Objective—To characterize gelatinases in bronchoalveolar
lavage fluid (BALF) and gelatinases produced
by alveolar macrophages of healthy calves.
Sample Population—Samples of BALF and alveolar
macrophages obtained from 20 healthy 2-month-old
Procedure—BALF was examined by use of gelatin
zymography and immunoblotting to detect gelatinases
and tissue inhibitor of metalloproteinase (TIMP)-1
and -2. Cultured alveolar macrophages were stimulated
with lipopolysaccharide (LPS), and conditioned
medium was subjected to zymography. Alveolar
macrophage RNA was used for reverse transcriptasepolymerase
chain reaction assay of matrix metalloproteinases
(MMPs), cyclooxygenase-2, and inducible
nitric oxide synthase.
Results—Gelatinolytic activity in BALF was evident at
92 kd (14/20 calves; latent MMP-9) and 72 kd (18/20;
latent MMP-2). Gelatinolytic activity was evident at 82
kd (10/20 calves; active MMP-9) and 62 kd (17/20;
active MMP-2). Gelatinases were inhibited by metal
chelators but not serine protease inhibitors.
Immunoblotting of BALF protein and conditioned
medium confirmed the MMP-2 and -9 proteins.
Endogenous inhibitors (ie, TIMPs) were detected in
BALF from all calves (TIMP-1) or BALF from only 4
calves (TIMP-2). Cultured alveolar macrophages
expressed detectable amounts of MMP-9 mRNA but
not MMP-2 mRNA.
Conclusions and Clinical Relevance—Healthy
calves have detectable amounts of the gelatinases
MMP-2 and -9 in BALF. Endogenous inhibitors of
MMPs were detected in BALF (ie, TIMP-1, all calves;
TIMP-2, 4 calves). Lipopolysaccharide-stimulated alveolar
macrophages express MMP-9 but not MMP-2
mRNA. The role of proteases in the pathogenesis of
lung injury associated with pneumonia has yet to be
determined. (Am J Vet Res 2004;65:163–172)
Objective—To determine the pharmacokinetic disposition of IV administered caffeine in healthy Lama spp camelids.
Animals—4 adult male alpacas and 4 adult female llamas.
Procedures—Caffeine (3 mg/kg) was administered as an IV bolus. Plasma caffeine concentrations were determined by use of high-performance liquid chromatography in 6 animals and by use of liquid chromatography-mass spectrometry in 2 llamas.
Results—Median elimination half-life was 11 hours (range, 9.3 to 29.8 hours) in alpacas and 16 hours (range, 5.4 to 17 hours) in llamas. The volume of distribution at steady state was 0.60 L/kg (range, 0.45 to 0.93 L/kg) in alpacas and 0.75 L/kg (range, 0.68 to 1.15 L/kg) in llamas. Total plasma clearance was 44 mL/h/kg (range, 24 to 56 mL/h/kg) in alpacas and 42 mL/h/kg (range, 30 to 109 mL/h/kg) in llamas.
Conclusions and Clinical Relevance—High-performance liquid chromatography and liquid chromatography-mass spectrometry were suitable methods for determination of plasma caffeine concentrations in alpacas and llamas. Plasma caffeine concentration-time curves were best described by a 2-compartment model. Elimination half-lives, plasma clearance, volume of distribution at steady state, and mean residence time were not significantly different between alpacas and llamas. Intravenous administration of caffeine at a dose of 3 mg/kg did not induce clinical signs of excitement.
Objective—To compare distributions of survivin among tissues from urinary bladders of dogs with cystitis, transitional cell carcinoma (TCC), or histologically normal urinary bladders.
Sample Population—24 archived and 7 fresh-frozen specimens of urinary bladders from dogs with cystitis.
Procedures—Immunohistochemical analysis of archived tissue specimens was performed to identify survivin protein in the nucleus and cytoplasm of cells by use of polyclonal rabbit anti-survivin antibody. Tissues that contained ≥ 5% immunoreactive cells were considered positive for survivin protein. Reverse-transcription PCR analysis was performed on fresh-frozen tissues to identify survivin mRNA. Data on tissues from dogs with TCC or histologically normal urinary bladders that were obtained during another study were used for statistical comparisons.
Results—Twelve of 24 (50%) cystitic tissues were positive for nuclear survivin, compared with 28 of 41 (68%) TCC tissues and 0 of 46 (0%) normal tissues. Two of 24 (8%) cystitic tissues were positive for cytoplasmic survivin, compared with 7 of 41 (17%) TCC tissues and 17 of 46 (37%) normal tissues. Proportions of specimens that contained nuclear or cytoplasmic survivin were significantly different between cystitic and normal tissues but not between cystitic and TCC tissues. Four of 7 cystitic tissues were positive for survivin mRNA, which was comparable with results for TCC and normal tissues.
Conclusions and Clinical Relevance—Nuclear survivin was detected in TCC and cystitic tissues but not in normal urinary bladder tissues. Additional studies are needed to determine whether nuclear survivin contributes to the development or progression of TCC.
Objective—To determine the effects of pasteurization
of colostrum on serum lactoferrin concentration and
neutrophil oxidative function by comparing values
from calves given pasteurized (76 C, 15 minutes)
colostrum versus calves given fresh frozen colostrum.
Animals—8 Holstein bull calves were used to study
the effects of pasteurization of colostrum on the
absorption of lactoferrin and neutrophil oxidative burst.
Three additional calves were used to study the effect
of exogenous lactoferrin on neutrophil oxidative burst.
Methods—Calves were fed fresh frozen or heat pasteurized
colostrum (76 C for 15 minutes) via
esophageal feeder within 4 hours of birth. Neutrophils
were isolated from whole blood samples. Neutrophil
oxidative burst was induced by phorbol ester (300
ng/ml) stimulation of cells (1 × 106 cells) at 37 C.
Serum lactoferrin concentrations were compared,
using immunoblot analysis. Serum IgG concentrations
were determined by radial immunoassay.
Comparisons were made between the use of the 2
types of colostrum in calves by measuring subsequent
serum IgG and lactoferrin concentrations and
neutrophil superoxide production.
Results—Serum IgG and lactoferrin concentrations
increased more in calves receiving fresh frozen
colostrum. Neutrophil superoxide production was higher
in neutrophils prepared from calves receiving fresh
frozen colostrum. Colostral lactoferrin addition to neutrophil
incubations resulted in increased oxidative burst.
Conclusions and Clinical Relevance—Compared
with calves given fresh frozen colostrum, calves given
pasteurized colostrum had decreased serum IgG and
lactoferrin concentrations and neutrophil superoxide
production 24 hours after administration. These
results suggest that pasteurizing bovine colostrum at
76 C for 15 minutes has substantial effects on passive
transfer of proteins and neutrophil function. (Am J Vet
Objective—To evaluate the effect of lactoferrin on lipopolysaccharide (LPS)-induced proliferation of bovine peripheral blood mononuclear cells (PBMCs), gene expression of inflammatory mediators, and production of prostanoids in vitro.
Sample Population—PBMCs isolated from 15 Holstein bull calves.
Procedures—Mixed populations of PBMCs were isolated by differential centrifugation. Proliferation assays were conducted in 96-well plates designed to allow addition of lactoferrin (200 ng/mL) with and without LPS (1 μg/mL) in a checkerboard design. Incorporation of 3H-thymidine was used to determine proliferation of PBMCs. Prostaglandin E2 production was determined in culture-conditioned medium by use of enzyme immunoassay. Effects of lactoferrin on LPS-induced gene expression of cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-9 were monitored by use of PCR assays.
Results—Lactoferrin supplementation significantly reduced LPS-induced incorporation of 3H-thymidine and production of prostaglandin E2 by PBMCs. Lactoferrin reduced LPS-induced expression of COX-2 and MMP-9 mRNA.
Conclusions and Clinical Relevance—Lactoferrin reduced LPS-induced cellular proliferation, inflammatory mediator gene expression, and prostaglandin E2 production by bovine PBMCs in vitro. These effects may be beneficial in reducing the impact of endotoxemia in neonates.