Effect of intravenous platelet-activating factor on bovine pulmonary mast cells.

The effect of platelet-activating factor (PAF) on bovine pulmonary mast cells (MCs) was investigated in calves aged 1-4 months. PAF was administered over a 5-min period at a rate of 50 ng/kg/min to six animals, which were killed approximately 10 min later. No macroscopical lesions were observed in the lung tissue of five control calves. On semi-thin toluidine-blue stained sections, MC cytoplasm showed profuse deep-blue granulation. In the lung tissues of PAF-treated calves, striking interstitial, alveolar and interlobular oedema and emphysema were observed. MC granules occurred at the periphery of the cytoplasm and MC nuclei were easily detectable. Moreover, the number of granules in the MC cytoplasm was remarkably low. A few granules with some metachromatic material were sometimes detected outside the cell limits. Ultrastructurally, the matrix pattern of the MC granules in lung tissues of control calves was either amorphous or granular. In lung tissues of the PAF-treated calves, the granular matrix pattern was pronounced, varying from fine- to coarse-grained, and the MCs were of reduced size, with many filiform processes and exocytotic vesicles. These findings indicate that pulmonary MC degranulation was induced by PAF in healthy calves.

Cytotoxicity of stimulated equine neutrophils on equine endothelial cells in culture.

We studied the interactions of isolated equine neutrophils with endothelial cells in culture, mimicking a situation of acute inflammation. Our main purpose was to demonstrate that the supernatant of activated neutrophils was sufficient to damage endothelial cells. Equine endothelial cells (from carotid arteries) were covered either with increased numbers of equine neutrophils stimulated by phorbol myristate acetate, or with the supernatant collected after an in vitro stimulation of the neutrophils. Cytotoxicity was estimated by the release of preincorporated 51Cr, and by light microscopy observations. To assert the specific role of reactive oxygen species, endothelial cells were treated by the hypoxanthine/xanthine oxidase (X/XOx) system (production of superoxide anion and hydrogen peroxide), and by hypochlorite (product of the activity of myeloperoxidase). A strong cytotoxicity was found with stimulated neutrophils; microscopic observations indicated a loss of 50% of the endothelial cells and morphological alterations in the remaining cells. The supernatant of stimulated neutrophils was cytotoxic, in correlation with the number of neutrophils used to obtain the supernatant, and with the supernatant concentration of myeloperoxidase. The cytotoxicity of the X/XOx system was weak, but was increased by myeloperoxidase. Hypochlorite was highly toxic. We concluded that the supernatant of stimulated neutrophils was sufficient to obtain cytotoxic effects on the endothelium, in the absence of a direct contact between endothelium and neutrophils, and that this cytotoxicity was mainly linked to the activity of myeloperoxidase. From these in vitro results, it can be extrapolated that in pathologies characterised by an important activation of neutrophils, damage can spread to cells and tissues away from the inflammation focus.

Inflammatory effect of intratracheal instillation of ultrafine particles in the rabbit: role of C-fiber and mast cells.

The effects of ultrafine polystyrene carboxylate-modified (fluorospheres) on inflammatory processes are being investigated in rabbit lungs. One milliliter of sterile NaCl (0.9%) containing 4 mg of ultrafine particles (UFP) was intratracheally instilled into anesthetized rabbits. The control animals were only instilled with sterile NaCl (0.9%). Twenty hours after being instilled, the rabbits were killed and their lungs were excised and then tracheally perfused with phosphate-buffered physiological solution (PBS). The lung effluents, collected from small holes made in the pleura, were analyzed for substance P (SP) and histamine content by radioimmunoassay (RIA) methods, after administration of drugs. In addition, in other groups of rabbits, the lung wet/dry (W/D) weight ratio was monitored, as were the cellular and protein contents in bronchoalveolar lavage (BAL). Electron microscopy examination was also performed. In tracheally superfused experiments, UFP induced a significant enhancement of both SP and histamine releases after administration of capsaicin (10(-4) M), to stimulate C-fiber, and carbachol (10(-4) M), a cholinergic agonist. A significant increase in histamine release was also recorded in the UFP-instilled group following the administration of both SP (10(-6) M) plus thiorphan (10(-5) M) and compound 48/80 (C48/80) (10(-3) M) to stimulate mast cells. In addition, the BAL fluid analysis of UFP groups showed an influx of neutrophils and an increase in total protein concentration. An increase in the lung WW/DW ratio was also recorded. Both epithelial and endothelial injuries were observed in the lungs of UFP-instilled rabbits. The pretreatment of rabbits in vivo with a mixture of either SR 140333 and SR 48368, a tachykinin NK(1) and NK(2) receptor antagonist, or a mixture of terfenadine and cimetidine, a histamine H(1) and H(2) receptor antagonist, prevented UFP- induced neutrophil influx and increased total proteins and lung WW/DW ratio. Therefore, it can be concluded that chemicaly inert, electrically charged UFP induce a pulmonary inflammatory process during which the release of SP and histamine from C-fibers and mast cells was enhanced after various stimuli. These latter mediators can also modulate the inflammatory process.

Morphometric study of the equine navicular bone: age-related changes and influence of exercise.

Navicular bones from the four limbs of 95 horses, classified in 9 categories, were studied. The effects of age on navicular bone morphometry and histomorphometry were estimated, after adjustment of the data to even out the effects of front and rear limbs, morphometrical type, sex, weight, and size. All the external measurements of the navicular bone decreased significantly with increasing age. From the histomorphometrical data, cortical bone volume decreased with age in most horses, whereas cancellous bone volume and, in particular, the marrow spaces increased. The increase in the cancellous bone volume could have resulted from tunnelling of the internal part of the cortex, which converted it progressively into a porous trabecular-like structure. Trabecular bone volume also decreased with age and the trabecular lattice changed dramatically to become disconnected in aged horses. These observations corresponded closely to those reported for ageing of the skeletal system in humans. However, in sporting horses, the navicular cortical bone volume increased with age and the cancellous bone volume decreased. Exercise appeared to have decreased bone resorption and increased bone formation at the endocortical junction. The cancellous bone architecture was also improved. in that the trabecular lattice and trabecular bone volume remained unchanged in aged sporting horses. Our findings confirmed that exercise may be good practice to prevent age-related bone loss.

Attachment but not penetration of bovine herpesvirus 1 is necessary to induce apoptosis in target cells.

Bovine herpesvirus 1 (BHV-1) induces apoptotic cell death in bovine peripheral blood mononuclear cells and B-lymphoma cells. Using a BHV-1 glycoprotein H null mutant, we have demonstrated that although penetration of BHV-1 is not required, attachment of BHV-1 viral particles is essential for the induction of apoptosis.

Morphometric study of the equine navicular bone: variations with breeds and types of horse and influence of exercise.

Navicular bones from the 4 limbs of 95 horses, classified in 9 categories, were studied. The anatomical bases were established for the morphometry of the navicular bone and its variations according to the category of horse, after corrections were made for front or rear limb, sex, weight, size and age. In ponies, navicular bone measurements were smallest for light ponies and regularly increased with body size, but in horses, navicular bone dimensions were smallest for the athletic halfbred, intermediate for draft horse, thoroughbreds and sedentary halfbreds and largest for heavy halfbreds. The athletic halfbred thus showed reduced bone dimensions when compared with other horse types. Navicular bones from 61 horses were studied histomorphometrically. Light horses and ponies possessed larger amounts of cancellous bone and less cortical bone. Draft horses and heavy ponies showed marked thickening of cortical bone with minimum intracortical porosity, and a decrease in marrow spaces associated with more trabecular bone. Two distinct zones were observed for the flexor surface cortex: an external zone composed mainly of poorly remodelled lamellar bone, disposed in a distoproximal oblique direction, and an internal zone composed mainly of secondary bone, with a lateromedial direction for haversian canals. Flexor cortex external zone tended to be smaller for heavy ponies than for the light ponies. It was the opposite for horses, with the largest amount of external zone registered for draft horses. In athletic horses, we observed an increase in the amount of cortical bone at the expense of cancellous bone which could be the result of reduced resorption and increased formation at the corticoendosteal junction. Cancellous bone was reduced for the athletic horses but the number of trabeculae and their specific surfaces were larger. Increased bone formation and reduced resorption could also account for these differences.

Ozone-induced stimulation of pulmonary sympathetic fibers: a protective mechanism against edema.

Tropospheric ozone exerts well-described toxic effects on the respiratory tract. Less documented, by contrast, is the ability of ozone to induce protective mechanisms against agents that are toxic to the lungs. In particular, interactions between ozone and the sympathetic nervous system have never been considered. Using a model of permeability edema in isolated perfused rabbit lungs, we report here that, immediately after exposure of rabbits to 0.4 ppm ozone for 4 hr, the pulmonary microvascular responses to acetylcholine and substance P are completely blocked. Several lines of evidence, including partial inhibition of the ozone-induced protective effect by several drugs (alpha2- and beta-adrenergic antagonists, neuropeptide Y antagonist, guanethidine), measured levels of released catecholamines in blood and urine and the in vitro response of isolated lungs exposed to 0.4 ppm ozone all seem to suggest that ozone can stimulate pulmonary adrenergic fibers and induce the local release of catecholamines and neuropeptide Y, this resulting in transient protection against pulmonary edema. We also showed that, 48 hr after the exposure, ozone increased the baseline microvascular permeability and the response to low concentrations of acetylcholine.

Morphometric study of the equine navicular bone: comparisons between fore and rear limbs.

Navicular bones collected from the four limbs of 95 sound horses were studied. The anatomic bases have been laid down about morphometry of the navicular bones and their variations according to limbs, after corrections have been made for morphologic type, gender, weight, size and age. All the dimensions of the navicular bone (except for the thickness) were larger in the fore limb. This phenomenon probably reflects an attempt to compensate for the greater forces exerted upon the fore limbs during exercise and at rest. Navicular bones collected from the four limbs of 61 sound horses were studied and the anatomic bases were described for histomorphometry of the fore and rear navicular bones. Fore navicular bones possess less cortical bone at the level of the articular surface, as well as at the level of the flexor surface and proximal border, but larger amounts of cancellous bone. Articular and flexor surface cortical bone show a larger porosity in the fore navicular bones and a larger amount of mineralized cartilage. The mineralized portion for distal impar- and collateral sesamoidean ligaments are also larger for the fore navicular bones. Two distinct zones are observed for the flexor surface cortex that have never been reported in the literature before: an external zone, which is mainly composed of poorly remodelled lamellar bone, arranged in a disto-proximal oblique direction, and an internal zone, which is mainly composed of secondary bone, with a latero-medial direction of Haversian canals. Bone architecture is discussed with regard to the mechanic load, encountered by the bone during locomotion.

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions.

We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.

Modulatory effect of neuropeptide Y on acetylcholine-induced oedema and vasoconstriction in isolated perfused lungs of rabbit.

1. The modulatory role of neuropeptide Y (NPY) on pulmonary oedema induced by acetylcholine and capsaicin was investigated. The effects of NPY on the haemodynamic response to acetylcholine, phenylephrine and substance P were also investigated. 2. Isolated, ventilated, exsanguinated lungs of the rabbit were perfused with a constant flow of recirculating blood-free perfusate. The double/arterial/venous occlusion method was used to partition the total pressure gradient (delta Pt) into four components: the arterial gradient (delta Pa), the pre- and post-capillary gradients (respectively delta Pa' and delta Pv') and the venous pressure gradient (delta Pv). Endothelial permeability was evaluated by measuring the capillary filtration coefficient (Kf,c). 3. Acetylcholine (10(-8) M to 10(-4) M) and substance P (SP, 10(-10) M to 10(-6) M) induced a concentration-dependent increase in the Kf,c. Capsaicin (10(-4) M) and 5-hydroxytryptamine (5-HT) (10(-4) M) also increased this parameter. NPY (10(-8) M) completely inhibited the effects of acetylcholine and capsaicin on the Kf,c, without preventing the effects of substance P and 5-HT. 4. Acetylcholine induced concentration-dependent vasoconstriction in the precapillary segment. The effect was inhibited by NPY and aspirin, an inhibitor of cyclo-oxygenase, while ketanserin, a 5-HT2 receptor antagonist, and SR140333, a new NK1 antagonist, had no protective effect. Phenylephrine increased delta Pa at high concentration, an effect also inhibited by NPY and aspirin. Substance P had no significant haemodynamic effect. When injected together with NPY, substance P (10(-6) M) induced a significant increase in the total pressure gradient. 5. It was concluded that NPY can protect the lung against acetylcholine- and capsaicin-induced oedemavia a prejunctional modulatory effect on the C-fibres. NPY also inhibits acetylcholine-evoked precapillary and phenylephrine-induced arterial vasoconstriction, probably by interfering with cyclo-oxygenase products synthesis.

Transgenic mice harboring Aujeszky's disease virus gD gene.

Plasmid pMLP10 gp50.5 (gD) (Eloit et al., 1990) provided the sequence to integrate. The construct has been injected into the male pronucleus of one-cell mouse embryos (CBA/C5B1). A total of 1,567 microinjected embryos were transferred into the oviduct of pseudopregnant females (Hogan et al., 1986). PCR revealed that 3 out of 10 mice were born with an integrated construct; among them, 2 mice have integrated the construct in their gonads because 20 out of 107 (18.7%) of their offspring were also transgenic. This low percentage could be explained by germline mosaicism and/or differential mortality between transgenic and non-transgenic embryos.

Immunocytochemical study of cell type distribution in the pituitary of Barbus barbus (Teleostei, Cyprinidae).

Antisera to mammalian pituitary and placental hormones have been used to identify and localize the different cell types in the pituitary of the barbel (Barbus barbus, L.). The immunocytochemical labeling employed the immunoperoxidase technique or the immunogold silver staining procedure. Corticotrophic and prolactin cells, visualized using antisera to human adrenocorticotropic hormone and ovine prolactin (PRL), respectively, occur in the rostral pars distalis (RPD). Antisera against mammalian gonadotropins [ovine follicle-stimulating hormone (FSH); bovine luteinizing hormone] or porcine growth hormone selectively cross-react with two different cell populations occupying the major part of the proximal pars distalis (PPD). Thyrotropic cells, stained by an antiserum to whole human thyroid-stimulating hormone preabsorbed with porcine FSH, are scattered throughout the PPD and found amongst growth hormone and gonadotrophic cells. The majority of pars intermedia cells are stained with anti-melanophore stimulating hormone whereas the scattered PAS positive cells are revealed by both anti-ovine PRL and anti-bovine placental lactogen (or chorionic somatomammotropin). The latter antiserum also cross-reacts with the PRL cells of the RPD. Our results indicate that the distribution of the different cell types in Barbus barbus is similar to that described in other families of teleosts. This report is also the first demonstration of antigenic similarity between mammalian placental lactogen and fish prolactin.