Amine modulation of the neurogenic \u3cem\u3eLimulus\u3c/em\u3e heart

(1) The biogenic amines octopamine (OCT), dopamine (DA), epinephrine (E), and norepinephrine (NE) cause dose-dependent increases in both the rate and amplitude of contractions of the isolated Limulus heart-cardiac ganglion. Their relative ability to produce this excitation is OCT \u3e DA ∼ E \u3e NE. (2) The excitatory effects of all these amines are antagonized by the α-adrenergic blocker phentolamine and the dopaminergic antagonist haloperidol. The β-adrenergic antagonist dichloroisoproterenol slightly reduces amine excitation, but is also a partial agonist. The β-adrenergic antagonist propanolol, the α-blocker phenoxybenzamine, and the serotonin antagonist metergoline are ineffective. (3) In addition to their excitatory effects, DA and, to a lesser extent, NE initially reduce contraction rate and amplitude. (4) The transient inhibition is eliminated selectively by metergoline and is unaffected by the other antagonists. (5) The amines all increase the frequency of cardiac ganglion electrical bursting activity, whether ganglia are isolated or attached to cardiac muscle. Dopamine and NE also transiently inhibit the cardiac ganglion. (6) The amines do not alter myocardial resting tension, contractility, or membrane potential. (7) These amines appear to exert their modulatory effects on Limulus heart by altering the properties of the neurons which comprise its cardiac ganglion

Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-α\alpha , IL-1β\beta , IL-12 and IFN-γ\gamma

After intramammary infection, polymorphonuclear neutrophil leukocytes (PMN) are the first cells recruited into the mammary gland. Rapid recruitment of and bacterial phagocytosis and killing by PMN are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innate immune response. We sought to determine whether bovine PMN produce cytokines in response to stimulation by lipopolysaccharide (LPS). To investigate the effects of LPS on the expression of cytokines secreted by bovine PMN, we measured the expression of tumor necrosis factor (TNF)-$\alpha$, interleukin (IL)-1$\beta$, IL-12, and interferon (IFN)-$\gamma$ by ELISA after stimulation with different concentrations of LPS, and secretion of IL-8 after co-stimulation with LPS and either TNF-$\alpha$ or IL-1$\beta$. Bovine PMN were shown to secrete TNF-$\alpha$, IL-1$\beta$, IL-12, IL-8 and IFN-$\gamma$ in response to LPS. Co-incubation of PMN with LPS and TNF-$\alpha$ increased secretion of IL-8 when compared to LPS alone. It was concluded that LPS stimulation up-regulates the secretion of cytokines by bovine PMN, and that co-incubation of LPS with TNF-$\alpha$ had an additive effect on the secretion of IL-8. These data show that bovine PMN, in addition to their phagocytic and bactericidal properties, may play a supportive role in the innate immune response to infection by Gram-negative bacteria through their ability to produce immuno-regulating cytokines

The production and characterization of anti-bovine CD14 monoclonal antibodies

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 µg) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-$\alpha$. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis

Shedding of sCD14 by bovine neutrophils following activation with bacterial lipopolysaccharide results in down-regulation of IL-8

CD14, the leukocyte co-receptor for lipopolysaccharide (LPS), is important in the response of bovine polymorphonuclear neutrophil leukocytes (PMN) to Gram-negative bacteria. In other species, the expression of CD14 on the surface of PMN was shown to increase after exposure to inflammatory stimuli. These newly expressed molecules may originate from either an intracellular pool or through new gene expression. We sought to characterize bovine PMN cell surface expression and shedding of CD14 molecules, and CD14's effect on secretion of the chemoattractants IL-8 and IL-1$\beta$ by PMN. Bovine PMN were incubated in RPMI for 20 h at 37 $^{\circ}$C with LPS (1, 10, 100 $\mu$g/mL). IL-8 release increased with treatment of 1 $\mu$g/mL LPS, but decreased 41.5 and 95% at the 10 and 100 $\mu$g/mL concentrations of LPS, respectively. In contrast, shedding of CD14 from the surface of PMN only increased at the highest concentration of LPS (100 $\mu$g/mL). Secretion of IL-1$\beta$ was similar regardless of the LPS concentration used to stimulate PMN. The effect of PMN concentration ($1 \times 10^{7}$, $2.5\times 10^{7}$, $5\times 10^{7}$, and $10\times 10^{7}$/mL) on CD14 cell surface expression and shedding of IL-8 and IL-1$\beta$ were also determined. Shedding of CD14 by PMN increased with increasing concentration of PMN after exposure to 0.1 and 10 $\mu$g/mL of LPS, while secretion of IL-8 decreased. IL-1$\beta$ increased at the highest concentration of PMN. The use of real time polymerase chain reaction showed that CD14 mRNA expression was not different between control and LPS-stimulated cells, indicating that the sCD14 came from either membrane bound CD14 or a preformed pool. Our results demonstrate that release of CD14 from PMN suppresses secretion of IL-8, and may be an important regulatory mechanism for controlling excessive migration of PMN into the bovine mammary gland

Characterization of monoclonal antibodies that recognize the Eimeria tenella Microneme Protein MIC2

The apicomplexan pathogens of Eimeria tenella cause coccidiosis, an intestinal disease of chickens that has a major economic impact on the poultry industry. Members of Apicomplexa share an assortment of unique secretory organelles (rhoptries, micronemes, and dense granules) that mediate invasion of host cells and the formation and modification of the parasitophorous vacuole. Among these, microneme protein 2 from E. tenella (EtMIC2) has a putative function in parasite adhesion to the host cell to initiate the invasion process. To investigate the role of EtMIC2 in host parasite interactions, the production and characterization of 12 monoclonal antibodies (mabs) produced\ud against recombinant EtMIC2 proteins is described. In an immunofluorescence assay, all mabs reacted with molecules belonging to the apical complex of sporozoites and merozoites of E. tenella, E. acervulina, and E. maxima. By Western blot analysis, the mabs identified a developmentally\ud regulated protein of 42 kDa corresponding to EtMIC 2 and\ud cross-reacted with proteins in developmental stages of E. acervulina. Collectively, these mabs are useful tools for the detailed investigation of the characterization of EtMIC2-related proteins in Eimeria species