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Abstract

Objectives

To clone equine interleukin 1 receptor antagonist (IL-1 ra) and determine its full-length cDNA sequence.

Procedure

A cDNA library derived from lipopolysaccharide-stimulated equine monocytes was screened by means of plaque hybridization to radiolabeled equine IL-1ra DNA probes generated by means of the polymerase chain reaction. The cDNA nucleotide sequence for equine IL-1ra was determined by use of the dideoxy chain termination technique, analyzed by use of computer software for sequence characteristics, and compared with sequences reported for IL-1ra of other species.

Results

The cDNA for equine IL-1ra was 1,614 base pairs in length with an ORF encoding a peptide of 177 amino acids with a predicted molecular mass of 20.427 kd. Similarity between the amino acid sequence of equine IL-1ra and sequences for human, murine, rat, and lapine IL-1ra was 76%. Similarity between sequence for equine IL-1ra and sequences for equine interleukin-1α and equine interleukin-1ß were 22.6 and 24.6%, respectively.

Conclusion

Comparison of the sequence for equine IL-1ra with sequences for IL-1ra of other species indicated a high degree of conservation.

Clinical Relevance

Results establish a basis for studying the roles of interleukin-1 in healthy and diseased joints in horses. (Am J Vet Res 1998;59:712-716)

Free access
in American Journal of Veterinary Research
Authors and

Abstract

Objective

To study the effects of 1,25(OH)2D3 and calcium (Ca) on splenocyte cytokine secretion during Mycobacterium paratuberculosis infection.

Design

Mice were assigned to the following treatments: 1—noninfected, 2—infected, 3—noninfected/1,25-(OH)2D3, 4—infected/1,25(OH)2D3, and 5—infected/low-Ca diet (0.15%).

Animals

Male beige mice averaging 6 weeks of age and 20 g in body weight.

Procedure

After acclimation to their diets, mice in treatments 2, 4, and 5 were inoculated IV with 108 colony-forming units of M paratuberculosis. At 1, 6, and 12 months after infection, mice in treatment groups 3 and 4 had miniosmotic pumps implanted subcutaneously that delivered 13 ng of 1,25(OH)2D3/day for 14 days. Treatment 5 was included as a control for comparison with treatment 4 because low dietary Ca should increase endogenous 1,25(OH)2D3 values. Splenocytes were isolated from mice at 1, 6, and 12 months and stimulated in vitro with medium alone (nonstimulated), lipopolysaccharide (LPS), concanavalin A, and M paratuberculosis whole-cell sonicate (MpS).

Results

Production of interleukin 6 after stimulation with LPS, concanavalin A, or MpS was higher (P < 0.05) for splenocytes isolated from mice fed the low-Ca diet, compared with control infected mice 1 and 6 months after infection. Interleukin 1 and tumor necrosis factor activities were increased (P < 0.05) in splenocytes cultured with LPS and MpS after isolation from mice of the low-Ca group. Mice infused with 1,25(OH)2D3 had higher (P < 0.05) interleukin 1 secretion after stimulation of splenocytes with LPS and higher (P< 0.05) tumor necrosis factor production after incubation with MpS.

Conclusion

1,25(OH)2D3 and low dietary Ca increase cytokine secretion in mice infected with M paratuberculosis. (Am J Vet Res 1996;57:825–829)

Free access
in American Journal of Veterinary Research

-α in bronchial epithelial and venous endothelial cells. 12 Interleukin-23, a member of the IL-12 family, is a heterodimer consisting of a p19 subunit that is closely related to the IL-12 p35 subunit and a p40 subunit that is common to IL-12

Full access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether cytokines of homologous species might mediate the stimulatory effects of endotoxin on release of luteinizing hormone (LH) from pituitary cells.

Sample Population

Cells from pituitary glands collected from 8- to 14-month-old wethers.

Procedure

Cells from the anterior pituitary gland were cultured in the presence of recombinant ovine or bovine cytokines (interleukin [IL]-1α, IL-1β, and IL-2), tumor necrosis factor-α (TNF), and interferon-γ (IFN-γ). Luteinizing hormone that was released into the medium was measured. Cells were also cultured with modulators of signal transduction pathways to evaluate the second messenger system used by IL-1α and IL-1β.

Results

Similar to effects of endotoxin, IL-1α and IL-1β stimulated release of LH. Interleukin 2, TNF, and IFN-γ did not have a detectable effect on release of LH. Stimulation of LH release by IL-1αand IL-1β required activation of voltage-dependent Ca2+ channels and appeared to involve protein kinase C.

Conclusions

IL-1αand IL-1β may mediate the direct stimulatory effect of endotoxin on release of LH in vitro. Interleukin 2, TNF, and IFN-γ do not have a direct effect on release of LH; therefore, they do not mediate this effect of endotoxin.

Clinical Relevance

Stressors, including infection, are often associated with reduced fertility. Infection resulting in endotoxin release, production of interleukins, or both, can lead to direct stimulation of LH release from the pituitary gland. Inopportune release of LH via cytokines may interfere with normal pulsatile release of LH, thereby suppressing gonadal function. (Am J Vet Res 1998;59:1488–1493)

Free access
in American Journal of Veterinary Research

. 12-14 Interleukin-23, a member of the IL-12 family, is important in the development of innate immunity, mucosal T-cell responses, and memory development of T cells. 14 Interleukin-23 consists of a unique p19 subunit and the p40 subunit that it

Full access
in American Journal of Veterinary Research

IL-1β and IL-6. 10,15 Such cytokines may also have anti-inflammatory properties. 4,16,17 Interleukin-6 is the primary chemical mediator involved in bone inflammation and bone pain. 10 Several drugs may be used for the treatment of pain. General

Full access
in American Journal of Veterinary Research

Abstract

Objectives—To evaluate the effects of equine recombinant interleukin-1α (rEqIL-1α) and recombinant interleukin- 1β (rEqIL-1β) on proteoglycan metabolism and prostaglandin E2 (PGE2) synthesis by equine articular chondrocytes in explant culture.

Sample Population—Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse.

Procedure—Expression constructs containing cDNA sequences encoding EqIL-1α and EqIL-1β were generated, prokaryotically expressed, and the recombinant protein purified. Near full-thickness articular cartilage explants (approx 50 mg) harvested from stifle joints of a 3-year-old and a 5-year-old horse were separately randomized to receive rEqIL- 1α or rEqIL-1β treatments (0 to 500 ng/ml). Proteoglycan release was evaluated by 1,9-dimethylmethylene blue spectrophotometric analysis of explant media glycosaminoglycan (GAG) concentration and release of 35S-sulfate-labeled GAG to explant media. Proteoglycan synthesis was assessed by quantification of 35S-sulfate incorporation into proteoglycan. Explant media PGE2 concentrations were evaluated using a PGE2-specific enzyme-linked immunoassay. Data were collected at 48-hour intervals and normalized by DNA content.

Results—Proteoglycan release was induced by rEqIL- 1α and rEqIL-1β at concentrations ≥ 0.1 ng/ml, with 38 to 76% and 88 to 98% of total GAG released by 4 and 6 days, respectively. Inhibition of proteoglycan synthesis (42 to 64%) was observed at IL-1 concentrations ≥ 0.1 ng/ml at 2 and 4 days. Increased PGE2 concentrations were observed at IL-1 concentrations ≥ 0.1 ng/ml at 2 and 4 days.

Conclusions and Clinical Relevance—The rEqIL-1 induced potent concentration-dependent derangement of equine chondrocyte metabolism in vitro . These findings suggest this model may be suitable for the in vitro study of the pathogenesis and treatment of joint disease in horses. (Am J Vet Res 2002; 63:551–558)

Full access
in American Journal of Veterinary Research

of intra-articular injections of IL-1 to induce articular inflammation —Interleukin-1 is the prototypical catabolic cytokine, which has a pivotal role in pathogenesis of joint disease. Stimulation of equine cartilage explants with IL-1 results in

Full access
in American Journal of Veterinary Research

Abstract

Objective

To quantitate nitric oxide synthase (NOS) activity in healthy and interleukin 1β (IL-1β)-exposed equine synovial membrane.

Animals

6 healthy horses, 2 to 8 years old.

Procedure

Recombinant human IL-1β (0.35 ng/kg of body weight) was injected intra-articularly into 1 metacarpophalangeal joint of each horse. The contralateral joint served as an unexposed control. All horses were euthanatized 6 hours after injection of IL-1β, and synovial membrane specimens were assayed for NOS activity by measuring conversion of arginine to citrulline. Severity of inflammation was semiquantitated by analysis of synovial fluids and histologic examination of synovial membrane.

Results

Equine synovial membrane had minimal NOS activity. A significant difference was not detected in NOS activity between control and IL-1β-exposed specimens. Histologic examination revealed a neutrophilic infiltrate in synovial membrane exposed to IL-1β. Synovial fluid from IL-1β-exposed joints had a moderate inflammatory response and significantly greater concentrations of IL-1β and interleukin-6 than fluid from healthy joints.

Conclusion

Healthy equine synovial membrane had low NOS activity that was not affected by exposure to IL-1β. (Am J Vet Res 1999;60:714-716)

Free access
in American Journal of Veterinary Research

healing is an extremely complex process that cannot be adequately described by the effects of the molecules measured in the present study. Moreover, proinflammatory cytokines (eg, interleukin-1, interleukin-6, or tumor necrosis factor-α) that might

Full access
in American Journal of Veterinary Research