Abundant molecular oxygen in the coma of comet 67P/Churyumov-Gerasimenko.

The composition of the neutral gas comas of most comets is dominated by H2O, CO and CO2, typically comprising as much as 95 per cent of the total gas density. In addition, cometary comas have been found to contain a rich array of other molecules, including sulfuric compounds and complex hydrocarbons. Molecular oxygen (O2), however, despite its detection on other icy bodies such as the moons of Jupiter and Saturn, has remained undetected in cometary comas. Here we report in situ measurement of O2 in the coma of comet 67P/Churyumov-Gerasimenko, with local abundances ranging from one per cent to ten per cent relative to H2O and with a mean value of 3.80 ± 0.85 per cent. Our observations indicate that the O2/H2O ratio is isotropic in the coma and does not change systematically with heliocentric distance. This suggests that primordial O2 was incorporated into the nucleus during the comet's formation, which is unexpected given the low upper limits from remote sensing observations. Current Solar System formation models do not predict conditions that would allow this to occur.

The loss of ions from Venus through the plasma wake.

Venus, unlike Earth, is an extremely dry planet although both began with similar masses, distances from the Sun, and presumably water inventories. The high deuterium-to-hydrogen ratio in the venusian atmosphere relative to Earth's also indicates that the atmosphere has undergone significantly different evolution over the age of the Solar System. Present-day thermal escape is low for all atmospheric species. However, hydrogen can escape by means of collisions with hot atoms from ionospheric photochemistry, and although the bulk of O and O2 are gravitationally bound, heavy ions have been observed to escape through interaction with the solar wind. Nevertheless, their relative rates of escape, spatial distribution, and composition could not be determined from these previous measurements. Here we report Venus Express measurements showing that the dominant escaping ions are O+, He+ and H+. The escaping ions leave Venus through the plasma sheet (a central portion of the plasma wake) and in a boundary layer of the induced magnetosphere. The escape rate ratios are Q(H+)/Q(O+) = 1.9; Q(He+)/Q(O+) = 0.07. The first of these implies that the escape of H+ and O+, together with the estimated escape of neutral hydrogen and oxygen, currently takes place near the stoichometric ratio corresponding to water.

Ion composition results during the international cometary explorer encounter with giacobini-zinner.

The International Cometary Explorer spacecraft passed through the coma of comet Giacobini-Zinner about 7800 kilometers antisunward of the nucleus on 11 September 1985. The ion composition instrument was sensitive to ambient ions with mass-to-charge ratios in the ranges 1.4 to 3 atomic mass units per electron charge (amu e(-1)) and 14 to 33 amu e(-1). Initial interpretation of the measurements indicates the presence of H(2)O(+), H(3)O(+), probably CO(+) and HCO(+), and ions in the mass range 23 to 24; possible candidates are Na(+) and Mg(+). In addition to these heavy ions, measured over the velocity range 80 to 223 kilometers per second, the instrument measured He(2+) of solar wind origin over the range 237 to 463 kilometers per second. The heavy ions have a velocity distribution which indicates that they have been picked up by the motional electric field, whereas the light ions are steadily decelerated as the comet tail axis is approached. These results are in agreement with the picture of a comet primarily consisting of water ice, together with other material, that sublimes, streams away from the nucleus, becomes ionized, and interacts with the solar wind. K. W. Ogilvie, NASA/Goddard Space Flight Center, Code 692, Greenbelt, MD 20771.

Induction of prostaglandin G/H synthase-2 in a canine model of spontaneous prostatic adenocarcinoma.

BACKGROUND: Prostate cancer is the most frequently occurring cancer in men in the United States, with an estimated 179 300 new cases in 1999. The induction of prostaglandin G/H synthase (PGHS), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in various cancers, most notably in colorectal cancers; however, the induction of PGHS expression in prostate cancer in vivo has not been reported for any species. The dog is the only nonhuman species that frequently develops spontaneous cancer of the prostate with increasing age, and the objective of this study was to determine whether PGHS isoenzymes were expressed in canine prostatic adenocarcinomas. METHODS: Four normal canine prostatic tissues and 24 canine prostatic adenocarcinomas were studied by means of immunohistochemistry and immunoblot analysis, using polyclonal antibodies specific for each of the two PGHS isoenzymes, PGHS-1 and PGHS-2. All P values were obtained by use of two-sided Fisher's exact tests. RESULTS: PGHS-1 immunostaining was localized to stromal fibroblasts and vascular endothelium in normal and cancerous prostates. PGHS-2 was not detected in normal prostates, but it was expressed by epithelial tumor cells in 18 (75%) of the 24 adenocarcinomas (P =.01). Immunoblot analysis confirmed the presence of PGHS-1 (69 000 molecular weight) in normal and cancerous tissues and the expression of PGHS-2 (72 000- to 74 000-molecular-weight doublet) only in prostatic adenocarcinomas. CONCLUSION: To our knowledge, these results demonstrate for the first time that PGHS-2 is induced in the majority of canine spontaneous prostatic adenocarcinomas and suggest that its expression may be involved in prostate cancer.

Identification and characterization of a bovine lipopolysaccharide-binding protein.

Endogenous regulatory mechanisms exist in mammals that enable a rapid response to lipopolysaccharide (LPS, endotoxin) stemming from gram-negative bacterial infections. Serum proteins and cell surface receptors exist that bind LPS, and this interaction may either aid in nonpathogenic removal of LPS from the body or potentiate the effects of LPS. We have used a photoreactive, thiol-cleavable, radiolabeled derivative of E. coli 0111:B4 LPS [LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide; 125I-ASD-LPS], to identify the presence of LPS-binding proteins (LBPs) in bovine serum. Ion exchange chromatography was used to fractionate bovine serum, and eluted protein was subsequently photoaffinity labeled using 125I-ASD-LPS. LBPs were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several LBPs including three with apparent molecular masses of 65, 60, and 50 kDa were variably present within the chromatography pools. A 22-residue NH2-terminal amino acid sequence of the 60-kDa protein showed 77% homology with human LBP and 68% with rabbit LBP within this region. Further purification utilizing high-performance liquid chromatography yielded a protein fraction that contained the 60-kDa protein and was distinctly more active than whole bovine serum in LPS-dependent macrophage activation assays (up to 1600-fold on a weight/volume basis). The LPS-mediated macrophage activation in concert with chromatographically purified serum protein in tissue factor assays was inhibitable using anti-CD14 monoclonal antibodies. The results indicate that an LPS-binding protein exists in samples of pooled bovine serum and that this protein has features in common with human and rabbit LBP.

Transendothelial migration of neonatal and adult bovine neutrophils in vitro.

We have compared and quantitated transendothelial migration of neonatal neutrophils (N-PMNs) and adult bovine peripheral-blood PMNs (A-PMNs) in vitro using monolayers of endothelium and a two-chamber apparatus. Bovine aortic endothelial cells were cultured to confluence on polycarbonate filters perforated with 3.0-micron-diameter pores. 51Cr-labeled PMNs were added to the upper chamber, with or without an anti-CD18 antibody (monoclonal antibody 60.3). Chemotactic stimuli in the lower chambers included recombinant human interleukin-8 (rhIL-8; 75 ng/ml), rhC5a (10(-7) M), and zymosan-activated bovine serum (ZAS; 10%). At 60 min incubation with rhIL-8, greater numbers (P < .01) of N-PMNs (24.70 +/- 5.95%) than of A-PMNs (15.77 +/- 3.66%) had migrated across the endothelial barrier, and a similar difference was present at 90 min. Migration rates of N-PMNs and A-PMNs were similar (P > .05) at all time points when using rhC5a and ZAS as stimuli. Anti-CD18 monoclonal antibody significantly decreased migration (P < .01) of both N-PMNs and A-PMNs to low levels when IL-8 and ZAS were used as stimuli. Because leukocyte integrin expression on PMNs affects transendothelial migration, we also compared surface expression of CD18, CD11a, and CD11c on PMNs from the two age groups. We found no significant quantitative differences in integrin expression between PMNs from the two age groups, regardless of whether the PMNs were incubated with buffer alone or with chemotaxins (rhIL-8, rhC5a, ZAS).

Modulation of an adhesion-related surface antigen on equine neutrophils by bacterial lipopolysaccharide and antiinflammatory drugs.

The essential role of the CD11/CD18 family of leukocyte adhesion molecules (LeuCams) in neutrophil-substrate adhesion is well documented. We have found that a monoclonal antibody designated 60.3 (MoAb 60.3) that recognizes the common beta-subunit (CD18) on human neutrophils (PMN) also recognizes a surface antigen on equine PMN. Antigen expression as assessed by immunofluorescence flow cytometry was enhanced by zymosan-activated serum (ZAS) or phorbol 12-myristate 13-acetate (PMA) stimulation. Pretreatment of equine PMN with MoAb 60.3 inhibited ZAS-stimulated aggregation, indicating that the monoclonal recognized a functional epitope on equine PMN involved in adhesion-related functions. Cells pretreated only with bacterial lipopolysaccharide (LPS; 1 microgram/ml) exhibited moderate increased binding of MoAb 60.3 as determined by fluorescence intensity. Preincubation of PMN with LPS resulted in a slight increase in MoAb 60.3 binding after subsequent ZAS stimulation, greater than that with either LPS or ZAS as sole stimulus. Similarly, enhanced binding of MoAb 60.3 was observed with LPS preincubation when PMA was used as a stimulus, but this effect was dose dependent and was observed at only one of three PMA concentrations tested (1 ng/ml). In other experiments, preincubation of PMN with antiinflammatory drugs inhibited 41.5-45.1% of ZAS-stimulated PMN adhesion to monolayers of equine endothelial cells. To determine whether modulation of expression of the adhesion-related antigen recognized by MoAb 60.3 correlated with these observed adhesive responses of PMN, we used immunofluorescence flow cytometry to assess expression of the antigen on drug-treated PMN. Using 10% ZAS as a stimulus, phenylbutazone (PBZ; 100 micrograms/ml) pretreatment of PMN reduced subsequent MoAb 60.3 binding by only 12.3%, and dexamethasone (DEX; 10(-5) M) reduced binding by only 1.0%; reductions of 16.4% with PBZ and 9.3% with DEX occurred when PMA (10 ng/ml) was used as the PMN stimulant. These data suggest that equine PMN express a functional adhesion molecule similar to those found on human PMN and that LPS may enhance the expression of this surface antigen. Expression of this adhesion-related surface antigen on equine PMN does not correlate well with levels of drug-induced diminished adhesion of PMN to endothelium in vitro.

Soluble CD14 and lipopolysaccharide-binding protein from bovine serum enable bacterial lipopolysaccharide-mediated cytotoxicity and activation of bovine vascular endothelial cells in vitro.

Bacterial endotoxin (lipopolysaccharide, LPS) has potent proinflammatory properties toward many cell types, including vascular endothelial cells. Bovine endothelial cells are often used for investigations involving the vascular endothelium in vitro, and other bovine products such as fetal bovine serum are also widely utilized in research laboratories. Evidence is presented that soluble CD14 (sCD14) is present in bovine serum and that LPS-mediated activation and cytotoxicity to bovine endothelial cells in vitro are dependent on sCD14. LPS-mediated activation of endothelial cells was quantitated by measuring tissue factor expression using an activated factor X-related chromogenic assay. Concentrations of 0.1-5.0% fetal bovine serum in the culture medium promoted LPS-induced tissue factor expression on bovine endothelial cells, and anti-CD14 monoclonal antibody (mAb) (20 micrograms/ml) inhibited tissue factor expression, whereas control antibodies did not. LPS-mediated damage to endothelial cells was assayed using the MTT tetrazolium assay. We found that either serum or recombinant human soluble CD14 (rsCD14, 20-2000 ng/ml) was required for LPS-related endothelial cell damage and that anti-CD14 mAb inhibited cytotoxicity. In addition, bovine LPS-binding protein (LBP, 20 ng/ml) purified from bovine serum had no effect on LPS-mediated cytotoxicity, but bovine LBP greatly enhanced the cytotoxic effect of LPS plus rsCD14. Western blot analysis performed on fractionated bovine serum samples with anti-CD14 mAb revealed immunoreactivity with a 50-55-kd protein, a size consistent with sCD14. Evidence of endothelial cell-associated CD14 was not detected using an immunofluorescence technique on cell preparations, nor by Northern blot analysis. These results indicate the existence of sCD14 in bovine serum and that soluble bovine serum factors including sCD14 and LBP facilitate presentation of LPS to receptive cells.

Serum components enhance bacterial lipopolysaccharide-induced tissue factor expression and tumor necrosis factor-alpha secretion by bovine alveolar macrophages in vitro.

We have compared the effect of bacterial lipopolysaccharide (LPS) in combination with normal adult bovine serum (NBS), fetal bovine serum (FBS), or a bovine serum fraction on tissue factor expression and tumor necrosis factor alpha (TNF-alpha) secretion by bovine alveolar macrophages. At a concentration of 1 ng/ml, bacterial LPS alone failed to induce measurable tissue factor expression by the macrophages, but the presence of FBS, NBS, or a fraction of normal pooled bovine serum isolated by ion-exchange chromatography (fraction 2) markedly potentiated the effect of LPS. A protein concentration of 64 micrograms/ml NBS, 192 micrograms/ml FBS, and only 640 ng/ml fraction 2 was required to induce maximal tissue factor expression on the macrophages in combination with 1 ng/ml LPS. Comparison of quantities of added serum protein required to induce maximal potentiating effects indicated that fraction 2 was 100 times more potent than whole NBS and 300 times more potent than whole FBS. We similarly found that TNF-alpha secretion by macrophages exposed to LPS was responsive to serum and was highly responsive to fraction 2. LPS alone (1 ng/ml) induced a relatively low level of TNF-alpha secretion by the macrophages, and the presence of FBS, NBS, or fraction 2 potentiated the effect of LPS. A concentration of 64.0 micrograms/ml NBS, 320.0 micrograms/ml FBS, and 3.2 micrograms/ml fraction 2 serum protein induced near-maximal TNF-alpha secretion by the macrophages. Comparison of the concentration of serum protein required to induce these potentiating effects indicated that fraction 2 was approximately 20 times more potent than whole NBS and 100 times more potent than whole FBS. The stimulatory effect of LPS plus fraction 2 serum proteins was dependent on the CD14 receptor, as monoclonal antibodies directed against CD14 (My4, 60bd; 10 micrograms/ml) inhibited tissue factor expression and TNF-alpha secretion by the macrophages.

Cometary science. 67P/Churyumov-Gerasimenko, a Jupiter family comet with a high D/H ratio.

The provenance of water and organic compounds on Earth and other terrestrial planets has been discussed for a long time without reaching a consensus. One of the best means to distinguish between different scenarios is by determining the deuterium-to-hydrogen (D/H) ratios in the reservoirs for comets and Earth's oceans. Here, we report the direct in situ measurement of the D/H ratio in the Jupiter family comet 67P/Churyumov-Gerasimenko by the ROSINA mass spectrometer aboard the European Space Agency's Rosetta spacecraft, which is found to be (5.3 ± 0.7) × 10(-4)­that is, approximately three times the terrestrial value. Previous cometary measurements and our new finding suggest a wide range of D/H ratios in the water within Jupiter family objects and preclude the idea that this reservoir is solely composed of Earth ocean-like water.

Molecular nitrogen in comet 67P/Churyumov-Gerasimenko indicates a low formation temperature.

Molecular nitrogen (N2) is thought to have been the most abundant form of nitrogen in the protosolar nebula. It is the main N-bearing molecule in the atmospheres of Pluto and Triton and probably the main nitrogen reservoir from which the giant planets formed. Yet in comets, often considered the most primitive bodies in the solar system, N2 has not been detected. Here we report the direct in situ measurement of N2 in the Jupiter family comet 67P/Churyumov-Gerasimenko, made by the Rosetta Orbiter Spectrometer for Ion and Neutral Analysis mass spectrometer aboard the Rosetta spacecraft. A N2/CO ratio of (5.70 ± 0.66) × 10(-3) (2σ standard deviation of the sampled mean) corresponds to depletion by a factor of ~25.4 ± 8.9 as compared to the protosolar value. This depletion suggests that cometary grains formed at low-temperature conditions below ~30 kelvin.

Cometary science. Time variability and heterogeneity in the coma of 67P/Churyumov-Gerasimenko.

Comets contain the best-preserved material from the beginning of our planetary system. Their nuclei and comae composition reveal clues about physical and chemical conditions during the early solar system when comets formed. ROSINA (Rosetta Orbiter Spectrometer for Ion and Neutral Analysis) onboard the Rosetta spacecraft has measured the coma composition of comet 67P/Churyumov-Gerasimenko with well-sampled time resolution per rotation. Measurements were made over many comet rotation periods and a wide range of latitudes. These measurements show large fluctuations in composition in a heterogeneous coma that has diurnal and possibly seasonal variations in the major outgassing species: water, carbon monoxide, and carbon dioxide. These results indicate a complex coma-nucleus relationship where seasonal variations may be driven by temperature differences just below the comet surface.

Co-expression of beta-adrenergic receptors and cyclooxygenase-2 in pulmonary adenocarcinoma.

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer and is highly resistant to conventional cancer therapy. A better understanding of the regulatory mechanisms which control the growth of this deadly malignancy are urgently needed to develop more effective cancer intervention strategies. Recent studies have shown that PAC frequently overexpresses cyclooxygenase-2 (COX-2). This enzyme converts arachidonic acid (AA) into several metabolites, some of which have been identified as modulators of mitogenesis and apoptosis. Accordingly, the AA cascade and COX-2 are currently widely studied as potential targets for lung cancer prevention. Recent studies by our research group have shown that cell lines derived from human PACs express beta1- and beta2-adrenergic receptors, which regulate the release of AA and DNA synthesis. Moreover, we have demonstrated that an antagonist for beta-adrenergic receptors or aspirin inhibited the development of experimentally induced PAC in a hamster model. These findings suggest that beta-adrenergic receptors may serve as upstream regulators of AA and COX-2-mediated PAC growth. However, no information is currently available on the expression of beta-adrenergic receptors and its possible correlation with the expression of COX-2 in tissue samples from human PAC, casting some doubt on the significance of these findings in vitro and in an animal model. In the current study, we have therefore analyzed tissue samples of human PACs for the expression of beta1-and beta2-adrenergic receptors as well as COX-2 by reverse transcription polymerase chain reaction (RT-PCR) or immunohistochemistry. Our data show that seven out of eight samples co-expressed COX-2 and one or both of these beta-adrenergic receptors, supporting the experimental evidence for a functional link between these neurotransmitter receptors and the AA cascade in the regulation of human PAC.

Comparison of 225actinium chelates: tissue distribution and radiotoxicity.

The biodistribution and tissue toxicity of intravenously administered 225-actinium (225Ac) complexed with acetate, ethylene diamine tetraacetic acid (EDTA), 1, 4, 7, 10, 13-pentaazacyclopentadecane-N, N', N", N"', N""-pentaacetic acid (PEPA), or the "a" isomer of cyclohexyl diethylenetriamine pentaacetic acid (CHX-DTPA), were examined. The percent of injected dose per organ and per gram of tissue for each chelate complex was determined. 225Ac-CHX-DTPA was evaluated further for radiotoxic effects. Mice receiving > or =185 kBq 225Ac-CHX-DTPA suffered 100% morbidity by 5 days and 100% mortality by 8 days postinjection, and all animals evaluated had significant organ damage. The in vivo instability of the 225Ac-CHX-DTPA complex likely allowed accumulation of free 225Ac in organs, which resulted in tissue pathology.

Secretory activity of equine polymorphonuclear leukocytes: stimulus specificity and priming effects of bacterial lipopolysaccharide.

Neutrophil (PMN) contributions to the acute inflammatory process and host defense include generation of bioreactive oxygen metabolites and secretion of granule enzymes. We assessed equine PMN secretion using several PMN stimuli, singly and in combination with bacterial lipopolysaccharide (LPS). LPS avidly associated with equine PMN, as shown by strong PMN labeling with FITC-conjugated LPS. LPS alone (1 or 10 micrograms ml-1) was a weak stimulus for PMN superoxide anion (O2-) generation, but preincubation with LPS followed by phorbol ester (PMA, 10 ng ml-1) significantly augmented (P less than 0.01) secretion of O2- (19.38 nmol O2- per 2 x 10(6) PMN per 5 min) over the amount generated by PMA stimulation alone (13.75 nmol O2-). A qualitatively similar, but smaller O2(-)-generation response occurred when either opsonized zymosan or recombinant human C5a was used as the PMN stimulus. Arachidonic acid (ArA; 50-200 microM) was a potent stimulus, with secreted O2- levels similar to those from PMA-stimulated PMN. Preincubation of PMN with either the formyl peptide, fMLP, or platelet-activating factor before stimulation with ArA did not significantly increase O2- generation over levels obtained using ArA alone. Release of PMN granule enzymes was also quantitated. A small amount of lysozyme secretion resulted when PMN were exposed to LPS alone (8.20% of total cell content), and PMA stimulation caused marked release of PMN lysozyme (44.45%). Non-specific proteolytic activity in PMN supernatants, assessed by cleavage of a collagen-rich substrate, was minimal with LPS as a sole stimulus (5.08%). There was significant proteolytic activity (P less than 0.01) in supernatants from PMA-stimulated PMN (27.21%), and preincubation with LPS followed by PMA stimulation slightly enhanced (P less than 0.05) the release of PMN proteases (34.62%). The activities of beta-glucuronidase, acid phosphatase, and alkaline phosphatase were minimal in PMN supernatants when using LPS and PMA as stimuli. The activity of PMN granule enzymes was found to be sensitive to the presence of normal equine serum, and proteolytic activity was markedly reduced (80.13% reduction) in the presence of 10% pooled serum.

Induction of procoagulant activity in virus infected bovine alveolar macrophages and the effect of lipopolysaccharide.

Three viruses known to be associated with the bovine respiratory disease complex were evaluated in vitro for potential impact upon the procoagulant activity (PCA) of bovine alveolar macrophages (bAM). Cultures of bAM were inoculated with bovine parainfluenza virus Type 3 (PI-3), cytopathic bovine viral diarrhea virus (cpBVDV), non-cytopathic BVDV (ncpBVDV), or bovine herpes virus Type 1 (BHV-1) and incubated for several time periods (24, 48, 72, 96 h). BAM were then exposed to E. coli lipopolysaccharide (LPS), or LPS with bovine serum. The amount of PCA expressed was quantified using a chromogenic assay. Viral inoculation increased bAM expression of PCA (P < 0.01). The increase in PCA expression was larger at higher rates of viral inoculation (P < 0.01). LPS enhanced PCA expression by bAM at low rates of viral inoculation (P < 0.01). The effect of LPS-serum treatment was greater than the LPS alone (P < 0.01). At high rates of viral inoculation, LPS had no enhancing effect on PCA expression. The effect of LPS on virus inoculated bAM varied with virus type, rate of inoculation, and duration of virus exposure (P < 0.01). The results suggest that these four viruses initiate the production of PCA by bAM independently of LPS. In the field situation, an initial viral infection may induce fibrin deposition in the pulmonary alveoli prior to the establishment of a secondary gram negative bacterial infection.

Purification of lipopolysaccharide-binding protein from bovine serum.

Lipopolysaccharide-binding protein (LBP) plays a central role in presentation of bacterial-derived lipopolysaccharide (LPS; endotoxin) to leukocytes such as macrophages and neutrophils. Interaction of LBP with LPS is significant because LBP-LPS complexes promote activation of leukocytes and the immune system, which results in enhanced secretion of a spectrum of proinflammatory cytokines. An improved, simplified method was used to purify bovine LBP from serum. Methodology consisted of ion-exchange chromatography using Bio-Rex 70 resin, followed by gel-filtration chromatography (Sephacryl S-200 resin) of a selected ion-exchange fraction (0.22-0.50 M NaCl). Densitometric scans on silver-stained polyacrylamide gels of chromatographically-derived proteins indicated up to 88.7% purity of the resultant 64kD protein (bovine LBP) in the cleanest fractions. The isoelectric point of bovine LBP was determined to be 6.8. Identity of the protein was substantiated by western-blot analysis, and by N-terminus amino acid sequence analysis with favorable comparison to published sequence data from rabbit, human, and murine LBP Identity was corroborated by use of purified bovine LBP in bioassays which demonstrated enhanced tissue factor expression of LPS (1 ng ml(-1)-stimulated bovine alveolar macrophages. Tissue factor expression was inhibitable in these assays using anti-CD14 monoclonal antibodies, which is also consistent with LBP-mediated activation of cells. When bovine LBP was heated at 56 degrees C for 30 min, the biological activity was reduced by 50% in the macrophage-based bioassays. Biological activity of bovine LBP was completely destroyed by heating at 62 degrees C for 30 min, which compared favorably with data resulting from use of fetal bovine serum.

Nitric oxide production and expression of inducible nitric oxide synthase by bovine alveolar macrophages.

Alveolar macrophages play a central role in host defense in the lower respiratory tract. Production of the reactive intermediate nitric oxide (NO), via expression of inducible nitric oxide synthase (iNOS) is an important microbicidal effector mechanism possessed by macrophages. In this study, cytokine regulation of NO production by bovine alveolar macrophages (bAM) was evaluated. Bovine alveolar macrophages were exposed to one or more of the following: recombinant human (rh) and recombinant bovine (rb) IFN gamma, rh- and rbIL-1 beta, rbGM-CSF, rhTNF alpha, rhIL-4, endotoxin (LPS), fetal bovine serum (FBS), mitogen-stimulated bovine splenic supernatant (SS), and purified human TGF beta-1. LPS alone, or in combination with SS, rbIFN gamma, or rbIL-1 beta stimulated production of NO in a time and dose dependent fashion. Recombinant bovine IFN gamma, rbIL-1 beta, and rhTNF alpha in combination produced maximal stimulation which was not further enhanced by LPS. Recombinant human IFN gamma, rhIL-1 beta, and rbGM-CSF had minimal effect either as single stimuli, or in combination with LPS, rbIFN gamma, rbIL-1 beta, or rhTNF alpha. Nitric oxide production was inhibited by rhIL-4, and the L-arginine analogue antagonists of iNOS, N-G-monomethyl-L-arginine (NGMMA) and aminoguanidine (AG). Purified human TGF beta-1 did not inhibit NO production. Messenger RNA for iNOS was maximally expressed by 8 h and remained detectable for at least 48 h. Expression of iNOS mRNA induced by cytokines and LPS varied with strength of the stimulus as determined by nitrite production in culture supernatant.

Interleukin-6 secretion by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

Interleukin-6 (IL-6) is a pluripotent cytokine that may play a role in pulmonary defense against bacterial pathogens. We have quantitated the response of bovine alveolar macrophages (bAM) to bacterial lipopolysaccharide (LPS; E. coli 055: B5) in vitro using the IL-6 sensitive 7TD1 cell line. Bacteria LPS in the absence of serum induced IL-6 secretion from bAM (1 x 10(6) ml-1) over a range of LPS concentrations from 10 ng ml-1 to 10 micrograms ml-1. This resulted in IL-6 levels ranging from approximately 5 to over 200 U ml-1.IL-6 secretion by from approximately 5 to over 200 U ml-1.IL-6 secretion by LPS-stimulated bAM was increased by 24 h poststimulation, and continued to increase up to 72 h after stimulation. Fetal bovine serum (FBS, 1% vol/vol; 320 micrograms ml-1) enhanced IL-6 secretion from macrophages in the presence of LPS by approximately 10-fold compared with LPS alone. A bovine serum fraction (1 microgram ml-1 protein) prepared using ion-exchange chromatography also markedly enhanced IL-6 secretion versus LPS alone. The stimulatory effect of IL-6-like activity in the bAM supernatants was neutralized by an anti-human IL-6 polyclonal antibody. Northern blot analysis revealed increased IL-6 mRNA at 2 h poststimulation with LPS + FBS, peak levels at 4 h, and levels were decreased by 6 h poststimulation. Results suggest that IL-6 is secreted by bovine alveolar macrophages, and that bacterial LPS and serum components synergize to produce this response.