Angela Szeto

Evaluation of enzyme immunoassay and radioimmunoassay methods for the measurement of plasma oxytocin

Objective: There is increased interest in measuring peripheral oxytocin levels to better understand the role of this peptide in mammalian behavior, physiology, and disease. The purpose of this study was to compare methods for plasma oxytocin measurement using a commercially available enzyme immunoassay (EIA) and radioimmunoassay (RIA), to evaluate the need for sample extraction, and to assess the immunospecificity of the assays. Methods: Oxytocin was measured in extracted and unextracted human plasma samples (n = 39). Oxytocin and its degradation products were separated by high-performance liquid chromatography and gel filtration chromatography and then assayed by EIA or RIA to identify oxytocin immunoreactive peaks. Results: Without extraction, plasma measured by EIA was more than 100-fold higher than in extracted plasma, and the correlation between oxytocin levels in extracted and unextracted plasma was minimal (Spearman &rgr; = −0.10, p = .54). Using the RIA, most samples (>90%) were below the level of detection with or without extraction. After chromatographic fractionation of sample extracts, multiple immunoreactive products were found to be present in addition to oxytocin, which casts doubts on the specificity of the assays. Conclusions: Changes in oxytocin levels have been reported in social and behavioral challenge studies. This study indicates that sample extraction is necessary to obtain valid assay results. Changes in oxytocin degradation products are likely to contribute to the previously observed responses in circulating oxytocin levels to behavioral and social challenge. There is a critical need for valid and reliable methods to measure oxytocin in biologic samples.Abbreviations: EIA = enzyme immunoassay; RIA = radioimmunoassay; HPLC = high-performance liquid chromatography; SEM = standard error of the mean; MW = molecular weight

Oxytocin attenuates NADPH-dependent superoxide activity and IL-6 secretion in macrophages and vascular cells

Oxytocin is synthesized and released in the heart and vasculature, tissues that also express oxytocin receptors. Although it has been established this intrinsic cardiovascular oxytocin system is important in normal homeostatic cardiac and vascular regulation, a role for this system in cardiovascular pathophysiology has not been investigated. The current study examined the influence of oxytocin on mechanisms in atherogenesis, oxidative stress, and inflammation in cultured human vascular cells, THP-1 monocytes, and macrophages. Oxytocin receptor protein and mRNA expression, NADPH-dependent superoxide activity, and interleukin-6 secretion were measured. Results demonstrated oxytocin receptor protein and mRNA in THP-1 monocytes and macrophages. Incubation of cells at physiological levels of oxytocin significantly decreased basal and stimulated NADPH-dependent superoxide activity in vascular cells, monocytes, and macrophages by 24-48%. Oxytocin also attenuated interleukin-6 secretion from stimulated THP-1 macrophages and endothelial cells by 56 and 26%, respectively. These findings suggest that oxytocin attenuates vascular oxidative stress and inflammation, two important pathophysiological processes in atherosclerosis. The fact that oxytocin receptors are found in monocytes and macrophages, and oxytocin decreases both superoxide production and release of a proinflammatory cytokine from these cells, suggests a potentially larger role for oxytocin in the attenuation of disease.

Oxytocin indexes relational distress following interpersonal harms in women

The hypothalamic neuropeptide oxytocin, known for its involvement in social affiliation and bonding in animals, has recently been associated with a host of prosocial behaviors that are beneficial for maintaining positive social relationships in humans. Paradoxically, however, people with high endogenous levels of oxytocin also tend to report relational distress and interpersonal difficulties in their everyday lives. To address these contradictory findings, oxytocin reactivity was measured in response to a well-defined laboratory task in young adult women following recent interpersonal harms. Elevated mean peripheral oxytocin reactivity (but not baseline levels of oxytocin or cortisol reactivity) was associated with increased post-conflict anxiety and decreased levels of forgiveness. These results corroborate previous research implicating oxytocin as a neuroendocrine marker of relational distress, but not general stress, and demonstrate the utility of studying oxytocin in response to naturally occurring relational events.

Circulating levels of glucocorticoid hormones in WHHL and NZW rabbits: circadian cycle and response to repeated social encounter

Social environment influences the progression of atherosclerosis in an important experimental model of disease, the Watanabe Heritable Hyperlipidemic rabbit (WHHL). Although the hypothalamic-pituitary-adrenocortical (HPA) system is likely to play an important role in the behavioral modulation of disease, relatively little is known about the glucocorticoid responses in these animals, or in other strains of rabbits. The purpose of the present study was to: (1) evaluate the rabbit glucocorticoid circadian rhythm, (2) compare plasma cortisol and corticosterone responses to social stress, and (3) examine strain differences (i.e., WHHL vs. New Zealand White (NZW)) in rabbit glucocorticoid responses to assess whether WHHLs have an aberrant HPA system. It was found that male rabbits secrete both corticosterone and cortisol in a circadian rhythm that peaks in the afternoon and reaches a nadir at 0600 h, i.e., approximately 12 h out-of-phase with the human glucocorticoid rhythm. Both glucocorticoids responded similarly to social stress induced by repeated daily 4 h pairings with another male rabbit; after 10 days of pairings, glucorticoid values were significantly correlated with the amount of defensive agonistic behavior exhibited. Finally, there were no significant strain differences in glucocorticoid circadian rhythms, baselines, or responses to social stress. These data suggest that glucocorticoid responses (i.e., circadian rhythms, responses to social stress) in the WHHL are similar to glucocorticoid responses in standard laboratory white rabbits.

Oxytocin attenuates atherosclerosis and adipose tissue inflammation in socially isolated ApoE-/- Mice

Objective: To determine the effect of exogenous oxytocin (OT) administration on inflammation and atherosclerosis in socially isolated apoE−/− mice. Hyperlipidemic animals housed in isolated or stressful social environments display more extensive atherosclerosis than those in an affiliative social environment. The neurohypophyseal peptide OT may be involved in both affiliative social behavior and cardiovascular homeostasis, suggesting a role in mediating the benefits of positive social interactions on atherosclerosis. Methods: A total of 43, 12-week-old, apoE−/− mice were surgically implanted with osmotic minipumps containing OT (n = 23) or vehicle (n = 20). Blood samples were taken at baseline and after 6 weeks and 12 weeks of treatment. After 12 weeks of treatment, animals were killed, and samples of adipose tissue were dissected from a subset of OT-treated (n = 12) and vehicle-treated (n = 12) animals and incubated in culture media for 6 hours. Media samples were analyzed for interleukin (IL)-6 concentration corrected by sample dry weight. Aortas were dissected, formalin-fixed, and stained with oil-red O for en face quantification of lesion area. t tests were used to compare group means on measures of percent lesion area and IL-6 concentrations. Results: There were no group differences in plasma lipids. Adipose tissue samples taken from OT-treated animals secreted significantly less IL-6 over 6 hours (p < .01). OT-treated animals displayed significantly less atherosclerosis in the thoracic aorta (p < .05). Conclusions: These results indicate that peripheral OT administration can inhibit atherosclerotic lesion development and adipose tissue inflammation, suggesting a potential role for this neuropeptide in mediating the benefits of stable group housing on atherosclerosis. OT = oxytocin; OTR = oxytocin receptor; SNS = sympathetic nervous system.

The Effect of Social Environment on Markers of Vascular Oxidative Stress and Inflammation in the Watanabe Heritable Hyperlipidemic Rabbit

Objective: Previous research demonstrated that social environment can influence progression of atherosclerosis in the Watanabe Heritable Hyperlipidemic (WHHL) rabbit. This study examined the effect of social environment on markers of oxidative stress and inflammation to clarify the physiological pathways potentially responsible for the influence of social environment on disease. Methods and Results: WHHL rabbits were assigned to 1 of 3 social groups: an unstable group, in which unfamiliar rabbits were paired daily, with the pairing switched each week; a stable group, in which littermates were paired daily; and an individually-caged group. The stable group engaged in more affiliative social behavior than the unstable group. The unstable group showed more agonistic behavior compared with the stable group and higher C-reactive protein levels than the individually caged group. The individually caged group was behaviorally sedentary, had higher 24-hour urinary catecholamine levels than the other groups, and exhibited higher NAD(P)H-oxidase activity in the aortic arch relative to the stable group. Conclusions: The results suggest that social environment creates distinct behavioral contexts that can affect markers of inflammation and oxidative stress early in the development of atherosclerosis. Specifically, physical inactivity associated with individual caging affects indices of oxidative stress and inflammation. These pathophysiological markers may help to explain behaviorally related differences in the extent of atherosclerosis observed in prior studies. ANG II = angiotensin II; ACE = angiotensin-converting-enzyme; CRP = c-reactive protein; HDL = high-density lipoprotein; IL = interleukin; LDL = low-density lipoprotein; ROS = reactive oxygen species; TNF = tumor necrosis factor; WHHL = Watanabe Heritable Hyperlipidemic.

Oxytocin Administration Attenuates Atherosclerosis and Inflammation in Watanabe Heritable Hyperlipidemic Rabbits

Oxytocin (OT) is a neurohypophyseal peptide traditionally associated with female reproductive functioning, and more recently with prosocial behavior. OT and its receptor are also expressed in the heart and vascular tissue and play a role in cardiovascular homeostasis. In vitro, it has been demonstrated that OT decreases NADPH-dependent superoxide production and pro-inflammatory cytokine release from vascular endothelial cells and macrophages, suggesting that OT may attenuate pathophysiological processes involved with atherosclerotic lesion formation. The present study sought to determine the effect of chronic exogenous OT administration on inflammation and atherosclerosis in an animal model of dyslipidemia and atherosclerosis, the Watanabe Heritable Hyperlipidemic (WHHL) rabbit. Twenty-two, 3-month-old WHHLs were surgically implanted with osmotic mini-pumps containing OT (n=11) or vehicle (n=11), and then were individually housed for the entire study. Blood and 24-h urine samples were taken at baseline and after 8 (midpoint) and 16 (endpoint) weeks of treatment. At endpoint, the aortas and visceral fat samples were dissected and stored for analyses. There were no group differences in body weight, serum lipids, plasma/urinary measures of oxidative stress, plasma cortisol or urinary catecholamines over the 16-week treatment. OT-treated animals exhibited significantly lower plasma C-reactive protein levels at midpoint and endpoint and developed significantly less atherosclerosis in the thoracic aorta relative to vehicle control animals at endpoint (p<0.05). Cytokine gene expression from visceral adipose tissue samples suggested that there was a decrease in adipose tissue inflammation in the OT-treated group compared to the vehicle control group, however these differences were not statistically significant. These results suggest that chronic peripheral OT administration can inhibit inflammation and atherosclerotic lesion development.

Social experience influences hypothalamic oxytocin in the WHHL rabbit

Social experience influences behavior and the progression of atherosclerosis in the Watanabe Heritable Hyperlipidemic (WHHL) rabbit, such that WHHL rabbits exposed to a consistent, stable social experience exhibited more affiliative social behavior and less aortic atherosclerosis compared to other social groups. Oxytocin (OT) has been implicated in the expression of social behavior, stress responses, and may provide a mechanism by which social experience influences atherogenesis in WHHL rabbits. The current study examined acute and chronic changes in central and peripheral OT before and after WHHL rabbits were exposed to one of three social conditions. Cannula implanted adjacent to the hypothalamic paraventricular nucleus (PVN) allowed chronic sampling of extracellular OT concentration via microdialysis. Rabbits were exposed to one of three social conditions: an Unstable group, with initially unfamiliar rabbits paired daily for 4h during the initial week and similarly paired with a different, unfamiliar rabbit each week; a Stable group; with the same 2 littermates paired daily for 4h the entire study; and an Individually Caged group. Dialysates from the PVN and blood from the marginal ear vein were collected twice, 20 days apart, from rabbits before and after 2h of exposure to their respective social condition. Dialysates were assayed for OT and plasma was assayed for OT, catecholamines and glucocorticoids. There were no changes in PVN OT in any group following the initial social experience. In contrast, after 20 consecutive days of exposure to their respective social condition, PVN OT increased significantly in the Unstable group, but was relatively unchanged in the Stable group following the social experience on day 22. Peripheral OT was not altered in any group following the 2h social experience on day 1 or 22. The concentration of peripheral OT was the highest in the Stable group at all times. The Stable group also exhibited significantly less aortic atherosclerosis, consistent with earlier findings from our laboratory. Data from the present study suggest that the type of social experience WHHL rabbits are exposed influences PVN OT, social behavior and the progression of atherosclerosis in the WHHL rabbit model of disease.

Stress buffering effects of oxytocin on HIV status in low-income ethnic minority women.

BACKGROUND Elevated perceptions of psychosocial stress and stressful life events are linked to faster disease progression in individuals living with HIV and these associations may be stronger for women from ethnic minority populations. Levels of neurohormones such as oxytocin (OT), cortisol, and norepinephrine (NE) have been shown to influence the effects of psychosocial stress in different populations. Understanding how intrinsic neuroendocrine substances moderate the effects of stressors in minority women living with HIV (WLWH) may pave the way for interventions to improve disease management. METHODS We examined circulating levels of plasma OT as a moderator of the effects of stress on disease status (viral load, CD4+ cell count) in 71 low-income ethnic minority WLWH. RESULTS At low levels of OT, there was an inverse association between stress and CD4+ cell counts. Counter-intuitively, at high levels of OT there was a positive association between stress and CD4+ cell counts. This pattern was unrelated to womens viral load. Other neuroendocrine hormones known to down-regulate the immune system (cortisol, norepinephrine) did not mediate the effects of OT and stress on immune status. CONCLUSIONS OT may have stress buffering effects on some immune parameters and possibly health status in low income ethnic minority WLWH reporting elevated stress.

Oxytocin, Social Support, and Sleep Quality in Low-Income Minority Women Living With HIV

Sleep disturbances are highly prevalent in women with HIV, and few studies examine potential protective factors that may reduce risk for sleep disturbances in this high-risk population. This study predicted that HIV-specific social support from various sources (i.e., friends, family members, and spouses), as well as oxytocin (OT), would explain sleep quality in 71 low-income minority women living with HIV. Social support from family members was associated with better sleep quality in women. For women with high OT, support from friends was associated with better sleep quality, whereas for women with low OT, support from friends was associated with poorer sleep quality. Women with low OT may not effectively interpret and utilize available support resources, which may be associated with sleep disturbances.