order, each horse was given endotoxin—1,000, 30, 1, and 0 ng/kg of body weight. Plasma il-6 activity was quantified as the ability to promote growth of the il-6-dependent B-cell hybridoma, B13.29 clone B9. Interleukin-6 activity (171 ± 10.2 U/ml) was found in all pretreatment plasma samples and was significantly (P < 0.05) increased above baseline from 2 to 12 hours after 1,000 ng of endotoxin/kg was given and at 3 hours after 30 ng of endotoxin/kg was given. After 1,000- or 30-ng/kg dosage of endotoxin, peak plasma il-6 activity (10,128 ± 4,096 and 1,555 ± 1,326 U/ml, respectively) was observed for 3 hours. The il-6 response of endotoxin-treated horses began about 1 hour after tumor necrosis factor appeared in the circulation, and its course closely approximated the endotoxin-induced febrile reaction. Significant increase in plasma il-6 activity was not detected in horses given 1 ng of endotoxin/kg or control buffer.

Free access
in American Journal of Veterinary Research

SUMMARY

A monoclonal antibody (mab) against equine tumor necrosis factor-α (Eq tnf) was used to investigate the role of tnf in cytokine, eicosanoid, and metabolic responses of Miniature Horses given endotoxin. Plasma concentrations of interleukin 6 (il-6), lactate, thromboxane A2 metabolite, and prostacyclin metabolite (6-keto-pgf ) were measured in 10 Miniature Horses given 0.25 µg of lipopolysaccharide (lps; Escherichia coli O55:B5)/kg of body weight. Five horses were given Eq tnf mab and 5 were given isotype-matched mab as control. All horses were given 1.86 mg of antibody/kg by iv infusion, 5 minutes before lps was given iv. Blood samples were taken 20 minutes before and at multiple intervals for 24 hours after lps was given. Interleukin 6 bioactivity in plasma was measured, using il-6-dependent cell line (B9). Eicosanoid activities were assessed by enzyme immunoassay, and plasma lactate concentration was determined enzymatically. Data were analyzed by anova and Tuke's honest significant difference test for significant (P < 0.05) effect of treatment. Horses given Eq tn mab had significantly (P < 0.050) lower peak mean ± sem il-6 (59 ± 29 U/ml), lactate (16 ± 2.00 mg/dl), and 6-keto-pgf (254 ± 79 pg/ml) values then did horses given control mab (880 ± 375 U/ml for il-6; 26 ± 0.04 mg/dl for lactate; and 985 ± 290 pg/ml for 6-keto-pgf ). There was no effect of anti-tnf treatment on lps-induced thromboxane A2 metabolite production. Tumor necrosis factor mediated il-6, lactate, and prostacyclin responses, without affecting thromboxane production in horses given lps.

Free access
in American Journal of Veterinary Research

Summary

Cytologic and bacteriologic responses, and changes in cytokine activity were evaluated in secretions of Staphylococcus aureus-infected mammary glands after treatment of heifers with recombinant bovine interferon gamma (rbIFNγ) or interleukin 2 (rbIL-2). Two groups of 4 heifers each, experimentally infected with 107 colony-forming units (cfu) of S aureus, were injected in 2 quarters via the teat canal, with 105 U of rbIFNγ (trial 1) or 7.5 × 105 U of rbIL-2 (trial 2) 2 weeks after experimentally induced infection; control quarters received phosphate-buffered saline solution. Mammary secretion samples were taken on days 0, 1, 2, 3, 4, 7, and 14 after cytokine infusion. Secretions were diluted 1:10 and used to perform somatic cell counts (scc), differential cell counts, and cfu enumerations, and to determine the number of leukocytes expressing major histocompatibility complex class-II (mhc II) antigens. In addition, mammary secretion samples taken on days 0, 1, and 2 were processed to obtain skimmed milk for evaluation of rbIFNγ- and rbIL-2-like activities. Treatment with rbIFNγ did not influence scc, or differential or bacteria counts, or the number of leukocytes expressing mhc II antigens. However, rbIL-2 stimulated leukocytosis, which may have reduced bacteria counts early in the trial; treatment with this cytokine also increased the neutrophil, macrophage, lymphocyte, and eosinophil counts in secretions. Similarly, numbers of mhc II-positive leukocytes were greater in rbIL-2-treated quarters vs controls. Compared with day 0, IFNγ-like activity was increased on only day 1 in both trials. Interleukin-2-like activity was not influenced in the rbIFNγ trial, but was increased on days 1 and 2 in the rbIL-2 trials. Results indicated that neither cytokine may have had a major influence on the course of established S aureus infections. However, the increased scc in rbIL-2-treated quarters may have accounted for the reduction in cfu throughout the trial after treatment with this cytokine. Greatest cytokine-like activity was observed on day 1; however, the consequences of cytokine activity, such as the sustained eosinophilia after rbIL-2 treatment, were detected over the 14-day trial period, indicating possible prolonged action.

Free access
in American Journal of Veterinary Research

Interleukin (IL)-8 concentration in cats with neoplasia, comparing those with metastatic disease to those without metastases. The concentration of Interleukin-8 was found to be higher in cats with radiologically suspected metastatic tumors compared with cats

Open access
in American Journal of Veterinary Research

Abstract

Objectives—To evaluate the role of interleukin (IL)-10 in the inability of monocyte-derived bovine macrophages to kill Mycobacterium avium subsp paratuberculosis organisms in vitro.

Sample Population—Monocytes were obtained from healthy adult Holstein dairy cows that had negative results when tested for infection with M avium subsp paratuberculosis.

Procedure—Monocyte-derived macrophages were incubated with M avium subsp paratuberculosisfor 2, 6, 24, 72, or 96 hours with or without addition of saturating concentrations of a goat anti-human IL-10 that has been documented to neutralize bovine IL-10 activity. Variables assessed included ingestion and killing of M avium subsp paratuberculosis; expression of tumor necrosis factor (TNF)-α, IL-12, IL-8, major histocompatability (MHC) class II, vacuolar H+ ATPase, and B cell CLL/lymphoma 2 (BCL-2); production of nitric oxide; acidification of phagosomes; and apoptosis of macrophages.

Results—Neutralization of IL-10 enabled macrophages to kill 57% of M avium subsp paratuberculosis organisms within 96 hours. It also resulted in an increase in expression of TNF-α, IL-12, IL-8, MHC class II, and vacuolar H+ ATPase; decrease in expression of BCL-2; increase in acidification of phagosomes; apoptosis of macrophages; and production of nitric oxide.

Conclusions and Clinical Relevance—The capacity of M avium subsp paratuberculosis to induce IL-10 expression may be a major determinant of virulence for this organism. (Am J Vet Res 2005;66:721–726)

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether matrix metalloprotease 13 (MMP-13; collagenase 3) is produced by equine chondrocytes and to investigate modulation of its expression by recombinant human interleukin β (rhIL-1β) and corticosteroids.

Procedure

Equine chondrocytes in monolayer culture were stimulated with rhIL-1β. Total RNA was extracted, purified, and reverse transcribed into DNA. Using appropriate primers, a putative MMP-13 fragment was amplified by polymerase chain reaction, and cloned into a bacterial vector. The resultant fragment was purified and sequenced, then was used to prepare a digoxigenin-labeled cRNA probe. Monolayer cultures of first-passage chondrocytes were treated with rhIL-1β in the presence or absence of dexamethasone (10-6 M) or methylprednisolone acetate (10-9 M to 10-5 M), in addition to positive and negative controls. Cellular RNA was extracted and resolved on agarose gels and subjected to northern blot analysis, using the equine MMP-13 probe.

Results

Reverse transcriptase-polymerase chain reaction enabled isolation of a 0.6-kb fragment of equine MMP-13 cDNA that had 93% homology with the human MMP-13 cDNA sequence. rhIL-1 significantly stimulated MMP-13 expression in the chondrocytes. Methylprednisolone acetate inhibited the stimulatory effects of rhIL-1 in dose-dependent manner that was statistically significant at 10-5 M.

Conclusions

Novel information was gained on the existence of MMP-13 and its expression in equine chondrocytes, which suggests a possible role for this enzyme in matrix degradation in horses with arthritis. (Am J Vet Res 1996;57:1631–1634)

Free access
in American Journal of Veterinary Research

Summary

The effect of exogenous hyaluronate on normal cartilage metabolism and interleukin-1 (il-1)-induced cartilage matrix degradation was investigated in a bovine cartilage explant culture system. Addition of hyaluronate at a concentration of 1.5 mg/ml to cartilage culture explants consistently decreased normal proteoglycan release from the matrix to a value less than that found in control cultures. Addition of 1.5 mg of hyaluronate/ml to il-1 stimulated cartilage culture systems reduced proteoglycan release from the matrix by 83 to 113%. The reduction in control and il-1-stimulated proteoglycan degradation by hyaluronate had a concentration-dependent trend.

Evaluation of alterations in protein (enzyme) release by il-1-stimulated chondrocytes after introduction of hyaluronate was evaluated by use of sodium dodecyl sulfate agar gel electrophoresis of cartilage-conditioned media. The quantity or the molecular weight profile of il-1-induced proteins did not differ after introduction of hyaluronate into the culture system.

Results indicate that introduction of high molecular weight hyaluronate into cartilage culture systems results in a decrease in proteoglycan release from the matrix in control systems, as well as in cultures incubated with il-1. Because il-1-stimulated protein synthesis by chondrocytes remains unchanged after addition of exogenous hyaluronate, the mechanism of inhibition of matrix degradation does not appear to be interference with binding of il-1 to chondrocytes or to be inhibition of the production of neutral metalloproteases, including stromelysin.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To determine whether small intestinal ischemia and reperfusion induces bacterial translocation and proinflammatory cytokine response in either the systemic or portal circulation in dogs.

Animals—17 healthy adult Beagles.

Procedure—The superior mesenteric artery (SMA) was occluded for 0 (group-3 dogs), 30 (group-1 dogs), or 60 (group-2 dogs) minutes, followed by reperfusion for 180 minutes; serum lactate and endotoxin concentrations and tumor necrosis factor-α (TNF-α), interleukin- 1β (IL-1β), and IL-6 activities in the systemic and portal circulation and intramucosal pH were measured at various time points.

Results—In group-2 dogs, TNF-α activity was found to be significantly increased in the portal circulation, peaking at 60 minutes of reperfusion; TNF-α activity, in the systemic circulation, gradually increased from 60 minutes of reperfusion to the end of the experiment; however, the increase was not significant. In group-1 and -2 dogs, IL-6 activities significantly and gradually increased in the systemic and portal circulation during the reperfusion phase, and the magnitude of these increases was dependent on the duration of the ischemic phase. There were no significant changes in IL-1β activity or endotoxin concentration in any dog group.

Conclusions and Clinical Relevance—Results of the our study indicate that intestinal ischemia and reperfusion leads to significant increases of the circulating TNF-α and IL-6 activities, depending on the duration of the ischemia phase, in the absence of detectable endotoxin in the circulation. This finding suggests that intestinal ischemia and reperfusion induces a systemic proinflammatory cytokine response in dogs. (Am J Vet Res 2002;63:1680–1686)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To characterize expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) and regulation of prostaglandin E2 (PGE2) production by equine articular chondrocytes.

Sample Population—Articular cartilage from the metacarpophalangeal joints of 7 adult horses.

Procedure—Equine chondrocyte monolayer cultures were stimulated with different concentrations (2.5, 5, 10, and 20 ng/mL) of recombinant human interleukin- 1β (rhIL-1β) for 24 hours and then with rhIL-1β (5 ng/mL) for 3, 6, 9, 12, and 24 hours. Concentration of PGE2 in the media was measured via radioimmunoassay. Total RNA was extracted from harvested chondrocytes, and regulation of COX-2 and mPGES-1 mRNA was studied via reverse transcriptase-polymerase chain reaction assay and Southern blot analysis with equine-specific probes. Western blot analyses were performed on cellular extracts to characterize expression of COX-2 and mPGES-1 protein.

Results—Stimulation with 5, 10, and 20 ng of rhIL- 1β/mL caused a significant increase in PGE2 concentrations in the culture media, and incubation of cells with rhIL-1β (5 ng/mL) for 6 to 24 hours increased PGE2 production significantly. The increase in prostaglandin production was associated with an induction of COX-2 and mPGES-1 transcripts. There also was an rhIL-1β–dependent induction in COX-2 and mPGES-1 protein expression.

Conclusions and Clinical Relevance—Collectively, results indicated that the rhIL-1β–dependent increase in PGE2 production in equine chondrocytes in monolayer culture was associated with coordinated upregulation of COX-2 and mPGES-1 expression. The pathophysiologic consequences of upregulated COX-2 and mPGES-1 expression and of PGE2 synthesis in rhIL-1β–stimulated equine chondrocytes remain to be elucidated. (Am J Vet Res 2005;66:1985–1991)

Full access
in American Journal of Veterinary Research

Abstract

Objective—To study expression of interleukin-1beta (IL-1β) in the digital laminae of horses in the prodromal stage of experimentally induced laminitis.

Animals—8 healthy adult horses with no signs of laminitis.

Procedure—Black walnut extract was administered via nasogastric tube to 4 horses, and water was administered to the remaining 4 (controls). Complete blood counts and physical examinations were performed every 30 minutes after administration of black walnut extract or water. General anesthesia was induced when total WBC count decreased by 30% in horses given the black walnut extract and 3 hours after water administration in control horses. The left forefoot was perfusion fixed with neutral-buffered 10% formalin, and paraffin-embedded sections of the digit were used for in situ hybridization with an equine-specific IL-1β probe.

Results—IL-1β mRNA expression was observed in perivascular cells of the small laminar venules and capillaries in all 4 horses given black walnut extract and in interstitial cells remote from the microvasculature in 1 of the 4. Other cellular components of the laminar tissue and cellular components of the digital arterioles and veins did not exhibit IL-1β mRNA expression. Expression of IL-1β mRNA was not detected in laminae from control horses.

Conclusions and Clinical Relevance—Results suggest that IL-1β mRNA is expressed by perivascular cells in the laminar tissues of horses in the prodromal stage of experimentally induced laminitis. This provides evidence of an inflammatory process during the prodromal stage of laminitis, indicating that local digital proinflammatory cytokine expression may be an initiating factor in laminitis.(Am J Vet Res 2001;62: 714–720)

Full access
in American Journal of Veterinary Research