Temporal pattern and effect of sex on lipopolysaccharide-induced stress hormone and cytokine response in pigs

The temporal pattern and sex effect of immune and stress hormone responses to a lipopolysaccharide (LPS) challenge were assessed using a pig model. Secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 increased in a time-dependent manner following LPS infusion. There was also a time-dependent increase in secretion of the stress-related hormones cortisol, epinephrine (E), and norepinephrine (NE) following LPS, with peak concentrations attained within 30 min. The magnitude of the TNF-α and IL-1β responses were both positively associated (P < 0.05) with the magnitude of cortisol response following LPS, whereas serum IL-1β and IL-6 were positively correlated with the magnitude of E and NE responses following LPS. Acute-phase protein production was also time-dependently increased following LPS. The concentration of immune cells in circulation was decreased (P < 0.05) at 5.5 h post-LPS and negatively correlated with pro-inflammatory cytokine production. By 24 h post-LPS, immune cell counts increased (P < 0.05) and were positively associated with both pro-inflammatory cytokine and stress hormone production. The amplitude of pro-inflammatory cytokine response following LPS was affected (P < 0.05) by sex classification; however, the magnitude of elevated cytokine concentrations was not. The magnitude of the NE response, but not of the E and cortisol responses, to LPS was influenced by sex (P < 0.05). Similar to the pro-inflammatory cytokines, the magnitude of exposure to the stress hormones following LPS was not influenced by sex. The production of serum amyloid A (SAA) was influenced by sex, with barrows producing more SAA than gilts at 24 h post-LPS (P < 0.05). Collectively, these results demonstrate sex-specific, concomitant temporal changes in innate immune- and stress-related hormones.     

Modeling the effects of estradiol and progesterone on the acute phase proinflammatory axis: variability in tumor necrosis factor-α, nitric oxide, and xanthine oxidase responses to endotoxin challenge in steers

The severity of host response in some diseases differs between sexes, and this dimorphism has been attributed to the immunomodulating effects of reproductive steroid hormones. In females, susceptibility to disease stress has been associated with reproductive status and attributed to prevailing progesterone (P4) or estrogen concentrations during different estrous cycle phases. Our objective was to clarify and define the effect of P4 or 17β-estradiol (E2) on the acute proinflammatory component of the innate immune system by administering these hormones to steers and evaluating initial and tolerance-associated concentration patterns of circulating proinflammatory immune response mediators after two consecutive lipopolysaccharide (LPS) challenges (LPS1 and LPS2, 6 d apart; 2.5 μg/kg BW, intravenously, Escherichia coli 055:B5). Plasma concentrations of the proinflammatory initiation cytokine tumor necrosis factor-α (TNF-α), nitrate+nitrite [NO(x), estimate of nitric oxide (NO) production], haptoglobin (HG; acute phase protein) and plasma xanthine oxidase activity (mediator of superoxide production) were measured. Crossbred steers (392 ± 7 kg) were fed a forage-concentrate diet (15% CP) to appetite and assigned to control (C; n = 7), P4 (n = 8), or E2 (n = 5) treatment. Jugular blood samples were obtained at 0, 1, 2, 3, 4, 7, and 24 h relative to each of the two LPS injections. For each proinflammatory biomarker, the area under the time by concentration curve (AUC) was used to evaluate and compare responses to the LPS challenge. Treatment with E2 disrupted LPS tolerance as observed in augmented plasma TNF-α (P < 0.01) and NO(x) (P < 0.01) responses to LPS2. Compared with C, P4 treatment decreased plasma NO(x) AUC after LPS2 (P < 0.05) and tended to reduce TNF-α AUC after LPS1 (P = 0.08). Plasma xanthine oxidase activity AUC was increased (P < 0.01) over C by E2 treatment after both LPS1 and LPS2. HG response to LPS1 within 24 h was not affected by any treatment. However, 6 d after LPS1 plasma HG concentration remained higher (P < 0.01) in steers treated with E2 than with C or P4. Results indicate that in cattle, P4 and E2, respectively, attenuate or amplify the response to LPS challenge at several points critical to the regulation of the progression of the proinflammatory cascade.     

Changes in ovine maternal temperature, and serum cortisol and interleukin-6 concentrations after challenge with Escherichia coli lipopolysaccharide during pregnancy and early lactation

Major changes in maternal physiology during pregnancy and lactation can have a large impact on the immune and neuroendocrine systems. One of the most significant changes, observed in rats and mice, is hyporesponsiveness of the hypothalamic pituitary adrenal axis (HPAA) in response to inflammation, restraint, and other psychological stressors during late pregnancy and lactation. This attenuation, however, has not been well characterized in ruminant animals and may be relevant to their susceptibility to inflammatory diseases during these periods. Thus, the intent of this study was to characterize responsiveness of the ovine HPAA to inflammatory challenge during pregnancy and lactation. Ewes from early (33 d), middle (55 d), and late (138 d) pregnancy, as well as early lactation (10 d), were challenged i.v. with a bolus dose of 400 ng of Escherichia coli lipopolysaccharide (LPS)/kg of BW or saline. A corresponding group of nonpregnant ewes was also challenged with LPS to serve as positive control animals for each pregnancy and lactation study. Responsiveness of the HPAA was assessed by measuring the 4-h change in serum cortisol concentration after LPS challenge. The cortisol increase after LPS challenge was elevated (P < 0.01) in pregnant ewes during late pregnancy over that of nonpregnant animals. In contrast, the characteristic temperature response associated with systemic LPS challenge was decreased (P < 0.01) during early pregnancy and lactation compared with nonpregnant or nonlactating animals. Serum IL-6 concentrations were measured to assess whether changes in HPAA responsiveness during pregnancy or lactation were attributed to changes in proinflammatory signaling to the HPAA. Interestingly, enhanced cortisol responsiveness during late pregnancy was correlated with increased (P < 0.01) serum IL-6 concentrations, indicating that IL-6 may contribute to enhanced HPAA responsiveness during this period. Serum IL-6 concentrations during early and midpregnancy did not increase in response to LPS challenge, indicating that HPAA activation during periods of pregnancy may be independent of IL-6 production.     

Effects of prenatal stress on cellular and humoral immune responses in neonatal pigs

In the present study, we investigated the effects of a daily 5 min restraint stress of pregnant sows in the last five gestational weeks on the development and reactivity of the immune system of the offspring. Maternal stress resulted in significant decreased serum immunoglobulin G (IgG) concentrations in suckling piglets at 1 and 3 days of age. Furthermore, the stress treatment of the sows had an immunosuppressive effect on lymphocyte proliferation in response to the T-cell mitogen concanavalin A (ConA) at postnatal days 1 and 7. A suppressive effect was also found in response to the B-cell mitogens lipopolysaccharid (LPS) at days 1 and 35 and pokeweed mitogen (PWM) at day 1 of life, whereas natural killer (NK) cell cytotoxicity was not altered by prenatal stress. The relative thymus weights were significantly reduced in prenatally stressed piglets on the first and 35th day of life and the morbidity and mortality during the suckling period were significantly increased in prenatally stressed litters, as shown by a higher frequency of diseased and died piglets per litter. In addition, the ConA-, LPS- and PWM-stimulated lymphocyte proliferation at the age of 7, 21 and 35 days, and the NK cell cytotoxicity at the age of 21 and 35 days decreased in prenatally stressed and in control piglets 1h after a corticotropin (ACTH) injection. However, the cellular immunity was always higher in the control piglets which might be a result of the weaker stress hormone reactivity in prenatally stressed animals. In conclusion, the results provide first experimental evidence that prenatal maternal stress during late gestation is able to impair both humoral and cellular immune function in suckling piglets. The data also suggest that gestational stress in pigs may affect the ontogeny of the foetal immune system with consequences on the susceptibility to diseases and immune responsiveness to stressful stimuli of the offspring.     

The impact of maternal overnutrition and obesity on hypothalamic-pituitary-adrenal axis response of offspring to stress

We evaluated the effect of maternal obesity before and throughout gestation on offspring hypothalamic-pituitary-adrenal axis function. Multiparous Rambouillet by Columbia crossbred ewes were fed either 100% of National Research Council (NRC) recommendations (control, C) or 150% of NRC recommendations (obese, OB) from 60 d before mating until lambing. Ten lambs born to OB ewes (five males and five females), and eight lambs born to C ewes (three male and five female) were studied. From delivery to weaning lambs were maintained with their mothers, who were all fed 100% NRC recommendations. After weaning, all lambs were group housed and fed the same diet to meet NRC requirements. At 19 mo of age lambs were placed in individual pens and fed a pelletized diet to meet maintenance requirements. Jugular vein catheters were placed and 2 d later lambs received an intravenous (i.v.) adrenocorticotropic hormone (ACTH) challenge followed by an i.v. corticotropin-releasing hormone (CRH)/arginine vasopressin (AVP) challenge 1 d later. Thirty d later offspring were again catheterized and placed into metabolism crates for 2 d before receiving an isolation stress test. ACTH and cortisol responses to the isolation stress test and CRH/AVP challenge and cortisol responses to ACTH challenge were determined. Cortisol was quantified via radioimmunoassay and ACTH was quantified using an Immulite 1000; both were analyzed using repeated measures using the MIXED procedure of SAS. Offspring from OB ewes had elevated basal plasma ACTH and cortisol compared with C offspring before all three challenges (P < 0.05). Offspring from OB mothers tended (P = 0.06) to have a greater ACTH response after an i.v. CRH/AVP injection than offspring from C mothers (12,340 ± 1,430 vs 8,170 ± 1,570 area under the curve, respectively). Cortisol response to the CRH/AVP and ACTH challenges was not influenced by maternal nutrition (P = 0.46) and averaged 4.77 ± 0.2 μg/dL and 1.94 ± 0.01 μg/dL, respectively. The ACTH response following the isolation stress test was also similar (P = 0.82) for OB and C offspring (147 ± 20 pg/mL), and cortisol response during the isolation stress test was similar between C and OB offspring (P = 0.64, 5.25 ± 0.3 μg/dL). These findings suggest that maternal obesity before and during gestation does not affect stress responses by the offspring, but has an impact on hypothalamic-pituitary-adrenal sensitivity. The lack of differences in cortisol release under the influence of difference concentrations of ACTH during the CRH/AVP challenge could indicate adrenal dysfunction in OB offspring.     

Effect of calf thymus extract and zinc supplementation on the cellular response of mice exposed to restraint stress

The studies were carried out on Balb/c mice exposed to restraint stress twice for 12 h at 24 h intervals. Prior to stress exposure, the mice were treated with calf thymus extract (TFX - Jelfa) i.p. at a dose of 10 mg/kg, ten times at 24 h intervals. TFX was used per se or with zinc ions interaction, by adding zinc ions (as sulfate salt) to drinking water at a dose of 72 microg/mouse per day. The results obtained show that restraint stress dramatically decreased the total number of thymocytes and splenocytes which is also accompanied by decreasing weight ratio of the thymus and spleen. The decreasing number of thymic and spleen cells corresponded to a diminishing percentage of immature, double-positive CD4+CD8+ thymocytes, mature single-positive CD4+ thymic cells and CD4+, CD8+ and CD19+ splenocytes. Changes in the number of thymic cells affect their activity, which is expressed as a decreased proliferative response of thymocytes stimulated in vitro with concanavalin A (Con A) and phytohaemagglutinin (PHA). Besides, exposure to the restraint stress decreased interleukin-1 (IL-1) production by murine intraperitoneal macrophages stimulated in vitro with lipopolisacharide (LPS) from E. coli. Previous treatment with TFX counteracted restraint stress-induced immunosuppression, which is expressed as partial normalisation of the total number of thymic and spleen cells, accelerated regeneration of these two lymphatic organs, shortned suppressive action of restraint stress on the percentage of immature CD4+CD8+ thymocytes and CD4+ splenocytes and in total normalisation of the CD4+ thymocytes and CD8+ splenocytes. TFX administered prior to restraint stress not only counteracted the suppresive effects of stress on the proliferative activity of thymic cells stimulated in vitro with Con A and PHA, but also augmented the proliferative response of these cells to two mitogens. The immunorestorative effect of TFX was augmented by zinc supplementation.     

Stress exposure during the preimplantation period affects blastocyst lineages and offspring development

We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life.     

Restraint effects on stress-related hormones and blood natural killer cell cytotoxicity in pigs with a mutated ryanodine receptor

A mutation in the ryanodine receptor gene (RYR1) of the calcium release channel is responsible for increased stress susceptibility in pigs. In the present study, the relation of a mutation in RYR1 with the neuroendocrine (stress-related hormone) response and the immune defense represented by natural killer cell cytotoxicity (NKCC) during a 4-h restraint and recovery phase in 60 male pigs was investigated. Blood samples were collected from pigs previously divided into RYR1 genotypes (nn, Nn, NN), based on PCR amplification and restriction analyses. The blood samples collected during the restraint and recovery phases of the experiment were used to determine NKCC (⁵¹Cr-release assay), large granular lymphocyte number (hematologic method), and plasma concentrations of prolactin (PRL), GH, ACTH, and cortisol (COR) (by specific RIA). The greatest degree of NKCC response (P < 0.05) to restraint stress relative to controls was observed for the stress-susceptible homozygote group (nn). Measures of stress-related hormones were positively correlated with NKCC during the entire experimental period (P < 0.001 for all investigated hormones) in the nn group. Immunostimulatory effects in the early (0–60 min) phase of restraint were associated with increased hormone responses, especially PRL and GH. In the late (180–240 min) phase of stress and the recovery phase (480 min), a decrease in immune response was accompanied by an elevated COR response in all RYR1 genotypes. Moreover, divergent responses of both PRL (greatest in nn, P < 0.001) and GH (greatest in NN, P < 0.001) to the 4-h restraint were observed. Our results suggest that stress-susceptible RYR1-mutated homozygotes develop a greater level of immune defense, including cytotoxic activity of NK cells, and accompanied by more pronounced stress-induced changes in neuroendocrine response than stress-resistant heterozygous (Nn) and homozygous (NN) pigs.     

牛膝多糖对免疫应激仔猪生长性能、炎性介质和内分泌激素的影响

试验旨在探讨牛膝多糖(ABPS)对脂多糖(LPS)刺激的断奶仔猪生长性能、炎性介质和内分泌激素的影响。选用48头(28±3)d(8.45±0.14)kg的杜洛克×长白×大白断奶仔猪,采用2×2因子设计,包括日粮处理(0或500 mg/kg ABPS)和免疫应激(注射LPS或生理盐水)。试验期28 d。在第14天和第21天,每日粮组一半的猪注射100μg/kg BW的LPS,另一半注射生理盐水作对照。注射后3 h,采血测定血浆肿瘤坏死因子-α(TNF-α)、前列腺素E2(PGE2)、皮质醇、生长激素(GH)和类胰岛素生长因子-I(IGF-I)含量。结果表明:LPS刺激显著降低了第14~21、22~28天的日增重(ADG)和日采食量(ADFI)(P<0.05或P=0.05);ABPS显著提高了22~28 d的ADG(P<0.05)。LPS刺激显著提高了14 d和21 d的TNF-α、PGE2和皮质醇的含量,降低了IGF-I的含量(P<0.01);ABPS显著缓解了LPS刺激导致的TNF-α(14 d和21 d)和皮质醇(14 d)含量的上升以及IGF-I(14 d和21 d)含量的下降(P<0.05)。说明ABPS缓解了免疫应激仔猪生长的抑制,其机制可能与其抑制了炎性介质的分泌有关。     

Exogenous testosterone modulates tumor necrosis factor-alpha and acute phase protein responses to repeated endotoxin challenge in steers

Clinical responses to some disease agents differ between sexes and this dimorphism has been attributed to the immunomodulating effects of steroid hormones. Our objective was to determine in steers the effect of testosterone on circulating concentrations of immune response mediators (tumor necrosis factor-alpha, TNF-alpha; serum amyloid-A, SAA; haptoglobin, HG; xanthine oxidase, XO; nitric oxide, NO) after two consecutive endotoxin challenges (LPS1 and LPS2, 5 days apart; 0.25 microg/kg BW). Sixteen crossbred steers (328+/-6 kg) were assigned to control (CON, n=8) or testosterone cypionate treatment (TES, n=8; 100 mg/m2 body surface; i.m. injection 12 and 2 days before LPS1). The response to LPS was calculated as area under the timexconcentration curve (AUC) for the parameter measured. After LPS1, TNF-alpha AUC was greater in TES than CON (P<0.05). Plasma HG and SAA concentrations increased (P<0.01) after LPS1 and LPS2. In all steers SAA AUC was greater after LPS1 than LPS2 (P<0.01) but the response was augmented over CON with testosterone treatment (P<0.05). HG response to LPS1 within 24 h was not affected by testosterone. However, 5 days after LPS1 mean plasma HG concentration remained higher in TES than CON (P<0.01). HG response to LPS2 was greater in TES than CON (P<0.01). Plasma nitrate+nitrite concentration (NO production marker) and XO activity increased after each LPS challenge but responses were not affected by testosterone treatment. Results indicate that the presence of circulating testosterone increases the magnitude of the TNF-alpha response to LPS challenge as well as the subsequent increases in acute phase proteins (APP). Effects of testosterone on increases in TNF-alpha and APP may underlie a differential presentation of disease symptoms between sexes or between steers and bulls. The data also suggest a role for testosterone in the development of tolerance to repeated immune challenge through its effect on the increased magnitude and duration of HG response.     

Staphylococcus aureus lipotechoic acid induces differential expression of bovine serum amyloid A3 (SAA3) by mammary epithelial cells: Implications for early diagnosis of mastitis

Mastitis is one of the most costly diseases of agriculturally important animals and is a common problem for lactating cows. Current methods used to detect clinical and especially subclinical mastitis are either inadequate or problematic. Pathogens such as the gram-positive bacterium Staphylococcus aureus or the gram-negative bacterium Escherichia coli typically cause mastitis. E. coli induces clinical mastitis, whereas, S. aureus causes a subclinical, chronic infection of the mammary gland. In this study we report the differential expression and secretion of mammary-derived serum amyloid A3 (SAA3) by bovine mammary epithelial cells following stimulation with the S. aureus cell wall component, lipotechoic acid (LTA). Two-dimensional immunoblot analyses confirmed that bovine SAA3 is the predominant SAA isoform produced by LTA stimulated mammary epithelial cells. Our previous study showed that bovine SAA3 is also differentially expressed in response to the gram-negative bacterial endotoxin lipopolysaccharide. Collectively, these data indicate that the local production of SAA3 by mammary epithelial cells in response to either gram-positive or gram-negative bacterial components may provide a sensitive indicator for early detection and treatment of mastitis in vivo, minimizing chronic cases of infection, the spread of mastitis to other animals, and economic losses.     

Characterisation of the acute phase response of heifers to a prolonged low dose infusion of lipopolysaccharide

The effect of a prolonged low dose infusion of bacterial lipopolysaccharide (LPs) on acute phase-like reactions was examined in heifers. LPS (2 μg kg⁻¹ dissolved in 100 ml water), or saline was infused (at 1 ml min⁻¹) intravenously for 100 minutes and blood samples were taken at various times before, during and after the infusion. The serum concentrations of tumour necrosis factor-alpha(TNFα), interleukin-Ibeta (IL-1β), interleukin-6 (IL-6) and serum amyloid A (SAA) and the rectal temperature increased in response to the LPS infusion. Serum TNFα increased before the increases in IL-1β and IL-6 and remained high from 20 minutes after the onset of the infusion until the end of the sampling period (six hours). The LPS-induced increases in serum IL-1β and IL-6 were biphasic. Plasma cortisol and lactate concentrations also increased, and plasma glucose and B-hydroxybutyrate concentrations decreased in response to the LPS infusion. The similarity of these reactions to changes observed in response to bacterial infections shows that the prolonged infusion of low doses of LPS is a good model for studying the acute phase response to Gramnegative bacterial infection in heifers.     

Effects of prenatal stress on corticosteroid receptors and monoamine concentrations in limbic areas of suckling piglets (Sus scrofa) at different ages

The present study was conducted in order to reveal the effects of prenatal stress on the central stress regulation in domestic pigs by measuring changes in corticosteroid receptor binding and monoamine concentrations in different limbic brain regions. Pregnant sows were subjected to a restraint stress for 5 min daily during the last 5 weeks of gestation. Maternal stress resulted in a significantly higher number of glucocorticoid receptors in the hippocampus, but decreased glucocorticoid receptors in the hypothalamus of the offspring at the first postnatal day. No alterations of hippocampal mineralocorticoid receptors were found. There was also no significant effect of prenatal stress on the brain monoamine concentrations. Prenatally stressed piglets showed lower basal plasma cortisol and increased corticosteroid binding globulin concentrations at the third postnatal day indicating decreased free cortisol concentrations after birth. Morbidity and mortality during the suckling period were significantly increased in prenatally stressed litters, as shown by a higher frequency of diseased and died piglets per litter. In conclusion, the results indicate that in pigs restraint stress during late gestation affects the ontogeny of the foetal neuroendocrine feedback system with consequences for the regulation of the hypothalamo-pituitary-adrenal function and the vitality of the offspring.     

Changes in blood biomarkers in Arabian horses with Clostridium difficile-induced enterocolitis

Clostridium difficile (CD) is considered a major health care problem both in developing and developed countries; frequently reported to be associated with enterocolitis and diarrhea in horses and other animals. In this study, we examined acute phase response (APR), cytokines response, neopterin (NP) procalcitonin (PCT) production and oxidative stress condition in horses and foals with C. difficile-induced enterocolitis (CDIE) and evaluated the effectiveness of these parameters as biomarkers for the disease. A total of 407 Arabian horses in 35 stables were examined between January 2017 to December 2018. Only 24 out of 407 horses showed two or more signs of CDIE. The blood level of serum amyloid A (SAA), haptoglobin (HP), proinflammatory cytokines (TNF-α, IL-6 and IL1-β), serum malondialdehyde (MDA), PCT and NPT in horses with CDIE were higher than in healthy horses. Nevertheless, the levels of nitric oxide (NO), superoxide dismutase (SOD) and total antioxidant concentration (TAC) were considerably lower in diseased horses compared to those that were healthy. The ROC curves for eleven selected blood parameters, both in healthy horses and horses with CDIE demonstrated that all examined blood markers had significant levels of differentiation between CDIE cases and healthy controls (AUC > 87.5). The data in this study suggest that the evaluation of acute-phase proteins, cytokines, PCT, NPT, and oxidative stress biomarkers may well be used as a tool for diagnosis and assessment of CDIE and in disease pathogenesis in Arabian horses.     

Inhibition of stress-induced adrenocorticotropin and prolactin secretion mediating hypophysiotropic factors by antagonist of AMPA type glutamate receptor

Glutamate is the dominant excitatory neurotransmitter in a large number of physiological processes including neuroendocrine regulation. Some pharmacological studies have shown that different subtypes of glutamate receptor, such as the N-methyl-D-aspartic acid (NMDA) and alpha-amino-3-hydroxy-5-methy-4-isoxazolepropionic acid (AMPA) receptors, are involved in stress-induced adrenocorticotropin (ACTH) and prolactin secretion. However, the roles of the respective glutamate receptors and the mechanism of ACTH and prolactin secretion during stress via these receptors have not been investigated in detail. In the present study, we evaluated the role of AMPA-type glutamate receptor in ACTH and prolactin regulation under restraint stress in adult male rats. Male rats pretreated with a selective AMPA receptor antagonist, 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 50 microg), through a lateral ventricle cannula were stressed by immobilization. Administration of NBQX inhibited ACTH and prolactin secretion in response to restraint stress. However, NBQX had no significant effects on the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, as measured by the accumulation of 3, 4-dihydroxyphenylalanine (DOPA). In addition, administration of NBQX suppressed stress-induced prolactin secretion in the male rats pretreated with alpha-MT, an inhibitor of dopamine synthesis, and infused with dopamine solution (2.5 microg/200 microl/10 min). These results indicated that the effects of NBQX on prolactin secretion might be mediated by non-dopamine mechanisms. The contents of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the median eminence (ME) of the male rats decreased during restraint stress; however, the fluctuations in CRH and AVP were eliminated by NBQX administration. These results suggest that stress-induced ACTH and prolactin release mediated by neurotransmission via AMPA receptors might be partly attributable to hypophysiotropic regulatory factors in the hypothalamus.     

Variability in tumor necrosis factor-α, nitric oxide,and xanthine oxidase responses to endotoxin challenge in heifers: Effect of estrous cycle stage

The severity of host response to some disease agents differs between sexes and this dimorphism has been attributed to the immunomodulating effects of steroid hormones. Our objective was to determine in heifers whether the phase of estrous cycle affected immune response mediators after endotoxin challenge (LPS, 2.5 μg/kg BW, i.v.). Sixteen beef heifers (426 ± 9 kg) were reproductively synchronized with the two-injection protocol of dinoprost tromethamine (Lutalyse^®, Pfizer) to establish diestrus and estrus stages of the estrous cycle. Heifers were challenged with LPS on day 3 (E, estrus; n = 8) or day 10 (D, diestrus, n = 8) after the last i.m. injection of Lutalyse^®. In all heifers, plasma concentrations of tumor necrosis factor-α (TNF-α) peaked 2 h after LPS treatment (P < 0.01) and returned to basal level by 7 h. However, the integrated TNF-α response (area under the time × concentration curve, AUC) was greater in E than in D (P < 0.05). Plasma concentrations of nitrate + nitrite (NO_x, an estimate of NO production) increased (P < 0.01) in all heifers at 7 and 24 h after LPS; plasma NO_x AUC after LPS was greater in E than D (P < 0.01). Plasma xanthine oxidase activity (XO, a mediator of superoxide production) responses were also greater in E than D (P < 0.05). A companion LPS challenge study in steers validated that the protocol for and use of Lutalyse^® did not affect any of the immune parameters studied in heifers in response to LPS. Results indicate that the underlying physiological attributes of the estrus and diestrus phases of the estrous cycle constitute a major source of variability in the magnitude of proinflammatory response to bacterial toxins like LPS.     

Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes

OBJECTIVE: To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. SAMPLE POPULATION: Venous blood samples obtained from 19 healthy horses. PROCEDURES: Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay. RESULTS: Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, and IL-10 and decreased LPS-induced expression of IL-6. CONCLUSIONS AND CLINICAL RELEVANCE: The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.     

人参多糖对脂多糖刺激小鼠巨噬细胞的免疫调控作用

本试验旨在研究脂多糖(LPS)刺激条件下人参多糖(GPS)对小鼠单核巨噬细胞形态及免疫功能的调节作用。采用LPS刺激小鼠巨噬细胞(RAW264.7),通过测量不同浓度(1、0.5、0.1 mg/mL)GPS对细胞形态、生物酶活性、促炎症因子分泌及TLR4/NF-κB信号通路mRNA表达量的影响来研究不同浓度的GPS对LPS引起小鼠巨噬细胞免疫应激的调控作用。结果表明:添加GPS能抑制由LPS引起的细胞形态和细胞增殖能力的变化;1 mg/mL GPS能够显著提高巨噬细胞酸性磷酸酶的活性;0.5、1 mg/mL GPS能够显著缓解由LPS刺激引起的碱性磷酸酶活性的降低;不同浓度GPS均能显著降低由LPS诱导的促炎症因子IL-1β、TNF-α水平;LPS刺激显著提高巨噬细胞TLR4、MyD88、NF-κB的mRNA表达量,而添加GPS后,巨噬细胞TLR4、MyD88、NF-κB的mRNA表达量均表现出不同程度降低(P<0.05)。结果显示,添加GPS可以改善细胞形态,恢复细胞增殖能力,GPS可通过调节TLR4/NF-κB信号通路降低促炎症因子IL-1β和TNF-α的分泌及表达,减少机体免疫应激反应。