Peripheral biomarkers of oxidative stress in dogs with acute pancreatitis.

BACKGROUND: Oxidative stress is considered a pathomechanism of acute pancreatitis (AP), but no studies have extensively characterized oxidant status in dogs with naturally-occurring AP. HYPOTHESIS OR OBJECTIVES: Evaluate measures of oxidant status in dogs with AP and explore whether these measures correlate with AP severity. ANIMALS: Fifteen dogs with AP and 9 control dogs. METHODS: Prospective, controlled observational study. Plasma reactive metabolite (RM) concentrations, antioxidant potential (AOP), and urinary F2 isoprostane concentrations were measured in AP dogs and healthy controls. Severity of AP was assessed by length of hospitalization and 3 disease severity indices: canine acute pancreatitis severity (CAPS), modified canine activity index (M-CAI), and the acute patient physiologic and laboratory evaluation score (APPLEfull ). RESULTS: Reactive metabolite (RM) concentrations (median, 65 relative fluorescent units [RFU]/µL; range, 20-331 RFU/µL) and RM:AOP (median, 7; range, 4-109) were higher in AP dogs than healthy controls (median RM, 25 RFU/µL; range, 16-41 RFU/µL; median RM:AOP, 4; range, 2-7; P < .001 for both comparisons). Reactive metabolite (rS  = 0.603, P = .08) and RM:AOP (rS  = 0.491, P = .06) were not correlated with the duration of hospitalization or disease severity indices evaluated. However, disease severity indices did not predict mortality in our study. Normalized urine 2,3-dinor-8-iso-prostaglandin F2α concentrations were correlated with C-reactive protein (CRP; rS  = 0.491, P = .03), canine specific pancreatic lipase (Spec cPL; rS  = 0.746, P = .002), and CAPS (rS  = 0.603, P = .02). CONCLUSIONS AND CLINICAL IMPORTANCE: Oxidant status is altered in dogs with naturally occurring AP, but the clinical relevance of this finding is unknown.

Postpartum meloxicam administration alters plasma haptoglobin, polyunsaturated fatty acid, and oxylipid concentrations in postpartum ewes.

BACKGROUND: Postpartum inflammation is a natural and necessary response; however, a dysfunctional inflammatory response can be detrimental to animal productivity. The objective of this study was to determine the effects of a non-steroidal anti-inflammatory drug (meloxicam) on ewe postpartum inflammatory response, ewe plasma polyunsaturated fatty acid and oxylipid concentrations, and lamb growth. RESULTS: After lambing, 36 Hampshire and Hampshire × Suffolk ewes were sequentially assigned within type of birth to control (n = 17) or meloxicam orally administered on d 1 and 4 of lactation (MEL; 90 mg, n = 19). Milk and blood samples were collected on d 1 (prior to treatment) and d 4. Milk glucose-6-phosphate was not affected by MEL. Plasma haptoglobin (Hp) concentrations were less for MEL ewes; control ewes with greater d 1 Hp concentrations had elevated Hp on d 4, but this was not the case for MEL-treated ewes. Treatment with MEL increased plasma arachidonic acid concentration by more than 4-fold in ewes rearing singles but decreased concentrations of 9,10-dihydroxyoctadecenoic acid, prostaglandin F2α, 8-iso-prostaglandin E2, and 8,9-dihydroxyeicosatetraenoic acid. Nine oxylipids in plasma had interactions of treatment with d 1 Hp concentration, all of which revealed positive associations between d 1 Hp and d 4 oxylipid concentrations for CON, but neutral or negative relationships for MEL. MEL decreased 13-hydroxyoctadecadienoic acid:13-oxooctadecadienoic acid ratio and tended to increase 9-hydroxyoctadecadienoic acid:9-oxooctadecadienoic acid ratio (both dependent on d 1 values), indicating progressive metabolism of linoleic acid-derived oxylipids occurred by enzymatic oxidation after MEL treatment. Meloxicam reduced oxylipids generated across oxygenation pathways, potentially due to an improved redox state. CONCLUSIONS: Postpartum MEL treatment of ewes decreased plasma concentrations of Hp and several oxylipids, with the greatest impact in ewes with biomarkers reflecting a greater inflammatory state before treatment. Anti-inflammatory strategies may help resolve excessive postpartum inflammation in some dams.

20-hydroxyeicosatetraenoic acid alters endothelial cell barrier integrity independent of oxidative stress and cell death.

Unregulated inflammation during bovine mastitis is characterized by severe mammary tissue damage with systemic involvement. Vascular dysfunction underlies tissue pathology because of concurrent oxidative stress mediated by several inflammatory mediators. We recently demonstrated increased production of 20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-derived (CYP) oxylipid that correlated with oxidative stress during severe bovine coliform mastitis. The hypothesis for this study was that 20-HETE-induced oxidative stress disrupts barrier function of endothelial cells. Primary endothelial cells from the bovine aorta were utilized to investigate the effects of 20-HETE on barrier integrity in an in-vitro model of oxidative stress. The effects of various antioxidants on modulating the 20-HETE barrier integrity effects also were investigated. Our results showed that 20-HETE decreased endothelial barrier integrity, which was associated with increased reactive metabolite production and decreased total glutathione. The antioxidant, vitamin E, partially delayed the loss of endothelial resistance upon exposure to 20-HETE but did not prevent complete loss of barrier integrity. The decrease in barrier resistance due to 20-HETE was neither associated with oxidative stress as assessed by oxidative protein or lipid damage nor endothelial cell apoptosis; however, selenium supplementation conferred resistance to loss of barrier integrity suggesting a role for shifts in redox status. Specific mechanisms by which 20-HETE alters vascular barrier integrity require further investigation to identify targets for therapy during inflammatory conditions with enhanced CYP450 activity.

Production of 15-F2t-isoprostane as an assessment of oxidative stress in dairy cows at different stages of lactation.

Oxidative stress contributes to dysfunctional immune responses and predisposes dairy cattle to several metabolic and inflammatory-based diseases. Although the negative effects of oxidative stress on transition cattle are well established, biomarkers that accurately measure oxidative damage to cellular macromolecules are not well defined in veterinary medicine. Measuring 15-F2t-isoprostane, a lipid peroxidation product, is the gold standard biomarker for quantifying oxidative stress in human medicine. The aim of our study was to determine whether changes in 15-F2t-isoprostane concentrations in plasma and milk could accurately reflect changes in oxidant status during different stages of lactation. Using liquid chromatography-tandem mass spectrometry, 15-F2t-isoprostane concentrations were quantified in milk and plasma of 12 multiparous Holstein-Friesian cows that were assigned to 3 different sampling periods, including the periparturient period (1-2 d in milk; n = 4), mid lactation (80-84 d in milk; n = 4), and late lactation (183-215 d in milk; n = 4). Blood samples also were analyzed for indicators of oxidant status, inflammation, and negative energy balance. Our data revealed that 15-F2t-isoprostane concentrations changed at different stages of lactation and coincided with changes in other gauges of oxidant status in both plasma and milk. Interestingly, milk 15-F2t-isoprostane concentrations and other indices of oxidant status did not follow the same trends as plasma values at each stage of lactation. Indeed, during the periparturient period, systemic 15-F2t-isoprostane increased significantly accompanied by an increase in the systemic oxidant status index. Milk 15-F2t-isoprostane was significantly decreased during the periparturient period compared with other lactation stages in conjunction with a milk oxidant status index that trended lower during this period. The results from this study indicate that changes in 15-F2t-isoprostane concentrations in both milk and plasma may be strong indicators of an alteration in redox status both systemically and within the mammary gland.

Differences in the Oxylipid Profiles of Bovine Milk and Plasma at Different Stages of Lactation.

Mastitis is caused by a bacterial infection of the mammary gland, which reduces both milk quality and quantity produced for human consumption. The incidence and severity of bovine mastitis are greatest during the periparturient period that results from dysfunctional inflammatory responses and causes damage to milk synthesizing tissues. Oxylipids are potent fatty acid-derived mediators that control the onset and resolution of the inflammatory response. The purpose of this study was to investigate how oxylipid profiles change in bovine milk at different stages of the lactation cycle. Results showed significantly lower concentrations of both milk polyunsaturated fatty acid content and total oxylipid biosynthesis during early lactation when compared to mid- or late-lactation. The only oxylipid that was higher during early lactation was 20-hydroxyeicosatetraenoic acid (HETE), which is often associated with inflammatory-based diseases. Milk oxylipid profiles during the different stages of lactation differed from plasma profiles. As such, plasma fatty acid and oxylipid concentrations are not a proxy for local changes in the mammary gland during the lactation cycle.

Duration of in vivo endotoxin tolerance in horses.

Endotoxemia models are used to study mechanisms and treatments of early sepsis. Repeated endotoxin exposures induce periods of endotoxin tolerance, characterized by diminished proinflammatory responses to lipopolysaccharide (LPS) and modulated production of proinflammatory cytokines. Repeated measure designs using equine endotoxemia models are rarely performed, despite the advantages associated with reduced variability, because the altered responsiveness would confound study results and because the duration of equine endotoxin tolerance is unknown. We determined the interval of endotoxin tolerance, in vivo, in horses based on physical, clinicopathologic, and proinflammatory gene expression responses to repeated endotoxin exposures. Six horses received 30 ng/kg LPS in saline infused over 30 min. Behavior pain scores, physical examination parameters, and blood for complete blood count and proinflammatory gene expression were obtained at predetermined intervals for 24h. Horses received a total of 3 endotoxin exposures. The first exposure was LPS 1, followed 7 days later by LPS 7 or 14-21 days later by LPS 14-21. Lipopolysaccharide exposures were allocated in a randomized, crossover design. Lipopolysaccharide produced clinical and clinicopathologic signs of endotoxemia and increased expression of tumor necrosis factor alpha (TNFα), interleukin (IL)-6 and IL-8, P<0.001. Horses exhibited evidence of endotoxin tolerance following LPS 7 but not following LPS 14-21. Horses had significantly lower pain scores, heart rates, respiratory rates and duration of fever, after LPS 7 compared to LPS 1 and LPS 14-21, P<0.001, and expression of TNFα was lower in the whole blood of horses after LPS 7, P=0.05. Clinical parameters and TNFα gene expression were similar or slightly increased in horses following LPS 14-21 compared to measurements made in horses following LPS 1, suggesting that endotoxin tolerance had subsided. A minimum of 3 weeks between experiments is warranted if repeated measures designs are used to assess in vivo response to endotoxin in horses.

Polyunsaturated fatty acids influence differential biosynthesis of oxylipids and other lipid mediators during bovine coliform mastitis.

Coliform mastitis is a severe and sometimes fatal disease characterized by an unregulated inflammatory response. The initiation, progression, and resolution of inflammatory responses are regulated, in part, by potent oxylipid metabolites derived from polyunsaturated fatty acids. The purpose of this study was to characterize the biosynthesis and diversity of oxylipid metabolites during acute bovine coliform mastitis. Eleven cows diagnosed with naturally occurring acute systemic coliform mastitis and 13 healthy control cows, matched for lactation number and days in milk, were selected for comparison of oxylipid and free fatty acid concentrations in both milk and plasma. Oxylipids and free fatty acids were quantified using liquid chromatography-tandem mass spectrometry. All polyunsaturated fatty acids quantified in milk were elevated during coliform mastitis with linoleic acid being the most abundant. Oxylipids synthesized through the lipoxygenase and cytochrome P450 pathways accounted for the majority of the oxylipid biosynthesis. This study demonstrated a complex and diverse oxylipid network, most pronounced at the level of the mammary gland. Substrate availability, biosynthetic pathways, and degree of metabolism influence the biosynthesis of oxylipids during bovine coliform mastitis. Further studies are required to identify targets for novel interventions that modulate oxylipid biosynthesis during coliform mastitis to optimize inflammation.

Effect of infection with bovine leukosis virus on lymphocyte proliferation and apoptosis in dairy cattle.

OBJECTIVE: To determine effects of infection with bovine leukosis virus (BLV) on lymphocyte proliferation and apoptosis in dairy cattle. ANIMALS: 27 adult Holstein cows. PROCEDURES: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from lactating Holstein cows seronegative for BLV (n = 9 cows), seropositive for BLV and aleukemic (aleukemic; 9), and seropositive for BLV and persistently lymphocytotic (PL; 9). Isolated PBMCs were assayed for mitogen-induced proliferation and were analyzed by means of flow cytometry. The PBMCs from a subset of each group were assayed for apoptosis, caspase-9 activity, and expression of selected genes related to apoptosis. RESULTS: PL cows had significantly higher total lymphocyte counts and significantly lower proportions of T-lymphocyte populations than did BLV-negative and aleukemic cows. Both groups of BLV-infected cows had significantly higher proportions of B cells and major histocompatibility complex II-expressing cells than did BLV-negative cows. Proliferation with concanavalin A was significantly lower for PL cows, compared with proliferation for BLV-negative cows. Pokeweed mitogen-induced proliferation was significantly higher for aleukemic and PL cows than for BLV-negative cows. Gene expression of apoptosis-inhibitory proteins BCL2 and BCL2L1 was significantly higher for aleukemic cows and expression of BCL2 was significantly higher for PL cows than for BLV-negative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Cattle infected with BLV had marked changes in PBMC populations accompanied by alterations in proliferation and apoptosis mechanisms. Because the relative distribution and function of lymphocyte populations are critical for immune competence, additional studies are needed to investigate the ability of BLV-infected cattle to respond to infectious challenge.

Selenium inhibits 15-hydroperoxyoctadecadienoic acid-induced intracellular adhesion molecule expression in aortic endothelial cells.

Increased intracellular adhesion molecule 1 (ICAM-1) expression and enhanced monocyte recruitment to the endothelium are critical steps in the early development of atherosclerosis. The 15-lipoxygenase 1 (15-LOX1) pathway can generate several proinflammatory eicosanoids that are known to enhance ICAM-1 expression within the vascular endothelium. Oxidative stress can exacerbate endothelial cell inflammatory responses by modifying arachidonic acid metabolism through the 15-LOX1 pathway. Because selenium (Se) influences the oxidant status of cells and can modify the expression of eicosanoids, we investigated the role of this micronutrient in modifying ICAM-1 expression as a consequence of enhanced 15-LOX1 activity. Se supplementation reduced ICAM-1 expression in bovine aortic endothelial cells, an effect that was reversed with 15-LOX1 overexpression or treatment with exogenous 15-hydroperoxyoctadecadienoic acid (15-HPETE). ICAM-1 expression increased proportionately when intracellular15-HPETE levels were allowed to accumulate. However, changes in intracellular 15-HETE levels did not seem to affect ICAM-1 expression regardless of Se status. Our results indicate that Se supplementation can reduce 15-HPETE-induced expression of ICAM-1 by controlling the intracellular accumulation of this fatty acid hydroperoxide in endothelial cells.

Relationship of body condition score and oxidant stress to tumor necrosis factor expression in dairy cattle.

The relationship between body condition score (BCS) with measures of oxidative status and TNF-alpha production was examined in 16 mid-lactation dairy cows. Cows were selected based on either a normal (2.5-2.7) or a high (> or =3.5) BCS using the standard five-point scaling system. The metabolic status of all cows was determined by plasma nonesterified fatty acid levels (NEFA). Plasma samples or white blood cell lysates also were analyzed for indices of oxidant stress and for the expression of TNF-alpha. Cows with a high BCS had significantly lower NEFA levels when compared to normal BCS cows and the over-conditioned animals were not in a state of negative energy balance. No significant changes in lipid hydroperoxide levels, glutathione peroxidase activity, or the ratio of reduced (GSH) to oxidized (GSSG) glutathione was detected with respect to BCS. However, high BCS cows did have a significantly lower overall antioxidant potential and reduced TrxR activities when compared with the normal BCS cows. Changes in the oxidative state of over-conditioned cows were accompanied by a significantly higher expression of TNF-alpha. Results from this study suggest that cows with a high BCS can experience oxidant stress in the absence of altered energy status. Increased TNF-alpha expression may be related to the pro-oxidant state of over-conditioned cows and possibly be a contributing factor to the enhanced susceptibility to disease in high BCS dairy cattle.