Effect of maternal restraint stress during gestation on temporal lipopolysaccharide-induced neuroendocrine and immune responses of progeny.

The impact of gestational dam restraint stress on progeny immune and neuroendocrine temporal hormone responses to lipopolysaccharide (LPS) challenge was assessed. Maternal stress (5-min snout snare restraint stress during days 84 to 112 of gestation) increased (P < 0.05) the magnitude of tumor necrosis factor (TNF)-α, interleukin-6, epinephrine (E), norepinephrine, and serum amyloid A (SAA) production following LPS infusion in the offspring. Moreover, these effects appear to be dependent on gender for TNF-α, E, and cortisol production. However, maternal stress did not affect (P > 0.05) the normalization of proinflammatory cytokines or neuroendocrine hormones produced following LPS. Collectively, these results indicate that maternal stress impacts aspects of the proinflammatory cytokine and stress hormone response in their progeny following LPS dosing of the offspring. This response is potentially responsible in part for the resultant changes to SAA production. Because several of the changes observed here are dependent on pig gender, these results are also the first evidence that inherent epigenetic factors coupled with maternal stress impact the cumulative response to stress and LPS in young pigs.

Oral administration of dehydroepiandrosterone-sulfate (DHEAS) increases in vitro lymphocyte function and improves in vivo response of pigs to immunization against keyhole limpet hemocyanin (KLH) and ovalbumin.

The present study tested the hypothesis that the oral administration of DHEAS enhances the in vitro and the in vivo immune response of young pigs. Crossbred, female pigs (80 days of age; 49+/-2 kg) were separated into two treatment groups (n=4/treatment) receiving either 0mg/kg (control) or 1mg/kg DHEAS twice daily (DHEAS) for 5 weeks. On day 7 pigs were immunized against KLH and ovalbumin. Body weight increased weekly throughout the study but did not differ between treatment groups. While white blood cell counts increased in response to immunization but did not differ between treatments, the neutrophil:lymphocyte ratio was enhanced (P<0.05) in DHEAS-supplemented pigs. Concanavalin A (ConA) induced an in vitro dose-dependent increase (P<0.05) in lymphocyte proliferation, but treatment did not affect proliferation prior to immunization. However, lymphocytes isolated from DHEAS-supplemented pigs displayed a greater increase in proliferation following immunization relative to control pigs (P<0.05). Dexamethasone (DEX) attenuated ConA-induced lymphocyte proliferation, with DHEAS-supplemented pigs retaining a greater proliferative response relative to control pigs (P<0.05). Serum IgG concentrations and relative concentrations of antigen-specific IgG increased after immunization with maximum values attained at 21 and 28 days for control and DHEAS-supplemented pigs, respectively. The DHEAS-supplemented pigs had greater (P<0.05) concentrations of IgG and relative concentrations of antigen-specific IgG compared to control pigs. Collectively these data suggest DHEAS supplementation increases the responsiveness of young pigs to antigenic challenge, and may be beneficial for improving their immune function.

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)-alpha, interleukin (IL)-1beta, 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-alpha and IL-1beta responses were both positively associated (P < 0.05) with the magnitude of cortisol response following LPS, whereas serum IL-1beta 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 5.5h post-LPS and negatively correlated with pro-inflammatory cytokine production. By 24h 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 24h post-LPS (P < 0.05). Collectively, these results demonstrate sex-specific, concomitant temporal changes in innate immune- and stress-related hormones.

Interrelationships among growth, endocrine, immune, and temperament variables in neonatal Brahman calves.

Interrelationships among growth, endocrine, immune, and temperament variables were assessed in neonatal Brahman calves. The velocity upon exiting a working chute (exit velocity) of an animal was measured and used as an objective indicator of temperament to classify calves as calm, intermediate, or temperamental. Calves (n = 116) were weighed weekly between d 0 and 21 to 24, and blood samples were collected for plasma and serum on d 0, 1, 2, 7, 14, and 21 to 24 after birth to measure concentrations of immunoglobulins, cortisol, and epinephrine (EPI). Body weight increased from d 0 through d 21 to 24 (P < 0.001) with bulls (n = 60) having greater BW than heifers (n = 56; P = 0.02). Serum concentrations of cortisol were greatest on d 0 before declining (P < 0.001) over the ensuing 21 to 24 d and were not related to temperament (P = 0.89) or sex (P = 0.97). Concentrations of EPI were affected by time, with an increase in EPI concentrations in temperamental bulls between 2 and 14 d of age (P < 0.008). Concentrations of EPI were not affected by temperament (P = 0.44) or sex (P = 0.68). Serum immunoglobulin concentrations peaked on d 1 before declining (P < 0.01) but were not related to temperament (P = 0.40 to 0.68). Of the stress hormones measured (cortisol and EPI), only cortisol was associated with the early performance of the calf. Calf BW at d 21 to 24 and BW gain were positively associated with serum immunoglobulin concentrations, yet negatively associated with concentrations of cortisol. Serum immunoglobulin concentrations were negatively correlated with cortisol concentrations (r = -0.28; P = 0.003), yet positively associated with EPI concentrations (r = 0.51; P = 0.003). During the neonatal period in this study, there was no relationship of temperament with passive immunity or stress hormone concentrations; however, growth was positively associated with passive immunity and negatively associated with stress hormones. Measuring exit velocity as early in life as d 21 to 24 fails to accurately predict temperament at weaning in over 40% of Brahman calves. Our conclusion is that measurement of exit velocity should be done nearer to the time of weaning than to birth. These data can be beneficial in developing best management practices for young calves.

Lithium chloride does not inhibit the proliferation of L6 myoblasts by decreasing intracellular free inositol.

We conducted a series of experiments to determine whether lithium chloride (LiCl) inhibited the proliferation of L6 myoblasts by reducing the availability of intracellular free inositol. After the myoblasts were plated in DMEM + 10% fetal bovine serum (FBS) for 24 h, medium was replaced with DMEM + 10% FBS containing 0 (control), 5, 10, or 20 mM LiCl. Cell number, protein content, and [3H]thymidine incorporation into DNA were determined at 24-h intervals. Control cells exhibited a 3.8-fold increase in cell number by 96 h in culture. Although 5 mM LiCl did not affect the rate or extent of proliferation, 10 and 20 mM LiCl caused 36 and 86% decreases, respectively (P < .05), in cell number by 96 h in culture. The effects of LiCl could not be overcome by the addition of free inositol (up to 20 mM) to the medium. Lithium chloride caused 4.6- and 7.3-fold increases (P < .05) in lactate dehydrogenase activity in culture media after 96 h of exposure to 10 and 20 mM LiCl, respectively, indicating loss of viability after chronic treatment. However, the acute effects of LiCl after 24 h of treatment were reversible, as indicated by a rapid resumption of proliferation following removal of LiCl. Concentrations of 5, 10, and 20 mM LiCl caused 4.7-, 8.2-, and 9.1-fold increases (P < .05), respectively, in the accumulation of [3H]inositol within the inositol monophosphate pool. Treatment of cells with 10 and 20 mM LiCl also increased (P < .05) label recovered as inositol bisphosphate. Rather than depress phosphoinositide synthesis, the addition of 10 and 20 mM LiCl dose-dependently increased (P < . 05) the incorporation of [3H]inositol into phosphatidylinositol and phosphatidylinositol-4-phosphate. These results indicate that LiCl does not decrease proliferation of L6 myoblasts via a depletion in the intracellular free inositol pool. Instead, LiCl may block the hydrolysis of phosphatidyl inositides.

Myelosuppression in the pig (Sus scrofa): uteroferrin reduces the myelosuppressive effects of 5-fluorouracil in young pigs.

The present study investigated the ability of uteroferrin to modulate the myelosuppressive effects of 5-fluorouracil (5-FU) in young pigs (Sus scrofa). Pigs (28-35 days of age; n = 6 per treatment) were infused with equal amounts of 5-FU on days 0 and 1 of the experimental period (37.5 mg/kg cumulative dose). Uteroferrin (100 micrograms/kg in 0.9% NaCl) or control (equivalent volume of 0.9% NaCl) was administered to pigs as intramuscular injections twice daily (08:00 and 20:00 hr) on days 1 through 21. Peripheral blood cell number, composition and progenitor cells were determined over 28 days. Treatment of pigs with 5-FU resulted in a rapid dose-dependent (P < 0.05) leukocytopenia. Concurrent treatment of pigs with uteroferrin reduced (P < 0.05) the rate of 5-FU-induced leukocytopenia (44 vs 77 +/- 7% decline from baseline on day 3) and enhanced (P < 0.05), the recovery from 5-FU on days 10 and 12 postinfusion. The positive effect of uteroferrin on leukocytes resulted primarily from a protection and/or enhanced recovery of neutrophils and monocytes. In addition, uteroferrin attenuated (P < 0.05) the suppression of red blood cell numbers after 5-FU administration (6.9 vs 6.1 +/- 0.2 x 10(6) cells/microliter on day 3), an affect reflected in increased hematocrit and hemoglobin concentrations. The effects of uteroferrin appeared to result from enhancement of the proliferation and/or differentiation of primitive pluripotent stem cells resistant to 5-FU, as concurrent treatment of pigs with uteroferrin resulted in a protection and/or enhanced recovery (P < 0.05) of CFU-GEMM, CFU-GM and BFU-E progenitor cells in the peripheral blood. These results are the first to demonstrate that uteroferrin can reduce the myelosuppressive effects of 5-FU in the pig and suggest that uteroferrin has hematopoietic growth factor activity in vivo.

Uteroferrin and recombinant bovine GM-CSF modulate the myelosuppressive effects of 5-fluorouracil in young female pigs (Sus scrofa).

The present study investigated the ability of uteroferrin and recombinant bovine granulocyte monocyte/macrophage-colony stimulating factor (rbGM-CSF) to modulate the myelosuppressive effects of 5-fluorouracil (5-FU) in young female pigs (Sus scrofa). Pigs (N = 3/treatment) were infused with 5-FU (32.5 mg/kg) on days 0 and 1 of the experimental period. Uteroferrin (100 micrograms/kg in 0.9% NaCl), rbGM-CSF (10 micrograms/kg in 0.9% NaCl), uteroferrin + rbGM-CSF (as above) or control (0.9% NaCl) were administered as intramuscular injections twice daily (0800 and 2000 hr). Peripheral blood cell number, composition, and progenitor cells were determined over 28 days. Treatment of pigs with 5-FU resulted in a rapid leukocytopenia and thrombocytopenia (nadirs on days 5 and 7, respectively) and a modest decrease (P < 0.05) in red blood cell (RBC) number (nadir on day 14). Although nor affecting RBC and thrombocytes, treatment of pigs with uteroferrin had an initial protective effect (P < 0.05) on the 5-FU-induced leukocytopenia (63 and 64 vs 48 and 39 +/- 6% of baseline on days 3 and 5, respectively). In contrast, rbGM-CSF enhanced (P < 0.05) the rate of the leukocytopenia and had only minor effects on thrombocyte numbers relative to controls. These effects appeared to be additive, as pigs treated with uteroferrin + rbGM-CSF had a reduced rate of leukocytopenia compared to pigs treated with rbGM-CSF alone. Uteroferrin + rbGM-CSF also attenuated (P < 0.05) the suppression and enhanced (P < 0.05) recovery of RBC and thrombocyte numbers following 5-FU treatment. In control pigs, a modest rebound leukocytosis (122 +/- 6% of baseline) and thrombocytosis (141 +/- 9% of baseline) was evident. Uteroferrin enhanced (P < 0.05) the rebound leukocytosis (135 +/- 6% of baseline), but attenuated (P < 0.05) the thrombocytosis. In contrast, rbGM-CSF enhanced (P < 0.05) the duration of the leukocytosis during the recovery phase, an effect augmented by the combination of uteroferrin + rbGM-CSF. In addition, treatment with uteroferrin + rbGM-CSF resulted in a sustained thrombocytosis (days 12 to 21). As indicated by changes in CFU-GM, BFU-E, and CFU-GEMM progenitor cells in peripheral blood, the effects of uteroferrin and rbGM-CSF appeared to reflect their ability to enhance the proliferation and/or differentiation of both similar and distinct hematopoietic progenitor cells.

The effect of uteroferrin and recombinant GM-CSF on hematopoietic parameters in normal female pigs (Sus scrofa).

The present study investigated the effect of uteroferrin and recombinant bovine granulocyte-monocyte/macrophage colony stimulating factor (rbGM-CSF) on hematopoiesis in young female pigs. Uteroferrin (100 micrograms/kg in 0.9% NaCl), rbGM-CSF (10 micrograms/kg in 0.9% NaCl), uteroferrin + rbGM-CSF (as above), or control (0.9% NaCl) were administered as intramuscular injections twice daily (0800 and 2000 hr). Peripheral blood cell number, composition, and progenitor cells were determined over 28 days. Uteroferrin had minimal effects on white blood cell (WBC) number, while rbGM-CSF caused both a rapid (days 2-7; maximum 122 +/- 8% of baseline) and late (days 16-28; maximum 133 +/- 8% of baseline) increase in WBC. Combination treatment with uteroferrin + rbGM-CSF abolished the initial increase in WBC number,but resulted in a prolonged increase in WBC number (days 14-28) relative to control. The rbGM-CSF-induced increase in WBC number resulted from rapid increases (P < 0.05) in monocytes and neutrophils. The addition of uteroferrin + rbGM-CSF enhanced (P < 0.05) the initial increase in the monocyte population and augmented the neutrophilia. In addition, uteroferrin + rbGM-CSF resulted in a dramatic eosinophilia (days 2-28), which was not detected in either the uteroferrin or rbGM-CSF treatments. Although not substantially affected by uteroferrin alone, rbGM-CSF caused an increase (P < 0.05) in thrombocyte numbers from days 1 through 9 (maximum 133 +/- 11% of baseline), an effect augmented by cotreatment with uteroferrin. The ability of these cytokines to modulate blood cell number and composition appeared to result from their effects on hematopoietic progenitor cells. Treatment of pigs with uteroferrin increased (P < 0.05) CFU-GEMM, CFU-GM, and BFU-E progenitor cells in peripheral blood, while rbGM-CSF caused increases (P < 0.05) relative to control in CFU-GM and CFU-GEMM. These effects were additive, as uteroferrin + GM-CSF augmented the increases in CFU-GM, BFU-E, and CFU-GEMM. Collectively, these results indicate that uteroferrin and rbGM-CSF can modulate hematopoiesis in young pigs. These effects were both additive and, in the case of neutrophils and eosinophils, synergistic. Hence, the mechanism(s) by which uteroferrin and rbGM-CSF modulate hematopoiesis appear to be different.

Alteration of glycerolipid and sphingolipid-derived second messenger kinetics in ras transformed 3T3 cells.

The effect of ras transformation (rasB fibroblasts) on basal and serum-stimulated diacylglycerol (DAG) composition and mass was examined over time with respect to changes in membrane phospholipid composition and ceramide mass. RasB cells vs. nontransformed control cells (rasD and NR6) had chronically elevated DAG levels (up to 240 min) following serum stimulation, indicating a defect in the recovery phase of the intracellular DAG pulse. Ras transformation also had a dramatic effect on DAG composition. Molecular species analysis revealed that DAG from unstimulated rasB cells was enriched in the delta 9 desaturase fatty acyl species (monoenoate 18:1(n - 7) and 18:1(n - 9)), and depleted in arachidonic acid (20:4(n - 6)). With the exception of glycerophosphoinositol (GPI), DAG remodeling paralleled the compositional alterations in individual phospholipid classes. Importantly, ras transformation altered the fatty acyl composition of sphingomyelin, a precursor to the ceramide second messenger. With the addition of serum, control cells (rasD) had a progressive increase in ceramide mass with levels approximately 5-fold higher by 240 min. In contrast, ceramide levels did not increase in rasB cells at either 4 or 240 min. These results demonstrate that ras-oncogene, in addition to its effects on DAG metabolism, can also abolish the cellular increase in ceramide mass in response to serum stimulation. Since DAG and ceramide may have opposing biological functions, the prolonged elevation of DAG and the suppression of ceramide levels would be consistent with an enhanced proliferative capacity.

Diacylglycerol and ceramide kinetics in primary cultures of activated T-lymphocytes.

T cell activation results in the generation of diacylglycerol (DAG), the physiological activator of protein kinase C. Recently, ceramide, a bioactive lipid intracellular second messenger, has been shown to play a positive role in T cell proliferation. Most studies examining mitogen induction of DAG and ceramide in T cells have been conducted in cell lines over short periods of time (0-30 min) relative to the 2-3-h time frame required for commitment to proliferation. Therefore, we examined T cell mitogen-induced DAG and ceramide kinetics under physiologically relevant conditions during the initial 2 h of culture. Freshly isolated murine splenic lymphocytes were stimulated with the T cell-specific mitogen, concanavalin A (Con A). Our results show that Con A induced a multiphasic DAG response with significant peaks in DAG mass occurring at 2, 20 and 120 min. Concomitantly, ceramide mass was significantly increased 2 min following Con A addition and remained elevated until 120 min. Addition of C8-ceramide (10 microM) to lymphocyte cultures significantly enhanced mitogen-induced proliferation. These results demonstrate that DAG is continuously produced by activated T lymphocytes in a multiphasic fashion, and that ceramide is a positive effector molecule with respect to murine T cell proliferation. These results establish a foundation for further examination of the relationship between DAG, ceramide and T cell activation.

Evidence for preadipocyte proliferation during culture of subcutaneous and intramuscular adipose tissues from Angus and Wagyu crossbred steers.

The primary objective of this study was to provide evidence for preadipocyte proliferation during culture of adipose tissue explants; a secondary objective was to compare the lipogenic activity and cellularity of adipose tissues from American Wagyu crossbred steers. Subcutaneous (s.c.) and intramuscular (i.m.) adipose tissues were obtained at slaughter from the 2nd to 6th lumbar region of the loin from Angus (n = 10) and Wagyu crossbred steers (n = 10) that had been fed for 552 d by typical Japanese production standards. Adipose tissue explants were incubated 36 h with [3H]thymidine in the absence and presence of aphidicolin (a specific inhibitor of genomic DNA replication). Adipocytes were liberated by collagenase treatment and [3H]thymidine incorporation into DNA was measured. Whereas there were no significant differences between adipose tissue depots, Wagyu s.c. and i.m. preadipocytes and stromal-vascular cells exhibited greater (P < .05) [3H]thymidine incorporation into DNA than adipocytes from Angus steers. Intramuscular adipose tissue from both breeds exhibited lower (P < .05) rates of lipogenesis from acetate both before and after long-term (36-h) incubation than s.c. adipose tissue. Furthermore, i.m. adipocytes were smaller (P < .05) than s.c. adipocytes. The activities of fatty acid synthetase and glucose-6-phosphate dehydrogenase were greater (P < .05) in Wagyu s.c. adipose tissue and less in Wagyu i.m. adipose tissue than in corresponding Angus tissues. There were no differences between breed types (P = .17) in rates of lipogenesis from acetate, either before or after explant culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Periodic changes in body composition and in priorities for tissue storage and retrieval in mature beef cows.

Sixty mature (4 to 6 yr), nonpregnant, nonlactating beef cows of two breed types, Angus (n = 30) and Simmental (n = 30), were used in a four-period, split-plot study (371 d) to determine the seasonal differences in body composition. Periods were chosen to represent each of the four seasons in one year that ran consecutively from June 14, 1986 to June 20, 1987. Initially, all cows were assigned randomly within breed type to one of four feeding levels based on the cows' estimated maintenance requirement (75, 87.5, 112.5, and 125% of weight maintenance) and were rotated such that all animals were fed at all four feeding levels during the course of the study. Body composition was estimated via isotope dilution initially and at the end of Periods 1 through 3 and via derived regression relationships after Period 4. Body composition differed by breed; Simmental cows had greater (P less than .05) amounts of empty body protein (79.4 vs 55.8 kg, respectively) and less (P less than .05) empty body fat (85.3 vs 93.9 kg, respectively) than the Angus cows. These differences also were apparent when expressed as a percentage of live weight. Season altered components of the empty body. On the average, both breeds mobilized (P less than .05) empty body protein (-3.6 and -5.4 kg for Angus and Simmental cows, respectively) in the summer while simultaneously gaining (P less than .05) empty body fat (15.3 and 30.1 kg for Angus and Simmental cows, respectively). Both breeds subsequently tended to gain empty body protein in the winter and spring.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of season on the maintenance requirements of mature beef cows.

Sixty mature, nonpregnant, nonlactating beef cows of two breed types, Angus and Simmental, were used in a four-period, split-plot study to determine the effects of season on maintenance requirements. Periods were chosen to represent each of the four seasons and ran consecutively from June 14, 1986 through June 20, 1987. All cows were fed at one of four feeding levels (75, 87.5, 112.5, and 125% of estimated ME required for maintenance) a cottonseed hull-based diet in each of the four seasons and were rotated such that all cows received all feeding levels during the study. Retained energy for all cows within periods was determined through changes in body composition determined initially and at the end of each period. Mean DM digestibility (DMD) was similar for both breeds (54.4%), and in both breeds DMD was similar in the summer, fall, and spring and lower (P less than .05) in the winter. Mean DE was 61.4% and also was similar between breeds. Season affected DE, which was greatest (P less than .05) for both breeds in the fall (62.5%). Simmental cows had a 16.1% greater (P less than .05) overall daily ME requirement for weight maintenance than Angus cows (123.5 vs 103.6 kcal/kg.75), and both breeds had greater (P less than .05) requirements during the summer and lower (P less than .05) requirements during the winter (122.6 vs 91.4 and 145.9 vs 109.3 kcal/kg.75 for Angus and Simmental cows, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)