Differential effects of heparin on NO and tumor necrosis factor-alpha production in bovine blood mononuclear cells stimulated with Salmonella typhimurium lipopolisaccharide.

We investigated the influence of heparin, one of the extracellular matrix (ECM) components, on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by bovine peripheral blood mononuclear cells (PBMC) and monocytes left to adhere for 2 (freshly adherent monocytes) and 48 h (resting monocytes), activated with Salmonella typhimurium lipopolysaccharide (LPS). After 24-h stimulation with LPS, heparin (100 microg/ml) increased (by about 40%) NO production by peripheral blood mononuclear cells and by freshly adherent monocytes. However, it did not change NO synthesis by the resting monocytes. Unlike its influence on NO level, heparin diminished TNF-alpha production by PBMC and monocytes stimulated with LPS. Microscopical examination of PBMC stained with biotin-labeled heparin, showed that both lymphocytes and monocytes were able to bind this glycosaminoglycan. We suggest that heparin, as a component of ECM, modulates the early response of monocytes to exogenous stimuli.     

Haematological and febrile response to Escherichia coli lipopolysaccharide in 12‐week‐old cockerels of genetically diverse layer lines fed diets with increasing L‐arginine levels

Due to its decisive function in the avian metabolic, endocrine and immune system L‐arginine (Arg) is dietary indispensable for chickens. In 12‐week‐old cockerels of two high‐ and two low‐performing purebred layer lines, the effects of increasing dietary Arg on the haematological and febrile response were studied over 48 h after single lipopolysaccharide (LPS) injection. The offered diets contained Arg equivalent to 70%, 100% and 200% of recommended supply. Pathophysiological alterations in weight gain, feed intake, body temperature and differential blood count were examined in comparison with their physiological initial values. Within the first 24 h after LPS injection, cockerels reduced feed intake and lost body weight subsequently. Thereby, low‐performing genotypes lost body weight to a lesser extent than high‐performing ones. The loss of body weight was further intensified by deficient dietary Arg. Within the following 24 h, cockerels recovered by improving feed intake and weight gain. Furthermore, LPS induced genotype‐specific fever response: both brown genotypes showed initial hypothermia followed by longer lasting moderate hyperthermia, whereas the white genotypes exhibited biphasic hyperthermia. Fever response was accompanied by significant changes in differential blood counts. Characterized by lymphopenia and heterophilia, a severe leucopenia was observed from 4 to 8 h after LPS injection and replaced by a marked leucocytosis with longer lasting monocytosis up to 48 h after LPS injection. Under given pathophysiological conditions, deficiently Arg‐supplied cockerels showed higher total leucocyte counts than adequately and excessively Arg‐supplied cockerels. However, deficient and surplus dietary Arg tended to cause higher ratios between heterophils and lymphocytes. To conclude, present results confirmed that LPS induced numerous immunological changes in 12‐week‐old cockerels and emphasized that chicken's genotype is a source of variation to be considered for immunological studies. Deficient dietary Arg intensified acute changes in differential blood counts and weight gain during LPS‐induced inflammation.     

Heat-tolerant versus heat-sensitive Bos taurus cattle: influence of air temperature and breed on the acute phase response to a provocative immune challenge

The difference in the acute phase response of a heat-tolerant and a heat-sensitive Bos taurus breed to a lipopolysaccharide (LPS) challenge when housed at different air temperatures (T_a) was studied. Angus (ANG; heat-sensitive; n = 11; 306 ± 26 kg BW) and Romosinuano (RO; heat-tolerant; n = 10; 313 ± 32 kg BW) heifers were transported from the USDA Agricultural Research Service SubTropical Agricultural Research Station in Florida to the Brody Environmental Chambers at the University of Missouri, Columbia. Heifers were housed in stanchions in 4 temperature-controlled environmental chambers. Initially, T_a in the 4 chambers was cycling at thermoneutrality (TN; 18.5°C–23.5°C) for a 1-wk adjustment period, followed by an increase in 2 of the 4 chambers to cycling heat stress (HS; 24°C–38°C) for 2 wk. On day 19, heifers were fitted with jugular catheters and rectal temperature (RT) recording devices. On day 20, heifers were challenged with LPS (0.5 μg/kg BW; 0 h), sickness behavior scores (SBSs) were recorded, and blood samples were collected at 0.5-h intervals from −2 to 8 h and again at 24 h relative to LPS challenge at 0 h. Serum was isolated and stored at −80°C until analyzed for cortisol and cytokine concentrations. A breed by T_a interaction (P < 0.001) was observed for RT such that the post-LPS average RT in RO heifers housed at TN was lower than the RT of all other treatment groups (P < 0.001), whereas ANG heifers housed at HS had greater post-LPS average RT than all other treatment groups (P < 0.001). In response to LPS, HS increased SBS after LPS in RO heifers compared to RO heifers housed at TN (P < 0.001), whereas HS decreased SBS after LPS in ANG heifers compared to ANG heifers housed at TN (P = 0.014). The cortisol response to LPS was greater in TN than in HS heifers (P < 0.01) and was also greater in RO than in ANG heifers (P = 0.03). A breed by T_a interaction (P < 0.01) was observed for tumor necrosis factor-α (TNF-α) concentration such that HS increased post-LPS serum concentrations of TNF-α in ANG heifers compared to ANG heifers housed at TN (P = 0.041), whereas HS decreased post-LPS concentrations of TNF-α in RO heifers compared to RO heifers housed at TN (P = 0.008). A tendency (P < 0.06) was observed for a breed by T_a interaction for IL-6 concentrations such that RO heifers had greater post-LPS concentrations of IL-6 than ANG heifers when housed at HS (P = 0.020). A breed by T_a interaction was observed for interferon-γ (IFN-γ; P < 0.01) concentrations such that HS decreased post-LPS concentrations of IFN-γ in ANG heifers compared to ANG heifers housed at TN (P < 0.001), and HS increased post-LPS concentrations of IFN-γ in RO heifers compared to RO heifers housed at TN (P = 0.017). These data indicate differences in the acute phase response between the heat-tolerant RO and heat-sensitive ANG heifers under different T_a which may aid in elucidating differences in productivity, disease resistance, and longevity among cattle breeds.     

Interleukin-10 inhibits lipopolysaccharide-induced upregulation of tissue factor in canine peripheral blood monocytes

The potentially fatal hemostatic disorder of disseminated intravascular coagulation (DIC) is initiated in bacterial sepsis by lipopolysaccharide (LPS)-induced tissue factor (TF) expression on monocytes. Interleukin-10 (IL-10) is a potent inhibitory cytokine that downregulates monocyte inflammatory and procoagulant responses. We hypothesized that canine recombinant IL-10 (rIL-10) would inhibit LPS-induced TF upregulation on canine monocytes in a dose-dependent manner. Canine peripheral blood mononuclear cells (PBMC), obtained by double-density gradient centrifugation, and monocytes, purified from PBMC by immunomagnetic bead separation with an anti-canine CD14 antibody (Ab), were stimulated in suspension with LPS (0.1-1000ng/mL) for various times. Recombinant IL-10 (10-5000pg/mL) was added with LPS or up to 2h later. Tissue factor procoagulant activity was measured by cleavage of a chromogenic substrate by activated Factor X generated by the TF-factor VII complex. We found that rIL-10, when given concurrently or 1h after LPS, strongly inhibited LPS-induced TF procoagulant activity in canine PBMC and monocytes. This inhibition was dose-dependent and blocked by an anti-canine IL-10 Ab. Our results indicate that rIL-10 effectively inhibits LPS-induced TF upregulation in canine monocytes and could potentially be useful in limiting the development of DIC in dogs with endotoxemia.     

Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris

Florida manatees (Trichechus manatus latirostris) are exposed to many conditions in their habitat that may adversely impact health and impair immune function in this endangered species. In an effort to increase the current knowledge base regarding the manatee immune system, the production of an important reactive nitrogen intermediate, nitric oxide (NO), by manatee peripheral blood mononuclear cells (PBMC) was investigated. PBMC from healthy captive manatees were stimulated with LPS, IFN-gamma, or TNF-alpha, either alone or in various combinations, with NO production assessed after 24, 48, 72, and 96 h of culture. NO production in response to LPS stimulation was significantly greater after 48, 72, or 96 h of culture compared to NO production after 24h of culture. A specific inhibitor of inducible nitric oxide synthase (iNOS), L-NIL (L-N(6)-(1-iminoethyl)lysine), significantly decreased NO production by LPS-stimulated manatee PBMC. Manatee specific oligonucleotide primers for iNOS were designed to measure expression of relative amounts of mRNA in LPS-stimulated manatee PBMC from captive manatees. NO production by PBMC from manatees exposed to red tide toxins was analyzed, with significantly greater NO production by both unstimulated and LPS stimulated PBMC from red tide exposed compared with healthy captive or cold-stress manatees. Free-ranging manatees produced significantly lower amounts of nitric oxide compared to either captive or red tide rescued manatees. Results presented in this paper contribute to the current understanding of manatee immune function and represent the first report of nitric oxide production in the immune system of a marine mammal.     

Antipyretic effect of oral sodium salicylate after an intravenous E. coli LPS injection in broiler chickens

A study was set up to investigate the influence of sodium salicylate on fever and acute phase reaction after lipopolysaccharide (LPS) injection in broiler chickens. An acute phase reaction was provoked through the intravenous injection of Escherichia coli LPS. Four oral doses of sodium salicylate were tested. Apart from body temperature, other inflammation indices, such as plasma corticosterone and ceruloplasmin, serum thromboxane B2 and zinc concentrations were monitored. Intravenous LPS induced a fever of about 1 degree C. A dose-dependent attenuation of the fever response of the chickens in the salicylate treated groups was observed. LPS-injected chickens also showed elevated plasma corticosterone and ceruloplasmin, while serum thromboxane and zinc concentrations decreased. Except for thromboxane B2, no linear relationship with increasing salicylate dose could be shown for the other blood variables. These data confirm that sodium salicylate is an effective antipyretic agent after injection of LPS in chickens, if used at an appropriate dosage. No dose-related change could be found for the other inflammation indices.     

Acute phase response in lactating dairy cows during hyperinsulinemic hypoglycaemic and hyperinsulinemic euglycaemic clamps and after intramammary LPS challenge

The link between energy availability, turnover of energy substrates and the onset of inflammation in dairy cows is complex and poorly investigated. To clarify this, plasma inflammatory variables were measured in mid‐lactating dairy cows allocated to three groups: hyperinsulinemic hypoglycaemic clamp, induced by insulin infusion (HypoG, n = 5); hyperinsulinemic euglycaemic clamp, induced by insulin and glucose infusion (EuG; n = 6); control, receiving a saline solution infusion (NaCl; n = 6). At 48 h after the start of i.v. infusions, two udder quarters per cow were challenged with 200 μg of E. coli lipopolysaccharide (LPS). Individual blood samples were taken before clamps, before LPS challenge (i.e. 48 h after clamps) and 6.5 h after. At 48 h, positive acute phase proteins (posAPP) did not differ among groups, whereas albumin and cholesterol (index of lipoproteins), negative APP (negAPP), were lower (p < 0.05) in EuG compared to NaCl and HypoG. The concentration of IL‐6 was greater in EuG (p < 0.05) but only vs. HypoG. At 6.5 h following LPS challenge, IL‐6 increased in the NaCl and EuG clamps (p < 0.05), while TNF‐α increased (p < 0.05) in the EuG only. Among the posAPP, haptoglobin markedly increased in EuG (p < 0.05), but not in NaCl (p = 0.76) and in HypoG; ceruloplasmin tended to decline during LPS challenge, the reduction was significant when all animals were considered (p < 0.05). Conversely, all the negAPP showed a marked reduction 6.5 h after LPS challenge in the three groups. In conclusion, EuG caused an inflammatory status after 48‐h infusion (i.e. decrease of negAPP) and induced a quicker acute phase response (e.g. marked rise of TNF‐α, IL‐6) after the intramammary LPS challenge. These data suggest that the simultaneous high availability of glucose and insulin at the tissue‐level makes dairy cows more susceptible to inflammatory events. In contrast, HypoG seems to attenuate the inflammatory response.     

Toxicokinetics and absolute oral bioavailability of melamine in broiler chickens

The objective of this study was to investigate the toxicokinetic characteristics of melamine in broilers due to the limited information available for livestock. Melamine was then administered to broiler chickens at an intravenous (i.v.) or oral (p.o.) dosage of 5.5 mg/kg of body weight, and plasma samples were collected up to 48 h. The concentration of melamine in each plasma sample was analyzed using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). Melamine was measurable up to 24 h after i.v. and p.o. administration. A one‐compartment model was developed to describe the toxicokinetics of melamine in broilers. Following i.v. administration, the values for the elimination half‐life (t_1/2β), the volume of distribution (V_d), and the clearance (CL) were 4.42 ± 1.02 h, 00.52 ± 0.18 L/kg, and 0.08 ± 0.01 L/h/kg, respectively. The absolute oral bioavailability (F) was 95.63 ± 3.54%. The results suggest that most of the administered melamine is favorably absorbed from the alimentary tract and rapidly cleared by the kidneys in broiler chickens.     

不同应激处理对猪血细胞参数和急性期蛋白浓度的影响

本试验模拟养猪生产中存在的不同应激条件,探讨应激类型与猪群血液急性期蛋白(acute phase proteins,APPs)浓度的关系,为评价猪群健康状态提供试验依据。利用猪多杀性巴氏杆菌病活疫苗(Pasteurella multocida vaccine,PMV)、猪乙型脑炎活疫苗(swine Japanese enceph-alitis vaccine,SJEV)及细菌脂多糖(lipopolysaccharide,LPS)分别模拟细菌性感染、病毒性感染及炎症状况的应激状态,研究不同应激状态下猪的行为学、血细胞参数及APPs浓度的变化。结果表明,LPS处理后仔猪出现呕吐、发烧和呼吸急促等现象。PMV处理后48 h中性粒细胞(neutro-phil,NEUT)和单核细胞(monocyte,MO)数量显著升高(P<0.05),LPS处理后24 h NEUT数量和处理后48 h MO数量显著升高(P<0.05);SJEV处理后48 h的血红蛋白(hemoglobin,HGB)含量和处理后24 h的血小板(platelet,PLT)数量显著降低(P<0.05);LPS处理后24和48 h红细胞(red blood cell,RBC)数量、HGB含量和PLT数量均显著降低(P<0.05)。触珠蛋白(hapto-globin,HPT)浓度在PMV处理后48 h和SJEV处理后72 h显著升高(P<0.05);主要急性期蛋白(major acute phase protein,MAP)浓度在PMV处理后72 h以及LPS处理后24、48和72 h显著降低(P<0.05),SJEV处理后24 h显著升高(P<0.05);载脂蛋白-A1(apolipoprotein-A1,Apo-A1)浓度在PMV处理后72 h、SJEV处理后48 h,LPS处理后24、48和72 h显著降低(P<0.05)。结果提示,正相急性期蛋白HPT浓度在细菌和病毒感染模型下显著升高,负相急性期蛋白Apo-A1浓度在本试验的3种感染模型下均显著降低,这初步证明了血液HPT和Apo-A1浓度可以作为评价猪只健康的指标。     

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.     

Ethyl pyruvate decreases proinflammatory gene expression in lipopolysaccharide-stimulated equine monocytes

Monocytes are among the initial cells that interact with circulating LPS. Binding of LPS to monocyte surface receptors triggers an intracellular signaling cascade and results in the production of proinflammatory cytokines. Ethyl pyruvate, a stable derivative of pyruvate, has been effective in mitigating LPS induced alterations in isolated human monocytes. We hypothesized that ethyl pyruvate would suppress proinflammatory gene expression in LPS-stimulated equine monocytes without affecting cell viability. Equine monocytes were isolated from whole blood using a sediment-gradient centrifugation protocol and enriched to 76% purity by adhesion to tissue culture dishes. Isolated monocytes were incubated with 0, 1, 5, 10 and 50 mM ethyl pyruvate. Cell viability, production of caspase 3/7, and caspase-3 gene expression were determined. In a separate experiment, monocytes were stimulated with LPS (0.1 ng/ml for 1h) followed by incubation with 0, 1, 5, or 10 mM ethyl pyruvate for 1 h. Proinflammatory gene expression was determined by real-time PCR. Ethyl pyruvate at 50 mM adversely affected monocyte viability. Ethyl pyruvate at 10mM or less had no significant effect on monocyte viability, and did not increase activity of caspase 3/7 nor caspase-3 gene expression. Incubation with LPS alone induced a significant upregulation in proinflammatory gene expression. Subsequent treatment of monocytes with ethyl pyruvate significantly reduced IL-8 expression in LPS stimulated monocytes at 5 mM, and IL-8, TNF-α and COX-2 at 10 mM. No beneficial effect on expression of IL-1β or IL-6 was detected. Overall, 10 mM ethyl pyruvate did not adversely affect monocyte viability and suppressed LPS-induced proinflammatory gene expression. Ethyl pyruvate may be a beneficial anti-inflammatory therapy in equine endotoxemia.     

In vitro lipopolysaccharide treatment alters regulatory T cell properties in chickens

The objective of this study was to identify the effect of in vitro lipopolysaccharide (LPS) treatment on regulatory T cells (Tregs) from chickens. Tregs had approximately 30-fold higher TLR 2-type 2 and six-fold higher TLR 4 mRNA content than CD4+CD25- cells. Tregs were treated with either 0 or 1 μg/ml LPS for 0, 2, and 4d. LPS treatment increased the IL-2 mRNA amount in Tregs at 2 and 4d post-LPS treatment. LPS treatment increased the IL-10 mRNA amount in Tregs at 4d post-LPS treatment. The total live cell numbers were approximately two-fold higher at 2d and three-fold higher at 4d in the 1 μg/ml LPS-treated groups than in the 0 μg LPS-treated controls. LPS treatment abrogated suppressive properties of Tregs at 2d post-LPS treatment. At 4d post-LPS treatment, Tregs became supersuppressive. In conclusion, chicken Tregs are differentially activated to facilitate immune response during the early stage of inflammation and to facilitate immune suppression at a later stage of inflammation.     

Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1beta in monocyte culture, whereas T(H)1 cytokines (IL-2 and IFN-gamma) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-gamma and the chemokine MIP-1beta, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T(H)1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.     

Changes in plasma alpha 1-acid glycoprotein concentration and selected immune response in broiler chickens injected with Escherichia coli lipopolysaccharide

1. Changes in plasma alpha1-acid glycoprotein (AGP) concentration and immune responses following Escherichia coli lipopolysaccharide (LPS) injection were studied in broiler chickens. 2. Higher plasma AGP concentrations were observed from 12 to 48 h after a single injection of LPS. 3. The highest concentration of plasma AGP was observed on day 2 followed by a gradual decrease in chicks injected with 150 mug/kg body weight of LPS every day for 13 d. 4. Plasma AGP concentration in chicks injected daily with LPS at 900 mug/kg body weight for 13 d increased on day 2, and decreased on day 4 to the concentration found before the injection. The concentration increased again on day 10. 5. Changes in plasma interleukin-1 (IL-1) like activity were similar to those in plasma AGP concentration when LPS was injected daily at 900 mug/kg body weight for 3 d. 6. Responses of blood mononuclear cell (MNC) proliferation to mitogen or concanavalin A, (Con A), and pokeweed mitogen (PWM) were positively correlated with changes in plasma AGP concentration. 7. The results suggest that plasma AGP concentration could be used as a positive indicator of changes in blood MNC proliferation to a mitogen and in plasma IL-1 like activity.     

Confocal Imaging of Trans‐epithelial Trafficking by Immune and Umbilical Cord Stem Cells in the Neonatal Porcine Intestine

Maternal colostral leucocytes (CL) and peripheral blood mononuclear cells (PBMC) enter the neonatal circulation after ingestion in pigs and cattle. Porcine umbilical cord matrix stem cells (PUCs) are relatively non‐immunogenic after initial allogeneic transplantation. Using intestinal explant cultures incubated with labelled cells and confocal microscopy, we demonstrated trans‐epithelial trafficking of exogenous CL, PBMC and PUCs below the luminal surface after 72 h of culture. We orally administered PBMC and PUCs to pre‐colostral neonatal pigs and tracked their location 8 or 24 h later. Both PBMC and PUCs were found in the intestinal wall of all samples. Exposure to 25% of acellular colostrum had no detected effect on trafficking. Labelled PUCs and PBMC were detected on the surface of the epithelium and in the lamina propria 8 h post‐treatment and PBMC were also in the superficial submucosa. At 24 h, PUCs and PBMC were observed on the surface of the epithelium, in the lamina propria, superficial submucosa and deep submucosa. Our findings show the potential of PUCs for allogeneic engraftment in the neonatal intestine and may lead to cell‐based delivery of therapeutics.     

Induction of proinflammatory cytokines and caspase-1 by leptin in monocyte/macrophages from holstein cows

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.     

Goat cathelicidin‐2 is secreted by blood leukocytes regardless of lipopolysaccharide stimulation

It has been reported that goat cathelicidin‐2, an antimicrobial peptide, localizes in leukocytes and is present in milk. Here, we examined whether cathelicidin‐2 is secreted by leukocytes. Different concentrations (10⁵–10⁸ cells/mL) of blood leukocytes were cultured for 0–48 h with or without lipopolysaccharide (LPS). After culture, the concentrations of cathelicidin‐2 in the conditioned media were measured. Blood was collected from male goats 0–24 h after the intravenous injection of Escherichia coli O111:B4 LPS. The plasma cathelicidin‐2 concentrations were determined and the blood leukocytes immunostained with anti‐cathelicidin‐2 antibody to calculate the proportion of cathelicidin‐2‐positive cells in the total leukocytes. When higher concentrations of leukocytes were cultured, the cathelicidin‐2 concentrations in the media increased significantly, whereas the addition of LPS to the media caused no further increase. The plasma cathelicidin‐2 concentrations did not increase with time after LPS infusion. The proportion of cathelicidin‐2‐positive cells in the total leukocytes was significantly reduced 1 h after LPS injection compared with that at 0 h, but increased again at 6 h and thereafter. These results suggest that cathlicidin‐2 is secreted by leukocytes even without LPS stimulation, whereas LPS may be required for cathelicidin‐2‐containing leukocytes to be recruited from the blood to tissues showing inflammation.     

Influence of procyanidin supplementation on the immune responses of broilers challenged with lipopolysaccharide

In the present study, the effect of dietary procyanidin (PCA, from pine needles) supplementation on the innate immunity of broilers were investigated. The experiment was designed as a 2 × 4 factorial arrangement (eight cages / treatment; six birds (one‐day‐old) / cage) with dietary PCA concentrations (0, 0.05, 0.075 and 0.1%) and two immune treatments (injection of lipopolysaccharide (LPS) (0.5 mg/kg body weight) or saline). LPS was dissolved in sterile 9 g/L (w/v) NaCl solution at 16, 18, 20 days of age to mimic immune stress. The remaining birds were injected with saline as a placebo. The results indicated that, prior to LPS challenge, the PCA diet had no significant effect on bird growth performance. The injection of LPS was also not associated with any significant changes in poultry performance. LPS injection increased the activity of nitrogen oxides (NOx) and the concentrations of inflammatory cytokines (interferon‐γ (IFN‐γ), interleukin‐1β (IL‐1β), IL‐2, IL‐4, IL‐6 and IL‐10) in serum; dietary PCA decreased these concentrations (P < 0.05) in the PCA 0.1% group, further illustrating the immune effect of PCA. In conclusion, PCA supplementation has a beneficial effect on LPS challenge, which may be associated with the inhibition of the secretion of cytokines and decrease in the proinflammatory marker NOx.     

Glucose transport and milk secretion during manipulated plasma insulin and glucose concentrations and during LPS‐induced mastitis in dairy cows

In dairy cows, glucose is essential as energy source and substrate for milk constituents. The objective of this study was to investigate effects of long‐term manipulated glucose and insulin concentrations in combination with a LPS‐induced mastitis on mRNA abundance of glucose transporters and factors involved in milk composition. Focusing on direct effects of insulin and glucose without influence of periparturient endocrine adaptations, 18 dairy cows (28 ± 6 weeks of lactation) were randomly assigned to one of three infusion treatments for 56 h (six animals each). Treatments included a hyperinsulinemic hypoglycaemic clamp (HypoG), a hyperinsulinemic euglycaemic clamp (EuG) and a control group (NaCl). After 48 h of infusions, an intramammary challenge with LPS from E. coli was performed and infusions continued for additional 8 h. Mammary gland biopsies were taken before, at 48 (before LPS challenge) and at 56 h (after LPS challenge) of infusion, and mRNA abundance of genes involved in mammary gland metabolism was measured by RT‐qPCR. During the 48 h of infusions, mRNA abundance of glucose transporters GLUT1, 3, 4, 8, 12, SGLT1, 2) was not affected in HypoG, while they were downregulated in EuG. The mRNA abundance of alpha‐lactalbumin, insulin‐induced gene 1, κ‐casein and acetyl‐CoA carboxylase was downregulated in HypoG, but not affected in EuG. Contrary during the intramammary LPS challenge, most of the glucose transporters were downregulated in NaCl and HypoG, but not in EuG. The mRNA abundance of glucose transporters in the mammary gland seems not to be affected by a shortage of glucose, while enzymes and milk constituents directly depending on glucose as a substrate are immediately downregulated. During LPS‐induced mastitis in combination with hypoglycaemia, mammary gland metabolism was more aligned to save glucose for the immune system compared to a situation without limited glucose availability during EuG.     

Bacillus subtilis-based direct-fed microbials augment macrophage function in broiler chickens

The present study was conducted to evaluate the function of Bacillus subtilis-based direct-fed microbials (DFMs) on macrophage functions, i.e., nitric oxide (NO) production and phagocytosis in broiler chickens. DFMs used in this study were eight single strains designated as Bs2084, LSSAO1, 3AP4, Bs18, 15AP4, 22CP1, Bs27, and Bs278, and one multiple strain DFM product (Avicorr™) containing equal amount of Bs2084, LSSAO1 and 15AP4. NO concentrations were monitored in plasma and in the supernatants from the peripheral blood-derived monocytic cells (PBMC)-derived macrophages stimulated by either chicken recombinant interferon gamma (IFNγ) or lipopolysaccharide (LPS) from Escherichia coli or Salmonella typhi. In addition, phagocytosis of fluorescent beads and green fluorescent protein (GFP)-labeled Salmonella by PBMC-derived macrophage was assayed. Plasma NO levels were significantly higher in groups given 3AP4 or Bs27 diets compared with the control group at days 7 and 14. NO production by PBMC-derived macrophages stimulated with IFNγ or LPS was apparent, although the effect was strain-dependent. Phagocytosis of fluorescent beads or GFP-labeled Salmonella by macrophages was augmented in groups on DFM-supplemented diets compared with those fed the control diet. This study describes the immunomodulatory effects of Bacillus-based DFMs on innate immunity in broiler chickens.