Jeffery C. Gandy

Evaluation of antioxidant and proinflammatory gene expression in bovine mammary tissue during the periparturient period

The incidence and severity of mastitis can be high during the period of transition from pregnancy to lactation when dairy cattle are susceptible to oxidative stress. Oxidative stress may contribute to the pathogenesis of mastitis by modifying the expression of proinflammatory genes. The overall goal of this study was to determine the relationship between critical antioxidant defense mechanisms and proinflammatory markers in normal bovine mammary tissue during the periparturient period. Mammary tissue samples were obtained from 12 cows at 35, 20, and 7 d before expected calving and during early lactation (EL, 15 to 28 d in milk). Enzyme activities for cytosolic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase were relatively low during the dry period, but increased during EL, whereas activity of thioredoxin reductase 1 did not change significantly as a function of time. In contrast, gene expression for these antioxidant selenoproteins and for heme oxygenase-1 gradually decreased as parturition approached and then increased during EL. The expression of intercellular vascular adhesion molecule-1 and vascular cell adhesion molecule-1 followed a similar trend where mRNA abundance gradually declined as parturition approached with a slight rebound in EL. Gene expression of the pro-oxidant, 15-lipoxygenase 1, which is known to increase during times of oxidative stress, also increased dramatically in mammary tissue from EL cows. Expression of the proinflammatory cytokines, IL-1beta, IL-6, and IL-8 did not change significantly during the periparturient period. Strong positive correlations were found between several antioxidant enzymes (cytosolic glutathione peroxidase, thioredoxin reductase 1, and heme oxygenase-1) and vascular adhesion molecules (intercellular vascular adhesion molecule-1, vascular cell adhesion molecule-1) suggesting a protective response of these antioxidants to an enhanced proinflammatory state. Ability to control oxidative stress through manipulation of key antioxidant enzymes in the future may modify the proinflammatory state of periparturient cows and reduce incidence and severity of some diseases such as mastitis.

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

OBJECTIVEnTo determine effects of infection with bovine leukosis virus (BLV) on lymphocyte proliferation and apoptosis in dairy cattle.nnnANIMALSn27 adult Holstein cows.nnnPROCEDURESnPeripheral 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.nnnRESULTSnPL 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.nnnCONCLUSIONS AND CLINICAL RELEVANCEnCattle 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.

15-F2t-Isoprostane Concentrations and Oxidant Status in Lactating Dairy Cattle with Acute Coliform Mastitis

Background Severe mammary tissue damage during acute coliform mastitis in cattle is partially caused by oxidative stress. Although considered a gold standard biomarker in some human conditions, the utility of 15‐F2t‐Isoprostanes (15‐F2t‐Isop) in detecting oxidative stress in dairy cattle has not been validated. Hypothesis Concentrations of 15‐F2t‐Isop in plasma, urine, and milk correlate with changes in oxidant status during severe coliform mastitis in cattle. Animals Eleven lactating Holstein‐Friesian dairy cows in their 3rd–6th lactation. Methods A case–control study using cows with acute coliform mastitis and matched healthy controls were enrolled into this study. Measures of inflammation, oxidant status, and redox status in plasma and milk samples were quantified using commercial assays. Plasma, urine, and milk 15‐F2t‐Isop were quantified by liquid chromatography/tandem mass spectrometry (LC‐MS/MS) and ELISA assays. Data were analyzed by Wilcoxon rank sum tests (α = 0.05). Results Plasma 15‐F2t‐Isop quantified by LC‐MS/MS was positively correlated with systemic oxidant status (r = 0.83; P = .01). Urine 15‐F2t‐Isop quantified by LC‐MS/MS did not correlate with systemic oxidant status, but was negatively correlated with redox status variables (r = −0.83; P = .01). Milk 15‐F2t‐Isop quantified by LC‐MS/MS was negatively correlated (r = −0.86; P = .007) with local oxidant status. Total 15‐F2t‐Isop in milk quantified by a commercial ELISA (cbELISA) was positively correlated with oxidant status in milk (r = 0.98; P < .001). Conclusions and Clinical Importance Free plasma 15‐F2t‐Isop quantified by LC‐MS/MS and total milk 15‐F2t‐Isop quantified by cbELISA are accurate biomarkers of systemic and mammary gland oxidant status, respectively. Establishing reference intervals for free and total 15‐F2t‐Isops for evaluating oxidative stress in dairy cows should currently be based on the LC‐MS/MS method.

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.

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.