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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

SUMMARY

Objective

To identify the effect of Pasteurella haemolytica lipopolysaccharide (LPS) and leukotoxin (LKT) on spontaneous and calcium ionophore-induced histamine and inflammatory mediator release from isolated bovine lung parenchyma.

Sample Population

Lungs from 8 healthy cattle.

Procedure

Isolated bovine lung parenchyma was incubated in vitro for 2 hours with LKT or LPS, and spontaneous and induced release of inflammatory mediators was determined.

Results

LKT and LPS increased spontaneous release of histamine and leukotriene B4. In addition, incubation with LPS increased spontaneous release of prostaglandin E2. Moreover, a differential effect of the 2 toxins on calcium ionophore-induced inflammatory mediator release was observed. LKT specifically primed isolated lung parenchyma to release leukotriene B4 and thromboxane B2 in response to calcium ionophore, whereas LPS did not alter the profile of prostanoids released by bovine lung tissue exposed to calcium ionophore.

Conclusions

Pasteurella haemolytica toxins have a direct effect on bovine lung parenchyma, causing release of inflammatory mediators, which contribute to response to infection. Furthermore, bacterial toxins (LKT in this study) may sensitize tissues to the effects of other irritant stimuli, amplifying the inflammatory response. (Am J Vet Res 1997;58:1227–1231)

Free access
in American Journal of Veterinary Research

SUMMARY

Cattle persistently infected with bovine viral diarrhea (bvd) virus have decreased neutrophil and lymphocyte functions. We reevaluated these functions and further characterized the inhibition of persistent bvd virus infection in neutrophils, using sensitive kinetic assays. In addition, the influence of in vitro incubation of neutrophils with recombinant bovine interferon gamma (rBoifn gamma) and in vitro incubation of lymphocytes with recombinant bovine interleukin-2 was evaluated.

Significant (P < 0.05) decrease in random migration under agarose, Staphylococcus aureus ingestion, cytochrome-C reduction, iodination, antibody-independent cell-mediated cytotoxicity, oxidant production, and cytoplasmic calcium flux were observed in neutrophils from cattle persistently infected with bvd virus, compared with noninfected control cattle. Incubation of neutrophils from noninfected controls with rBoifn gamma significantly (P < 0.05) decreased random migration under agarose, cytochrome-C reduction, and cytoplasmic calcium flux. Neutrophils from cattle persistently infected with bvd virus also had decreased random migration under agarose after incubation with rBoifn gamma; in addition, antibody-independent cell-mediated cytotoxicity, elastase release, and cytoplasmic calcium flux were significantly enhanced. The rBoifn gamma induced significantly (P < 0.05) different effects on chemotaxis, cytochrome-C reduction, iodination, and cytoplasmic calcium flux of neutrophils from infected and control cattle. The rBoifn gamma was more effective at improving the function of neutrophils from cattle persistently infected with bvd virus, compared with neutrophils from controls.

Lymphocytes from infected cattle had decreased histogenesis in response to phytohemagglutinin, concanavalin A, and pokeweed mitogen. Incubation of those lymphocytes with recombinant bovine interleukin-2, with no mitogen present, significantly (P < 0.05) increased incorporation of [3H]thymidine. However, the response of lymphocytes to mitogen stimulation was not significantly increased by the presence of recombinant bovine interleukin-2, indicating that depression of in vitro lymphocyte histogenesis in the cattle persistently infected with bvd virus is not attributable to decreased production of interleukin-2.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine functional responses of neonatal chicken and turkey heterophils to various inflammatory agonists.

Animals

100 one-day-old chickens and turkeys.

Procedure

Blood heterophils were isolated and stimulated for 30 minutes at 39 C with ionomycin, phorbol myristate acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucyl-phenylalanine (FMLP). Functional responses (shape change, adherence, phagocytosis, influx of intracellular calcium, and oxidative burst) of stimulated heterophils were measured and compared with responses of unstimulated (control) heterophils.

Results

Turkey and chicken heterophils did not respond to FMLP stimulation. Stimulation of chicken and turkey heterophils with ionomycin resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, intracellular calcium concentration, and oxidative burst. Turkey heterophils did not respond to PMA stimulation, whereas stimulation of chicken heterophils with PMA resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, and oxidative burst but not intracellular calcium concentration. Stimulation of chicken and turkey heterophils with OZ resulted in significant increases in oxidative burst.

Conclusions

Mechanisms regulating initiation of heterophil activation in neonatal chicken and turkey heterophils are consistent with those described for heterophils isolated from mature birds. The biochemical and cytoskeletal systems of neonatal avian heterophils undergo functional alterations following stimulation with inflammatory agonists.

Clinical Relevance

Understanding heterophil activation and regulation should eventually lead to methods for controlling bacterial diseases in poultry. (Am J Vet Res 1998;59:1404–1408)

Free access
in American Journal of Veterinary Research

Summary

The immunomodulating polypeptide interleukin 1β (il-1β) has been shown to be homologous to osteoclast-activating factor and is capable of stimulating increased osteoclastic bone resorption. This effect prompted an investigation into the potential use of il-1β for prevention of parturient paresis, a disease of dairy cows characterized by hypocalcemia and poor osteoclastic resorption of bone. Six nonpregnant cows were treated with a high dosage of il-1β (166 ng/kg of body weight) every 8 hours for 4 days. The il-1β treatment significantly (P < 0.05) increased urinary hydroxyproline excretion, an index of osteoclast activity, indicating that bone calcium resorption might be stimulated by il-1β treatment of cows. However, il-1β treatment also caused transient fever, inappetence, increased pulse and respiratory rate, and diuresis. The acute, but transient, effect of il-1β treatment was to cause a decrease in plasma calcium and phosphorus concentrations. The pleiotropic effects of il-1β administration negated the positive effects on osteoclastic bone resorption, and indicates that this cytokine may be of minimal benefit for prevention of parturient paresis.

Free access
in American Journal of Veterinary Research

SUMMARY

Objective

To characterize 2 bovine neutrophil monoclonal antibodies (MAB) as to effects on bovine neutrophil function and their binding antigens on the cell surface of bovine neutrophils.

Animals

16 healthy, lactating Holstein cattle, 1 calf with leukocyte adhesion deficiency, and 1 age-matched control calf, 2 healthy ewes, and 2 healthy human beings as neutrophil sources.

Procedure

Neutrophil chemotactic and respiratory burst activities and calcium influx, and binding properties of the 2 MAB were determined. Molecular mass of corresponding cell surface antigens also was determined, as was binding of human L-selectin MAB DREG56 to molecules recognized by MAB 11G10 and 2G8 on the surface of bovine neutrophils.

Results

MAB 11G10 and 2G8 inhibited chemotactic activity of bovine neutrophils, up-regulated amplitude of native chemiluminescence, and shortened the time to reach maximal chemiluminescence induced by serum-opsonized zymosan. Crosslinking both MAB with a second antibody induced rapid increase in intracellular free calcium concentration. Binding density of MAB 11G10 and 2G8 to bovine neutrophils treated with trypsin was increased (P < 0.05), compared with that of untreated neutrophils. Neutrophils treated with phosphatidylinositol-specific phospholipase C had decreased (P < 0.05) binding density of MAB 11G10 and 2G8. Binding of the various MAB to neutrophils from calves with bovine leukocyte adhesion deficiency was lower (P < 0.05) than binding to neutrophils from healthy calves. Expression of antigens recognized by the aforementioned MAB on the surface of bovine neutrophils was decreased (P < 0.05) within 10 minutes.

Conclusion

MAB 11G10 and 2G8 recognized L-selectin molecules on bovine neutrophil membrane. L-Selectin (CD62L) is involved in low-affinity adhesion reactions between leukocytes and L-selectin ligand on postcapillary venular endothelial cells. (Am J Vet Res 1997;58:1392–1401)

Free access
in American Journal of Veterinary Research

Summary

Objectives of this investigation were to extract and isolate protein fractions inhibitory to the cytotoxic properties of tumor necrosis factor-α (tnf-α). In this context, mixed populations of wbc were harvested from equine blood and were stimulated with a combination of a synthetic chemotactic peptide and a calcium ionophore. Several methods were subsequently applied for the initial preparation of cell-free crude protein extracts, including fractional precipitation with gradient concentrations of ammonium sulfate and preparative-scale isoelectric focusing. In addition, protein fractions were harvested from extracts of concentrated equine urine. Protein extracts of urinary origin were further separated by gel-filtration column chromatography.

Identification of protein fractions possessing properties inhibitory to the cytotoxic characteristics of tnf-α was facilitated by a tissue culture-based technique for the biological assay of tnf-α-mediated cytotoxicity. Purified protein extracts possessed a marked ability to inhibit or neutralize the cytotoxic properties of tnf-α, on the basis of survival of murine fibrosarcoma cell populations, compared with appropriate negative and positive reference controls. Relative purity of inhibitors and estimation of approximate molecular weight were established by conventional reducing and nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. Equine inhibitory protein fractions from mixed wbc populations, purified in the manner described, had molecular weights of 70,000 to 80,000 and 28,000. An analogous protein fraction of 28 kDa also was isolated from equine concentrated urine. Estimated isoelectric point of tnf-α inhibitor protein fractions was between pH of 5.5 and 6.1. These physical characteristics of equine tnf-α inhibitor protein fractions were similar to those described for a membrane-associated tnf-α receptor protein shed from chemotaxin- and calcium-ionophor-stimulated human wbc populations.

Free access
in American Journal of Veterinary Research

Abstract

Objective

To study binding of purified complement component C3b to bovine blood and mammary neutrophils (PMN) after various treatments and determine their ability to modulate receptor numbers.

Design

Cell isolation, activation, and flow cytometric studies.

Animals

Healthy lactating Holstein cattle.

Procedure

Complement component C3b (18,300 kd) was isolated from bovine serum by column chromatography, and flow cytometric assays using fluorescein isothiocyanate-labeled C3b were developed to evaluate binding to PMN complement receptor 1. Multiple substances were tested to determine their overall effect on C3b binding to PMN. Blood and milk PMN were isolated by differential centrifugation and exposed to optimal concentrations of recombinant human C5a, formyl-methyl leucyl phenylalanine, recombinant bovine interferon-γ, variable concentrations of phorbol myristate acetate (0.01 to 100 ng), calcium ionophore A23187, serum-opsonized zymosan, zymosan-activated serum (ZAS), zymosan-activated plasma (ZAP), and hydrocortisone acetate (25 and 70 ng). Additionally, mammary and blood PMN were preincubated in skim milk and whey.

Results

Variable concentrations of phorbol myristate acetate caused a dose-dependent increase in percentage of PMN binding C3b, and increased the amount of C3b bound per cell. Significant increases were observed after PMN treatment with calcium ionophore, serum opsonized zymosan, ZAS, and ZAP; conversely, incubation of PMN with hydrocortisone acetate resulted in reduced overall binding of C3b. Mammary PMN consistently bound more C3b, which was attributed to their activation during migration into the mammary gland. Binding of C3b was inhibited by skim milk. Activation of blood PMN with PMA, ZAS, and ZAP elicited larger responses than those observed for mammary PMN.

Conclusions

Modulation of complement receptors on bovine PMN is possible. Additionally, significant difference between the level of binding of C3b to blood and milk PMN, with milk PMN having higher binding, may be attributable to migration of PMN into the mammary gland, causing increased receptor expression.

Clinical Relevance

Contribution to a greater understanding of the role of complement in bovine immunologic systems, leading to testing for in vivo enhancement of bovine immune responses to invading pathogens.

Free access
in American Journal of Veterinary Research

SUMMARY

Neutrophils from newborn calves have been shown to be deficient in ability to generate superoxide anion (O2 -) after stimulation of the respiratory burst enzyme with the phorbol ester, phorbol 12-myristate 13-acetate (pma). This compound activates the O2 --generating enzyme of bovine neutrophils through a pathway involving protein kinase C (pkc). To investigate the biochemical basis underlying this functional difference between neutrophils from newborn and adult cattle, we measured and compared the activity of the enzyme pkc in nonstimulated and pma-stimulated bovine neutrophils. Neutrophils from newborn calves (n = 5) and adult cows (n = 5) were stimulated with various concentrations of pma (0, 10, 100, and 500 ng/ml) for 3 minutes, and pkc activity was assayed in the cytosolic and the membrane fractions. In nonstimulated cells, most pkc activity was detected in the cytosolic fraction of neutrophils from newborn and adult cattle. Activity ofpkc in the cytosol was dependent on the presence of added calcium and phospholipids, whereas membrane-associated pkc in nonstimulated cells did not have such dependence. Significant differences in pkc activity were not observed between newborn and adult cattle in either the cytosolic or the membrane fractions from nonstimulated cells. Stimulation with pma caused redistribution of pkc activity in the cell (translocation) in newborns and adults, consisting of decrease in cytosolic pkc activity and increase in membrane-associated pkc activity. Similar to that in nonstimulated cells, pkc activity in cytosolic fractions from pma-stimulated neutrophils was dependent on the presence of cofactors (calcium and phospholipids), whereas pkc activity in the membrane did not have such requirement. Translocation of pkc from the cytosol to the membrane was expressed as percentage of decrease of activity in the cytosol and increase of activity in the membrane, compared with that in nonstimulated cells. Overall, significant differences were not evident in the ability of neutrophils from newborn and adult cattle to translocate pkc after stimulation with various concentrations of pma. Results indicate that differences in redistribution of pkc activity are not responsible for the difference observed in O2 - production between neutrophils from newborn calves and adult cattle after cells are stimulated with pkc agonists.

Free access
in American Journal of Veterinary Research

SUMMARY

Stimulation of bovine alveolar macrophages with calcium ionophore A23187 resulted in marked production of leukotriene (lt)B4 and a lesser increase in thromboxane (tx)B2, whereas opsonized zymosan (opz) resulted in production of txb 2 and relatively small increases in ltb 4 and prostaglandin (pg)F. Alveolar macrophages incubated with recombinant bovine interferon-γ or lipopolysaccharide, and subsequently stimulated with A23187 or opz, had altered arachidonic acid metabolism, producing markedly increased amounts of txb 2 and pgf , and slightly increased ltb 4. Incubation of alveolar macrophages with lipopolysaccharide had a more profound effect on the increased amounts of txb 2 and pgf , observed in response to stimulation with A23187 or opz, than did incubation with interferon-γ. Alveolar macrophages incubated with recombinant bovine interferon-α1-1 also produced slightly increased amounts of ltb 4 when stimulated with A23187 or opz. Altered arachidonic acid metabolism by alveolar macrophages exposed to interferons and lipopolysaccharide may contribute to the development of pulmonary inflammation, such as in the early stages of bacterial pneumonia following viral infections that induce interferon production.

Free access
in American Journal of Veterinary Research