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Abstract

Objective—To identify and characterize a platelet activating factor (PAF) receptor in bovine neutrophils by use of radioligand binding, reverse transcription-polymerase chain reaction (RT-PCR) assay, and western blot analysis.

Animals—4 healthy adult cows.

Procedure—Bovine neutrophil membranes were isolated for association, dissociation, and saturation binding experiments with PAF labeled with hydrogen 3 (3H-PAF). The RT-PCR assay was performed with appropriate human primers, and western blot analysis was developed with a polyclonal antibody obtained from a peptide of bovine PAF receptor.

Results—Analysis of kinetic binding data supported a single class of PAF receptor. Binding of 3H-PAF to membrane preparations was selectively displaced by PAF and a nonhydrolyzable analogue of guanine triphosphate (Gpp[NH]p) and by lyso-PAF (a biologically inactive analogue of PAF) to a lesser extent. Among other PAF receptor antagonists, 14-deoxyandrographolide and WEB 2086 were the most effective in inhibiting 3H-PAF binding sites in neutrophil membranes; 2 lignans, schisandrin-A and γ-schisandrin were also effective, but 2 gingkolides (BN52020 and BN52021) only mildly inhibited 3H-PAF binding. Results of RT-PCR assay and western blot analysis of neutrophil crude membranes confirmed the presence of a PAF receptor.

Conclusions and Clinical Relevance—Results indicated that bovine neutrophils express only 1 type of PAF receptor, and it is likely that this receptor is involved in inflammatory responses. The most effective PAF antagonists were 14-deoxyandrographolide and WEB 2086; these PAF antagonists may be potentially useful in the treatment of inflammatory processes in cattle. ( Am J Vet Res 2004;65:628–636)

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in American Journal of Veterinary Research

SUMMARY

Objective

To investigate whether platelet-activating factor (PAF) primes the porcine pulmonary response to lipopolysaccharide (LPS), and what effect PAF priming has on porcine neutrophil superoxide (SO) release.

Animals

8- to 10-week old pigs.

Procedures

After pigs were anesthetized with sodium pentobarbital and instrumented for standard cardiopulmonary hemodynamic measurements, they were randomly assigned to receive PAF (0.1 ng/kg of body weight/min, 0 to 2 hours) plus saline solution (2 to 6 hours), saline solution (0 to 2 hours) plus LPS (0.25 μg/kg/h, 2 to 6 hours), or PAF plus LPS. Cardiopulmonary variables were measured throughout the study. Neutrophils were isolated from saline- or PAF-treated pigs at 0 (baseline) and 2 hours, and the effect of in vivo PAF exposure on ex vivo phorbol myristate acetate (PMA)-induced SO release was measured. Additionally, neutrophils isolated from immune-naive pigs were primed in vitro for 10 minutes with 10 μM PAF, and PMA-induced SO release was measured.

Results

PAF infusion significantly enhanced the increase in mean pulmonary arterial pressure, pulmonary vascular resistance, and hypoxemia associated with LPS administration. The infusion increased ex vivo neutrophil SO release, and in vitro PAF exposure primed neutrophils for enhanced SO release that was inhibited by pretreatment of cells with indomethacin.

Conclusions

PAF primes the porcine pulmonary system for the response to LPS. It primes porcine neutrophils in vivo and in vitro for PMA-induced SO release, and in vitro priming is mediated by cyclooxygenase products of arachidonic acid metabolism.

Clinical Relevance

PAF may modulate the porcine inflammatory response by acting as a priming agent, making pigs more responsive to the negative effects of bacterial LPS. (Am J Vet Res 1997;58:1386–1391)

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in American Journal of Veterinary Research

Abstract

Objective

To determine the role of platelet activating factor (PAF) in lipopolysaccharide (LPS)-induced thrombocytopenia and neutropenia in dogs.

Animals

42 dogs.

Procedures

Blood samples were obtained from dogs given LPS (40 µg/kg of body weight; n = 16), PAF (1 µg/kg; 6), PAF (5 µg/kg/h for 90 minutes; 4), or physiologic saline (0.9% NaCl) solution (0.1 ml/kg/h for 90 minutes; 3) IV to monitor changes in blood cell counts, using automated counters and blood smears stained with Giemsa. Blood samples were also obtained from dogs given LPS (40 µg/kg) that had (n = 5) or had not (6) been treated beforehand with TCV-309, a potent PAF antagonist. Concentration of PAF in blood was determined by use of 125I-radioimmunoassay in dogs given LPS at 1 mg/kg (n = 3) and 40 µg/kg (9).

Results

Thrombocytopenia and neutropenia were found in all dogs except those given saline solution. The LPS-induced thrombocytopenia was significantly suppressed by prior treatment with TCV-309. The PAF concentrations increased markedly 1 hour after injection of 1 mg/kg of LPS and increased slightly but significantly 10 minutes after injection of 40 µg/kg of LPS.

Conclusion and Clinical Relevance

PAF plays an important role in the development of LPS-induced thrombocytopenia and neutropenia in dogs. Control of PAF production, PAF-induced effects, or both may be important in the treatment of dogs with gram-negative bacterial infections and associated thrombocytopenia and neutropenia. (Am J Vet Res 1999;60; 216-221)

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in American Journal of Veterinary Research

Abstract

Objective—To determine whether the nonsteroidal anti-inflammatory drugs (NSAIDs) carprofen, flunixin meglumine, and phenylbutazone have cyclooxygenase (COX)-independent effects that specifically inhibit activation of the proinflammatory transcription factor nuclear factor kappa B (NfκB).

Study Population—Purified ovine COX-1 and -2 and cultures of RAW 264.7 murine macrophages.

Procedure—The COX-1 and -2 inhibitory effects of the NSAIDs were tested in assays that used purified ovine COX-1 and -2. Prostaglandin production was analyzed by use of a radioimmunoassay. Inhibitory effects of these drugs on lipopolysaccharide (LPS)- induction of inducible nitric oxide synthase (iNOS) and LPS-stimulated translocation of NfκB were determined by use of RAW 264.7 murine macrophages.

Results—Flunixin meglumine and phenylbutazone were selective inhibitors of COX-1. Carprofen and flunixin meglumine, but not phenylbutazone, inhibited LPS-induction of iNOS. Carprofen and, to a lesser degree, flunixin meglumine had inhibitory effects on NFκB activation.

Conclusions and Clinical Relevance—The ability of drugs such as carprofen and flunixin meglumine to inhibit activation of NfκB-dependent genes such as iNOS, in addition to their effects on COX, suggests an additional mechanism for their anti-inflammatory effects and may explain the ability of flunixin meglumine to be an effective inhibitor of the effects of endotoxin in horses with endotoxemia. (Am J Vet Res 2003;64:211–215)

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in American Journal of Veterinary Research

with naturally occurring SCIs and in animals with experimentally induced SCIs, including axon destruction, demyelination, and centrally oriented necrosis and cavitation. 8 Spinal cord lesions in affected dogs contain activated microglia 9 ; have

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in American Journal of Veterinary Research

, platelet activation has preceded the onset of lameness. 5 One measure of platelet activation in experimentally induced acute laminitis is an increase in circulating concentrations of serotonin, which is detectable in the interval between oligofructose

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in American Journal of Veterinary Research

Abstract

Objective—To evaluate platelet surface-associated P-selectin, mean platelet component concentration (MPC), mean platelet component distribution width (MPCDW), mean platelet volume (MPV), and platelet distribution width (PDW) for detection of activated platelets in dogs with septic and nonseptic inflammatory disease.

Animals—20 healthy dogs and 20 dogs with septic and nonseptic inflammatory disease.

Procedures—Platelet surface-associated P-selectin (expressed as the median fluorescence intensity [MFI] of the platelet population), MPC, MPCDW, MPV, and PDW were determined in 20 healthy adult dogs, and reference ranges were calculated. These parameters were also determined in 11 dogs with nonseptic and 9 dogs with septic inflammatory disease and evaluated to determine which parameters were useful for detection of activated platelets.

Results—12 dogs with inflammatory disease had Pselectin greater than the upper limit of the reference range, whereas 16 dogs with inflammatory disease had MPC lower than the lower limit of the reference range. All dogs in which P-selectin was greater than the upper limit of the reference range had MPC lower than the lower limit of the reference range. The correlation coefficient for P-selectin and MPC was 0.62. Differences in the MPCDW, MPV, and PDW in most dogs with inflammatory disease (compared with healthy dogs) were found; however, the correlation coefficients for P-selectin and MPCDW, MPV, and PDW were low.

Conclusions and Clinical Relevance—Platelet surface- associated P-selectin and MPC appeared to be useful to detect activated platelets in most dogs with septic and nonseptic inflammatory disease. (Am J Vet Res 2005;66:325–329)

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in American Journal of Veterinary Research

Abstract

Objective

To test the hypothesis that platelet-activating factor (PAF) induces inositol phosphate turnover through a receptor-linked, pertussis toxin-sensitive guanine nucleotide-binding (G) protein-dependent pathway in porcine alveolar macrophages.

Design

Randomized complete block design was used with 2 or 3 replicates/block.

Animals

Porcine alveolar macrophages were obtained by lavage of excised lungs from Yorkshire-type pigs (mean ± SEM, 21 ± 2 kg).

Procedure

Phospholipase C activation was assessed, using anion exchange chromatography to measure accumulation of inositol phosphates in [3H)myo-inositol-labeled alveolar macrophages. Macrophages were incubated with saline solution, pertussis toxin (4.75 nM), or B-oligomer (4.75 nM) for 2 hours. Cells then were washed and incubated for 5 minutes with PAF (0, 0.1, 1.0, or 10 μM; n = 15). Results were expressed as total inositol phosphates (inositol monophosphate, bisphosphate, trisphosphate, and tetrakisphosphate).

Results

Concentrations of total inositol phosphates were significantly (P < 0.05) increased to 162 ± 7, 172 ± 4, and 194 ± 9% of control in response to 0.1, 1.0, and 10 μM PAF, respectively. Pertussis toxin attenuated the PAF-induced increase in total inositol phosphates by approximately 50% (P < 0.05). The B-oligomer of pertussis toxin failed to modify PAF-induced increases in total inositol phosphates. The specific PAF receptor antagonist WEB 2086 markedly attenuated PAF-induced (10 μM) increase in inositol phosphates.

Conclusions

We conclude that PAF stimulates accumulation of inositol phosphates through a specific receptor and that a pertussis toxin-sensitive G protein is involved in the signal transduction process leading to activation of phospholipase C in porcine alveolar macrophages. (Am J Vet Res 1996;57:574-579)

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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

Objective—To evaluate splenic mast cell tumors (MCT) of cats for activating mutations in the protooncogene c-kit.

Sample Population—10 formalin-fixed, paraffinembedded splenic MCT from cats in the pathology database of the Veterinary Medical Teaching Hospital at the University of California, Davis.

Procedure—Genomic DNA was isolated from tumor specimens, and the polymerase chain reaction (PCR) procedure was performed for exons 11, 12, and 17. The PCR products were analyzed by use of agarose gel electrophoresis and then directly sequenced.

Results—We did not identify mutations in the juxtamembrane domain (encoded by exons 11 and 12) or catalytic domain (encoded by exon 17) of c-kit in any of the splenic MCT specimens.

Conclusions and Clinical Relevance—Although mutations in the proto-oncogene c-kitoccur frequently in naturally developing MCT in dogs and aggressive mastocytosis in humans, the data reported here documented that dysregulation of Kit function through activating mutations is unlikely in splenic MCT of cats. Therapeutic strategies aimed at inhibiting Kit signaling (ie, kinase inhibitors such as imatinib [STI571]) may not be of benefit for the treatment of this disease in cats. (Am J Vet Res 2002;63:1129–1133).

Full access
in American Journal of Veterinary Research