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  • Author or Editor: David J. McClenahan x
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Abstract

Objective—To define the cytokine response of a cultured mammary gland epithelial cell line (ie, Mac-T) when incubated with Escherichia coli or its products.

Sample Population—Mac-T cells and E coli from cows with mastitis.

Procedure—Mac-T cells were incubated with E coli or its products. The cytokine response of Mac-T cells to these treatments was quantified by measuring mRNA content of interleukin (IL)-1α, IL-1β, IL-8, and tumor necrosis factor (TNF)-α by use of a quantitative reverse transcriptase-polymerase chain reaction assay. The amount of TNF-α secreted was also measured.

Results—Treatment with E coli or its products resulted in significant increases in IL-1α, IL-8, and TNF-α mRNA content in Mac-T cells. This increase was reversible when culture filtrate was incubated with polymyxin B. The amount of IL-1β mRNA in Mac-T cells increased only slightly over baseline after treatment with E coli or its products, but this increase was not diminished by incubation of E coli filtrate with polymyxin B.

Conclusions and Clinical Relevance—Incubation of Mac-T cells with E coli or its products resulted in increased amounts of IL-1α, IL-8, and TNF-α mRNA. Inhibition of this response by incubation of culture filtrate with polymyxin B suggested that lipopolysaccharide was the main bacterial product that stimulated the cytokine response. The small increase in IL-1β content in Mac-T cells incubated with E coli or its products suggested that this cytokine had a smaller role in the Mac-T cell response to E coli. (Am J Vet Res 2005;66:1590–1597)

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in American Journal of Veterinary Research

Abstract

Objective—To determine whether platelet-activating factor (PAF) is involved in acute lung microvascular injury associated with pneumonic pasteurellosis in calves.

Animals—15 healthy 2- to 4-week-old male Holstein calves.

Procedure—Calves were anesthetized and inoculated intrabronchially with saline (0.9% NaCl) solution (n = 5) or 1 × 109 Pasteurella haemolytica organisms (n = 10). Of the 10 calves inoculated with P haemolytica, 5 also were treated with WEB 2086, a potent inhibitor of PAF, and 5 were treated with vehicle. Blood and bronchoalveolar lavage samples were collected before and 1, 2, 4, and 6 hours after inoculation of P haemolytica. Blood samples were analyzed to evaluate total number and differential counts of leukocytes, dilute whole-blood leukocyte deformability, size of neutrophils, and neutrophil CD11b expression. Bronchoalveolar lavage samples were analyzed for total number and differential counts of nucleated cells, total protein concentration, and hemoglobin concentration. Size and gross and histologic appearance of lung lesions also was determined.

Results—Treatment of calves with WEB 2086 reduced size of lung lesions, attenuated the increase in microvascular permeability, and reduced neutrophil infiltration in the first 4 hours after inoculation. Treatment with WEB 2086 also attenuated a decrease in leukocyte deformability, increase in size of neutrophils, and CD11b expression by circulating neutrophils.

Conclusions and Clinical Relevance—It appears that PAF is a major mediator for altered lung microvascular permeability and activation of circulating neutrophils in the first 4 hours after onset of pneumonic pasteurellosis in calves. (Am J Vet Res 2000;61: 248–254)

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in American Journal of Veterinary Research

Abstract

Objectives

To determine the structural and functional alterations in circulating neutrophils that may lead to sequestration in lung microvasculature and endothelial injury in calves with experimentally induced pneumonic pasteurellosis.

Animals

10 healthy, 2- to 4-week-old male Holstein calves.

Procedures

Holstein calves were anesthetized and inoculated intrabronchially with Dulbecco phosphate buffered saline (0.9% NaCl) solution (DPBSS; 5 control calves) or 1 × 109 Pasteurella haemolytica organisms (5 infected calves). Blood samples were collected before and 1, 2, 4, and 6 hours after inoculation. Total and differential WBC count, dilute whole blood leukocyte deformability, neutrophil size distribution, and neutrophil surface CD11b expression were measured in blood samples.

Results

A progressive decrease in leukocyte deformability and increase in neutrophil size was detected 1, 2, 4, and 6 hours after inoculation of P haemolytica. Neutrophil surface CD11b expression was greater than baseline values at 6 hours after inoculation of P haemolytica. Two populations of neutrophils with an increase in size were detected in P haemolytica-infected calves. Both subpopulations had increased CD11b expression, compared with neutrophils that were typical in size.

Conclusions and Clinical Relevance

Neutrophils circulate in an activated and nondeformable state in calves with experimentally induced pneumonic pasteurellosis. A decrease in neutrophil deformability and neutrophil aggregation may contribute to neutrophil trapping in the lung microvasculature during pneumonic pasteurellosis in calves. (Am J Vet Res 1999;60:1307–1311)

Free access
in American Journal of Veterinary Research

Abstract

Objective—To investigate rheologic properties of bovine neutrophils that may result in adhesion molecule- independent sequestration of neutrophils in inflamed lungs of cattle.

Animals—Healthy 2- to 4-week-old male Holstein calves.

Procedures—Neutrophil deformability, filamentous actin (F-actin) content, and CD11b expression was determined for unstimulated bovine neutrophils and bovine neutrophils incubated with the inflammatory mediators tumor necrosis factor-α (TNF), platelet-activating factor (PAF), interleukin-8 (IL-8), zymosan-activated plasma (ZAP), Pasteurella haemolytica-derived lipopolysaccharide (LPS), and P haemolytica leukotoxin. Neutrophils were separated into 3 subpopulations on the basis of size. The F-actin content and CD11b expression were evaluated by use of flow cytometry. Leukocyte deformability was evaluated by filtration of dilute whole blood.

Results—The subpopulation of the smallest-sized neutrophils (> 90% of neutrophils) contained little F-actin. A subpopulation of slightly larger neutrophils had a profound increase in F-actin content and CD11b expression. The subpopulation of the largest neutrophils had increased F-actin content and CD11b expression, compared with those for both subpopulations of smaller neutrophils. Incubation of neutrophils with PAF and ZAP, but not TNF, IL-8, LPS, or leukotoxin, resulted in decreased neutrophil deformability and increased F-actin content. Incubation with PAF and TNF induced an increase in size of neutrophils.

Conclusions and Clinical Relevance—Size can be used to identify subpopulations of large and rigid neutrophils in blood samples from healthy calves. Platelet-activating factor and activated complement fragments are potent inducers of F-actin formation and neutrophil rigidity. Physical changes in neutrophils may impede their transit through lung microvasculature and result in leukocyte trapping independent of adhesion molecule interactions with endothelial cells. (Am J Vet Res 2000;61:380–386)

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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)

Full access
in American Journal of Veterinary Research

SUMMARY

Objectives

To determine whether platelets are hyperaggregable or form platelet-neutrophil aggregates during the prodromal stages of acute laminitis of ponies.

Animals

Healthy adult ponies: 8 experimental and 6 control.

Procedures

Acute laminitis was induced by oral administration of corn starch and wood flour to 8 ponies, and indices of platelet activation were evaluated. Blood samples were collected before and at 4, 8, 12, 24, 28, and 32 hours after carbohydrate administration, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, and platelet-neutrophil aggregates were determined. When lameness was first detected, ponies were euthanatized and arteriography and histologic examination of hooves were performed.

Results

Carbohydrate overload was associated with hyperaggregability of platelets throughout the prodromal stages of laminitis and increased numbers of platelet-neutrophil aggregates. Reduction of blood supply to affected hooves was variable, and blood clots were found in 6 of 11 laminitis-affected hooves.

Conclusions

Platelets were hyperaggregable throughout the prodromal stages of carbohydrate-induced laminitis and formed platelet-neutrophil aggregates. Platelet-neutrophil aggregates may initiate or contribute to development of acute laminitis.

Clinical Relevance

Anti-platelet therapy may be useful for treatment of acute alimentary laminitis in horses. (Am J Vet Res 1997;58:1376–1380)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether plasma protein concentrations were altered in ponies with alimentary laminitis.

Animals

12 adult ponies.

Procedure

Acute laminitis was induced in 6 ponies by oral administration of carbohydrate (85% corn starch, 15% wood flour); the other 6 ponies were used as controls. A physical examination was performed and blood samples were collected immediately before and 4, 8, 12, 24, and 28 hours after administration of carbohydrate. Plasma protein concentrations were determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Results

19 plasma proteins ranging from a molecular weight of 24,000 to a molecular weight of 350,000 were identified in all 12 ponies. Plasma concentrations of proteins with molecular weights of 350,000 (fibrinogen), 130,000 (ceruloplasmin), 118,000 (c-reactive protein), 67,000 (α1-antitrypsin I), 65,000 (α1-antitrypsin II), 50,000 (haptoglobulin), and 45,000 (acid glycoprotein) were significantly increased in ponies with laminitis, compared with concentrations in control ponies.

Conclusion

Changes in plasma protein concentrations are detectable within 4 hours after the onset of alimentary laminitis in ponies.

Clinical Relevance

Measurement of plasma protein concentrations may be useful in monitoring the progression of laminitis in ponies. (Am J Vet Res 1998;59:1234–1237)

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To determine whether platelets become activated and form platelet-platelet or platelet-neutrophil aggregates, or both, when subjected to shear.

Sample Population

Blood obtained from 3 Thoroughbreds.

Procedures

Blood, with PCV adjusted to 32 (low hematocrit) or 60 (high hematocrit)%, was subjected to shear rates of 11.25, 22.5, 45, 90, 225, and 750/s for 3 minutes by use of a cone-plate viscometer. Flow cytometric techniques were used to identify activated platelets, platelet-platelet aggregates, and platelet-neutrophil aggregates.

Results

Shear resulted in decreased platelet count, increased mean platelet volume, platelet activation, and formation of platelet-platelet and platelet-neutrophil aggregates. These changes occurred at lower shear rates in blood with high hematocrit. Platelet-neutrophil aggregate formation was inhibited by blocking P-selectin, but not CD11/CD18 receptors.

Conclusions

Shear-induced platelet activation and aggregate formation occur at physiologic shear rates.

Clinical Relevance

Shear-induced platelet activation may explain the exercise-associated platelet-neutrophil aggregates observed in Thoroughbreds undergoing treadmill exercise. (Am J Vet Res 1998;59:1243-1246)

Free access
in American Journal of Veterinary Research

SUMMARY

Objectives

To determining whether inhibition of platelet aggregation prevents development of carbohydrate overload-induced alimentary laminitis.

Animals

22 healthy adult ponies.

Procedures

Acute laminitis was induced by oral administration of corn starch/wood flour to 16 ponies, 8 of which were treated with a synthetic analogue of the platelet fibrinogen receptor antagonist peptide (RPR) RGDS (arginine-glycine-aspartic acid-serine) 110885; 6 ponies served as negative controls. Blood was collected before and at 4, 8, 12, 24, 28, and 32 hours after administration of carbohydrate overload, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, in vivo platelet activation, and platelet-neutrophil aggregates were evaluated.

Results

Of 16 ponies given carbohydrate, 6 of 8 untreated ponies developed laminitis and 0 of 8 ponies treated with RPR 110885 developed laminitis. The RPR 110885 treatment attenuated the increase in platelet-neutrophil aggregates observed in untreated ponies.

Conclusions

Platelets are involved in the pathogenesis of equine alimentary laminitis.

Clinical Relevance

Platelet aggregation inhibitors may be useful for prevention or treatment of laminitis, or both. (Am J Vet Res 1998;59:814–817)

Free access
in American Journal of Veterinary Research