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Aim: To investigate the usefulness of assaying endotoxin in non-directed bronchial lavage fluid (NBL), bronchoscopic bronchoalveolar lavage fluid (BAL), and sera as a means of diagnosing Gram negative, ventilator associated pneumonia., Methods: Samples from 64 patients were investigated. Fifty nine BALs and 92 NBLs were assayed in total including specimens taken during 28 episodes of clinical ventilator associated pneumonia (VAP)., Results: The concentration of endotoxin in BAL from patients with VAP developing within four days of commencing ventilation was significantly higher than in those without VAP (p = 0.015). There was no significant difference in endotoxin concentration in NBL or serum when comparing patients with and without VAP. A BAL endotoxin concentration of 6 EU/ml yielded the optimal operating characteristics (sensitivity, 81%; specificity, 87%; positive predictive value, 67%; negative predictive value, 95%). However, Gram stain of BAL provided the same information as quickly as the endotoxin assay and is considerably cheaper., Conclusions: Despite its accuracy and rapidity, the BAL endotoxin assay must be shown to alter clinical management and patient outcome to be cost effective.

Th1 cells exposed to Ag and the G(1) blocker n-butyrate in primary cultures lose their ability to proliferate in Ag-stimulated secondary cultures. The ability of n-butyrate to induce anergy in Ag-stimulated, but not resting, Th1 cells was shown here to be blocked by cycloheximide. Subsequent experiments to delineate the nature of the protein apparently required for n-butyrate-induced Th1 cell anergy focused on the role of cyclin-dependent kinase (cdk) inhibitors p21(Cip1) and p27(Kip1). Normally, entry into S phase by Th1 cells occurs around 24 h after Ag stimulation and corresponds with relatively low levels of both p21(Cip1) and p27(Kip1). However, unlike control Th1 cells, anergic Th1 cells contained high levels of both p21(Cip1) and p27(Kip1) when examined 24 h after Ag stimulation. The increase in p21(Cip1) observed in Ag-stimulated anergic Th1 cells appeared to be initiated in primary cultures. In contrast, the increase in p27(Kip1) observed in these anergic Th1 cells appears to represent a re-expression of the protein much earlier than control cells following Ag stimulation in secondary cultures. The anergic Th1 cells contained functionally active cdk inhibitors capable of inhibiting the activity of both endogenous and exogenous cdks. Consequently, it appears that n-butyrate-induced anergy in Th1 cells correlated with the up-regulation of p21(Cip1) and perhaps the downstream failure to maintain low levels of p27(Kip1). Increased levels of both p21(Cip1) and p27(Kip1) at the end of G(1) could prevent cdk-mediated entry into S phase, and thus help maintain the proliferative unresponsiveness found in the anergic Th1 cells.

The regulation of pro- and anti-mediator release from cells within the alveolar space would represent a desirable mechanism serving to protect this delicate gas-exchanging region of the lung. This study investigates the effect of alveolar surfactant lipids on the regulation of tumour necrosis factor alpha (TNF-alpha), a potent inflammatory cytokine, and prostaglandin E(2)(PGE(2)), a lipid mediator with anti-inflammatory properties. The results of this investigation reveal a marked effect on the release of these two important mediators from a monocytic cell line, MonoMac 6 (MM6), by phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol (Chol) and sphingomyelin (SM). PC, PE and Chol demonstrated marked downregulation of TNF-alpha production at lipid concentrations of 125 and 250 microg/ml. Interestingly, SM significantly up regulated the release of TNF-alpha at these concentrations. However, the release of PGE(2)in MM6 cells incubated with the same lipids was significantly increased with PC and Chol, and significantly decreased in cells pre-treated with SM. This indicates a role for these lipids in alveolar immunoregulation., (Copyright 2000 Academic Press.)

Objectives: First, we sought to study the effects of short- and long-term vitamin C therapy on oxidative stress and endothelial dysfunction in chronic heart failure (CHF), and second, we sought to investigate the role of neutrophils as a cause of oxidative stress in CHF., Background: Oxidative stress may contribute to endothelial dysfunction in CHF. Vitamin C ameliorates endothelial dysfunction in CHF, presumably by reducing oxidative stress, but this is unproven., Methods: We studied 55 patients with CHF (ischemic and nonischemic etiologies) and 15 control subjects. Flow-mediated dilation (FMD) in the brachial artery was measured by ultrasound wall-tracking, neutrophil superoxide anion (O2-) generation by lucigenin-enhanced chemiluminescence and oxidative stress by measurement of free radicals (FRs) in venous blood using electron paramagnetic resonance (EPR) spectroscopy and plasma thiobarbituric acid reactive substances (TBARS). Measurements were performed at baseline in all subjects. The effects of short-term (intravenous) and long-term (oral) vitamin C therapy versus placebo were tested in patients with nonischemic CHF., Results: At baseline, FRs were higher in patients with CHF than in control subjects (p < 0.01), TBARS were greater (p < 0.005), neutrophil O2- -generating capacity was enhanced (p < 0.005) and FMD was lower (p < 0.0001). Compared with placebo, short-term vitamin C therapy reduced FR levels (p < 0.05), tended to reduce TBARS and increased FMD (p < 0.05), but did not affect neutrophil O2- -generating capacity. Long-term vitamin C therapy reduced FR levels (p < 0.05), reduced TBARS (p < 0.05) and improved FMD (p < 0.05), but also reduced neutrophil O2- -generating capacity (p < 0.05). Endothelial dysfunction was not related to oxidative stress, and improvements in FMD with vitamin C therapy did not relate to reductions in oxidative stress., Conclusions: Oxidative stress is increased in ischemic and nonischemic CHF, and neutrophils may be an important cause. Vitamin C reduces oxidative stress, increases FMD and, when given long term, decreases neutrophil O2- generation, but the lack of a correlation between changes in endothelial function and oxidative stress with vitamin C implies possible additional non-antioxidant benefits of vitamin C.

In mammals, including rats and mice, the development of pulmonary alveolar septa is primarily limited to late gestation and the early periods of postnatal life. Before this time, the rat lung contains a relatively large supply of endogenous retinyl ester that, together with its metabolite retinoic acid, has been shown to increase elastin gene expression and the number of alveoli. We have hypothesized that mice bearing a deletion of one or more genes encoding for retinoic acid receptors (which are DNA binding proteins that alter transcription of retinoic acid-responsive genes) may demonstrate abnormalities in retinoid-mediated alveolar formation. Our studies demonstrate that the absence of the retinoic acid receptor-gamma (RARgamma) is associated with a decrease in the steady-state level of tropoelastin messenger RNA in a subpopulation of lung fibroblasts at Postnatal Day 12. RARgamma gene deletion also resulted in a decrease in whole lung elastic tissue and alveolar number, and an increase in mean cord length of alveoli (L(m)) at Postnatal Day 28. The additional deletion of one retinoid X receptor (RXR)alpha allele resulted in a decrease in alveolar surface area and alveolar number, and an increase in L (m). These data indicate that RARgamma is required for the formation of normal alveoli and alveolar elastic fibers in the mouse, and that RAR/RXR heterodimers are involved in alveolar morphogenesis.

Background: Exaggerated postprandial lipemia (PPL) is a factor in atherogenesis, involving endothelial dysfunction and enhanced oxidative stress. We examined the effect of ciprofibrate therapy on these parameters in type 2 diabetes mellitus., Methods and Results: Twenty patients entered a 3-month, double-blind, placebo-controlled study. Each subject was studied fasting and after a fatty meal, at baseline, and after 3 months of treatment. Glucose and lipid profiles were measured over an 8-hour postprandial period. Endothelial function (flow-mediated endothelium-dependent vasodilatation [FMD]) and oxidative stress (electron paramagnetic resonance spectroscopy) were measured after fasting and 4 hours postprandially. At baseline, both groups exhibited similar PPL and deterioration in endothelial function. After ciprofibrate, fasting and postprandial FMD values were significantly higher (from 3.8+/-1. 8% and 1.8+/-1.3% to 4.8+/-1.1% and 3.4+/-1.1%; P<0.05). This was mirrored by a fall in fasting and postprandial triglycerides (3. 1+/-2.1 and 6.6+/-4.1 mmol/L to 1.5+/-0.8 and 2.8+/-1.3 mmol/L, P<0. 05). Fasting and postprandial HDL cholesterol was also elevated (0. 9+/-0.1 and 0.8+/-0.1 mmol/L and 1.2+/-0.2 and 1.2+/-0.1 mmol/L, P<0. 05). There were no changes in total or LDL cholesterol. Fasting and postprandial triglyceride enrichment of all lipoproteins was attenuated, with cholesterol depletion of VLDL and enrichment of HDL. There were similar postprandial increases in oxidative stress in both groups at baseline, which was significantly attenuated by ciprofibrate (0.3+/-0.6 versus 1.5+/-1.1 U, P<0.05)., Conclusions: This study demonstrates that fibrate therapy improves fasting and postprandial endothelial function in type 2 diabetes. Attenuation of PPL and the associated oxidative stress, with increased HDL cholesterol levels, may be important.

Trichloroethylene (1,1,2-trichloroethene) is a major environmental contaminant. There is increasing evidence relating exposure to trichloroethylene with autoimmunity. To investigate potential mechanisms, we treated the autoimmune-prone MRL +/+ mice with trichloroethylene in the drinking water at 0, 2.5 or 5.0 mg/ml and sacrificed them at 4, 8 and 22 weeks. As early as 4 weeks of treatment, Western blot analysis showed a dose-dependent increase in the level of trichloroethylene-modified proteins, indicating that a reactive metabolite of trichloroethylene was formed. Significant increases in antinuclear antibodies (ANA) and total serum immunoglobulins were found following 4-8 weeks of trichloroethylene treatment, indicating that trichloroethylene was accelerating an autoimmune response. Investigation into possible mechanisms of this autoimmune response revealed that trichloroethylene treatment dramatically increased the expression of the activation marker CD44 on splenic CD4+ T cells at 4 weeks. In addition, splenic T cells from mice treated for 4 weeks with trichloroethylene secreted more IFN-gamma and less IL-4 than control T cells, consistent of a T-helper type 1 (Th1) type immune or inflammatory response. A specific immune response directed against dichloroacetylated proteins was found at 22 weeks of trichloroethylene treatment. Taken collectively, the results suggest that trichloroethylene treatment accelerated an autoimmune response characteristic of MRL +/+ mice in association with nonspecific activation of Th1 cells. In addition, long-term treatment with trichloroethylene led to the initiation of a trichloroethylene-specific immune response.

Oxygen free radicals are highly reactive species that are produced in increased quantities during strenuous exercise and can damage critical biological targets such as membrane phospholipids. The present study examined the effect of acute ascorbic acid supplementation on exercise-induced free radical production in healthy subjects. Results demonstrate increases in the intensity of the alpha-phenyl-tert-butylnitrone adduct (0.05 +/- 0.02 preexercise vs. 0.19 +/- 0.03 postexercise, P = 0.002, arbitrary units) together with increased lipid hydroperoxides (1.14 +/- 0.06 micromol/l preexercise vs. 1.62 +/- 0.19 micromol/l postexercise, P = 0.005) and malondialdehyde (0.70 +/- 0.04 micromol/l preexercise vs. 0.80 +/- 0.04 micromol/l postexercise, P = 0.0152) in the control phase. After supplementation with ascorbic acid, there was no significant increase in the electron spin resonance signal intensity (0.02 +/- 0. 01 preexercise vs. 0.04 +/- 0.02 postexercise, arbitrary units), lipid hydroperoxides (1.12 +/- 0.21 micromol/l preexercise vs. 1.12 +/- 0.08 micromol/l postexercise), or malondialdehyde (0.63 +/- 0.07 micromol/l preexercise vs. 0.68 +/- 0.05 micromol/l postexercise). The results indicate that acute ascorbic acid supplementation prevented exercise-induced oxidative stress in these subjects.

The effects of the surfactants, alcohol ethoxylate, amine ethoxylate, amine oxide and SDS on cell membranes were investigated using the lipid soluble spin label 5-doxyl stearic acid (5-DS). Electron paramagnetic resonance (EPR) spectroscopy revealed that the action of the surfactants was to significantly increase membrane fluidity of Proteus mirabilis, Staphylococcus aureus and Saccharomyces cerevisiae. The action of these surfactants as biocides was investigated and found to be dependent on the type of organism tested. There was, however, no direct correlation between enhanced membrane fluidity observed due to the action of the surfactants and biocidal activity. Data presented suggest that perturbing the fluidity of the cytoplasmic membrane is not immediately responsible for cell death.

Whole cells and lipopolysaccharides (LPSs) extracted from Burkholderia cepacia, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli were compared in their ability to stimulate tumor necrosis factor alpha (TNF-alpha) from the human monocyte cell line MonoMac-6. B. cepacia LPS, on a weight-for-weight basis, was found to have TNF-alpha-inducing activity similar to that of LPS from E. coli, which was approximately four- and eightfold greater than the activity of LPSs from P. aeruginosa and S. maltophilia, respectively. The LPS-stimulated TNF-alpha production from monocytes was found to be CD14 dependent. These results suggest that B. cepacia LPS might play a role in the pathogenesis of inflammatory lung disease in cystic fibrosis, and in some patients it might be responsible, at least in part, for the sepsis-like cepacia syndrome.

The acquisition of Burkholderia cepacia in some cystic fibrosis patients is associated with symptoms of acute pulmonary inflammation that may be life threatening. The ability of lipopolysaccharide (LPS) from B. cepacia to prime a monocyte cell line for enhanced superoxide anion generation was investigated and compared with the priming activities of LPSs from Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Escherichia coli. The human monocyte cell line MonoMac-6 (MM6) was primed overnight with different LPSs (100 ng/ml), and the respiratory burst was triggered by exposure to opsonized zymosan (125 micrograms/ml). Superoxide generation was detected by enhanced chemiluminescence with Lucigenin. B. cepacia LPS was found to prime MM6 cells to produce more superoxide anion than P. aeruginosa or S. maltophilia LPS, and this priming response was CD14 dependent. In addition, the inhibition of respiratory burst responses in monocytes by a bacterial melanin-like pigment purified from an epidemic B. cepacia strain was investigated. The melanin-like pigment was isolated from tyrosine-enriched media on which B. cepacia had been grown and was purified by gel filtration, anion ion-exchange chromatography, and ethanol precipitation. The scavenging potential of the melanin-like pigment for superoxide anion radical (*O2-) generated during the respiratory burst was confirmed with superoxide produced from a cell-free system with xanthine-xanthine oxidase and detected by electron paramagnetic resonance spectroscopy with the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-n-oxide. The addition of melanin during the LPS priming stage had no effect on the subsequent triggering of the respiratory burst, but melanin inhibited *O2- detection when added at the triggering stage of the respiratory burst. We conclude that melanin-producing B. cepacia may derive protection from the free-radical-scavenging properties of this pigment.

Free radicals or oxidants are continuously produced in the body as a consequence of normal energy metabolism. The concentration of free radicals, together with lipid peroxidation, increases in some tissues as a physiological response to exercise - they have also been implicated in a variety of pathologies. The biochemical measurement of free radicals has relied in the main on the indirect assay of oxidative stress by-products. This study presents the first use of electron spin resonance (ESR) spectroscopy in conjunction with the spin-trapping technique, to measure directly the production of radical species in the venous blood of healthy human volunteers pre- and post-exhaustive aerobic exercise. Evidence is also presented of increased lipid peroxidation and total antioxidant capacity post-exercise.

A method to amplify and detect TNF-alpha mRNA from primed Mono Mac 6 cells is described. A silica-based extraction system was utilised for preparation of cell extracts and specific oligonucleotide primers were designed for amplification of TNF-alpha mRNA by the NASBA process. Amplification products were detected using either a liquid hybridisation assay, with analysis by polyacrylamide gel electrophoresis, or a plate hybridisation system. The method has many potential applications for the study of inflammatory cytokines and cellular mRNAs in cell culture and clinical samples.

During the alveolar stage of lung development, lipid droplet-laden interstitial cells are present at the base of elongating alveolar septa. These cells that have been named lipid interstitial cells or lipofibroblasts (LFs) may supply lipids for surfactant production, the synthesis of membrane phospholipids, and/or energy metabolism. They also have myofibroblastic characteristics and participate in the generation of the interstitial elastic fiber network, that is, in the pulmonary alveolar septum. To understand how this cell regulates its lipid-storing and elastin-producing properties, we have examined the effects of peroxisome proliferators on the expression of the genes that are associated with an elastin-producing myofibroblastic phenotype or an adipocyte-like phenotype. Two known ligands for peroxisome proliferator-activated receptors, 5,8,11,14-eicosatetraynoic acid (ETYA) and 15-deoxy-delta-12,14-prostaglandin J2 (15-dPGJ2), decrease elastin gene transcription and the steady-state levels of tropoelastin (TE) and alpha-smooth muscle actin mRNAs in cultured LFs. Concurrently, cultured LFs increase the expression of adipocyte lipid binding protein, which is regarded as an adipocyte-specific protein, and accumulate lipid droplets. Their abilities to store lipids and express desmin intermediate filaments, alpha-smooth muscle actin, and smooth muscle myosin heavy chain in contractile filaments in vitro illustrate similarities among the pulmonary LF, the hepatic lipocyte, and the contractile interstitial cell, which contribute to the repair reaction in the lung after pulmonary injury.

During late gestation, the lungs of rats contain retinyl esters, but their concentration decreases considerably at the time of birth. The regulation of the acquisition and utilization of these stored retinoids remains poorly understood, although it has been hypothesized that they are involved in surfactant production and alveolar septal formation. Previous investigations demonstrated that exogenous retinoic acid increases elastin production in cultured neonatal lung fibroblasts and increases the number of alveoli when it is administered to neonatal rats. It has been hypothesized that these pulmonary stores of retinyl esters may regulate the perinatal expression of various genes in the lung, including elastin. To test this hypothesis, inhibitors of retinoid metabolism were used to reduce the flux of retinyl esters to retinoic acid, and the effects of this maneuver on elastin gene expression were analyzed. Inhibitors of alcohol and aldehyde dehydrogenases and of retinyl ester hydrolases decreased the steady-state level of tropoelastin mRNA without reducing alpha 1(I) procollagen mRNA. The magnitude of the effects of the inhibitors was retinol dependent and was significantly reduced in lung tissue that was obtained from vitamin A-deficient fetuses. These findings suggest that the late gestational pulmonary stores of retinoids may increase elastin gene expression during the fetal and early postnatal life in the rat.

Elastin, an important structural protein of the extracellular matrix, confers elastic properties on the pulmonary alveolar interstitium. In the alveolar wall, elastin is primarily produced postnatally by fibroblasts. The mechanisms that regulate lung fibroblast (LF) elastin gene expression have not been completely defined, although both transcriptional and posttranscriptional mechanisms appear to be involved. Transforming growth factors-beta (TGF-beta s) have been shown to increase elastin production by cultured neonatal rat LF. Analyses of elastin gene transcription and mRNA stability indicate that exogenous TGF-beta 1 increases the half-life of tropoelastin mRNA by 1.5-fold and does not alter elastin gene transcription. Interference with the functions of endogenous TGF-beta 1 in cultured LF, through the addition of neutralizing antibodies or antisense oligodeoxynucleotides, decreases tropoelastin and tropoelastin mRNA production by these cells. The content of total (latent plus active) TGF-beta s was approximately 4.5-fold greater in lungs obtained from rats on postnatal day 8 than in lungs obtained from adults. These findings indicate that endogenous TGF-beta s, in cultured LF, regulate elastin gene expression, most likely by a posttranscriptional mechanism. Since others have shown that elastin mRNA appears to have a longer half-life in neonatal than in adult rat lungs, we hypothesize that the higher content of TGF-beta s could contribute to the greater elastin mRNA stability in neonatal lungs.

The role of leukotrienes (LTs) in asthma is reviewed, and research to develop anti-LT agents for this condition is described. Greater understanding of the role played by inflammatory cells and their mediators in the pathophysiology of asthma has shifted the emphasis of research from the bronchoconstriction component of the disease to the inflammatory one. LTs are believed to play a key role in the complex interplay of inflammatory cells that occurs in asthma. Inhibiting the production of LTs or blocking their receptor sites may decrease the inflammatory response and thereby provide a useful therapeutic modality. Three approaches have been used in attempts to affect the activity of LTs: inhibition of 5-lipoxygenase, inhibition of 5-lipoxygenase-activating protein, and LTD4-receptor antagonism. Investigational agents that have undergone clinical trials include zileuton (a 5-lipoxygenase inhibitor), MK-591 (a 5-lipoxygenase-activating protein inhibitor), and zafirlukast, pranlukast, and verlukast (three LT-receptor antagonists). Many studies suggest that these orally administered agents are effective, particularly with respect to the early asthmatic response to allergens and other challenges, and have a low adverse-effect profile; the reaction of greatest concern is elevation of liver enzymes, especially with zileuton. Larger trials conducted over longer periods, as well as comparative trials, will be necessary to delineate the ultimate role of these agents in asthma therapy. Because of the complexity of the inflammatory process in asthma, anti-LT agents are likely to become part of multidrug regimens. Using drugs to interfere with leukotrienes may prove beneficial in the treatment of asthma.

Clinical strains of Stenotrophomonas (Xanthomonas) maltophilia often show large, growth temperature-dependent, variations in their susceptibility (TDVS) to aminoglycoside antibiotics. Strains showing more than a fourfold increase in susceptibility between 30 degrees and 37 degrees C (TDVS+ strains; n = 23) were contrasted with those showing lesser variation (TDVS- strains; n = 15) in studies of growth temperature-dependent variation in protein and cell-wall lipopolysaccharide (LPS) electrophoresis patterns in an attempt to determine the mechanism of TDVS. Several proteins showed increased intensity with increasing growth temperature. These comprised bands at c. 65, 55, 42.5, 26 and 21.5 kDa in the whole cell proteins, an outer membrane protein band at c. 21.5 kDa, and cytoplasmic membrane protein bands at c. 42.5 and 27 kDa. Two whole cell protein bands at c. 30 and 24 kDa and three outer membrane protein bands at c. 45, 30 and 24 kDa decreased in intensity with increasing growth temperature. However, there was no correlation with the extent of variation in susceptibility, either in the extent of temperature dependent changes in protein banding patterns, or the presence or absence of specific protein bands. By contrast, temperature-dependent variation in LPS patterns correlated well with TDVS. TDVS+ strains yielded intense ladder patterns of more than 30 discrete bands, and the mean molecular weight of the ladder pattern was markedly higher at growth temperatures < or = 30 degrees C, than at > or = 37 degrees C. TDVS- strains gave a clearly distinct high mol. wt LPS banding pattern showing fewer, less intense bands and a smaller and less consistent shift in mean molecular weight with temperature. Strains which were clearly resistant at 30 degrees and 37 degrees C, had a high mol. wt. polysaccharide component but an absence of the typical LPS-ladder pattern. We conclude that the temperature-dependent variation in the aminoglycoside susceptibility of this species was not correlated with any detectable change in protein composition, but correlated well with changes in LPS structure.

The rate of oxygen consumption in the human acute monocytic leukemia-derived cell line, Mono Mac 6, in response to lipopolysaccharide (LPS) in vitro was measured by electron paramagnetic resonance spectroscopy using an oxygen-sensitive spin-label, 4-oxo-2,2,6,6-tetramethylpiperidine-d16-1-oxyl (15N-PDT). Lipopolysaccharide impaired oxygen consumption in a dose-dependent manner which was shown to be mediated by mitochondrial dysfunction and could be augmented by pretreatment of the cells with interferon-gamma. Treatment of the cells with anti-CD14 monoclonal antibody failed to inhibit the LPS-induced effects on cellular respiration. These results suggest that LPS can directly reduce normal cellular oxygen consumption possibly via a CD14-independent pathway. This alteration of mitochondrial function by LPS may be responsible for the observed cell damage during sepsis.

Interleukin-4 (IL-4) and interleukin-10 (IL-10) were evaluated for their ability to inhibit the production of nitric oxide (NO) by interferon-gamma (IFN-gamma)- or lipopolysaccharide (LPS)-activated murine macrophages (RAW 264.7 and J774.2). Macrophages pre-treated with IL-4 and then stimulated with IFN-gamma or LPS showed significant inhibition in their ability to produce NO as measured by nitrite production. Simultaneous treatment of IL-4 pre-incubated cells with IFN-gamma and LPS together augmented nitrite accumulation. On the other hand, similar exposures of the macrophages to IL-10 followed by IFN-gamma or LPS treatments resulted in significantly increased NO production. Thus IL-10 failed to suppress IFN-gamma or LPS-induced NO production and showed opposite effects in these experiments to IL-4. We conclude that the two lymphokines have differing roles in the control of production of NO and might act to control the secretion of nitric oxide in vivo.

Electron Paramagnetic Resonance (EPR) oximetry was used to measure tissue oxygen tension (pO2-partial pressure of oxygen) simultaneously in the kidney cortex and outer medulla in vivo in mice. pO2 in the cortex region was higher compared to that in the outer medulla. An intravenous injection of endotoxin resulted in a sharp drop in pO2 in the cortex and an increase in the medulla region, resulting in a transient period of equal pO2 in both regions. In control kidneys, functional Magnetic Resonance (MR) images showed the cortex region to have high signal intensity (T2*-weighted images), indicating that this region was well supplied with oxygenated hemoglobin, whereas the outer medulla showed low signal intensity. After administration of endotoxin, we observed an immediate increase in signal intensity in the outer medulla region, reflecting an increased level of oxygenated blood in this region. Pretreatment of mice with NG-monomethyl-L-arginine prevented both the changes in tissue pO2 and distribution of oxygenated hemoglobin, suggesting that localized production of nitric oxide has a critical role to play in renal medullary hemodynamics. In combining in vivo EPR with MR images of kidneys, we demonstrate the usefulness of these techniques for monitoring renal pO2 and changes in the distribution of oxygen.

The effects of interferon (IFN)-gamma and IFN-beta on the incorporation of 14C-linoleic acid into J774.2 cell membrane phospholipids were examined. Interferon-gamma induced a statistically significant increase in incorporation of 14C-linoleic acid into all the major phospholipid classes. In contrast, IFN-beta induced a slightly reduced incorporation of this fatty acid into the phospholipids. Neither IFN-gamma nor IFN-beta had any effect on the incorporation of the saturated fatty acid 14C-stearic acid into the cellular phospholipids. Interferon-gamma had no effect on the metabolism of 14C-linoleic acid in the fibroblast cell line L929. Macrophage membrane fluidity was assessed by spin-label ESR spectroscopy after incubation with either IFN-gamma or IFN-beta. Interferon-gamma significantly increased membrane fluidity whereas IFN-beta significantly decreased the fluidity. The findings of this study reveal that IFN-gamma might act on the enzymes controlling the labelling of the sn2 position of phospholipids (linoleic acid) but not the sn1 position (stearic acid), and this increases the polyunsaturated fatty acid content of macrophage membranes. This increase in polyunsaturation is reflected in the increased membrane fluidity. We also conclude that IFN-beta and IFN-gamma have different mechanisms of action on macrophage membrane lipid metabolism.

The early postnatal life of most mammals marks a period of extensive enlargement of the alveolar surface area and increase in the elastin content of the lung. The factors that regulate the onset and abatement of this burst of elastin synthesis have not been identified. Previous studies of lipid-laden rat pulmonary interstitial fibroblasts (LIF) have shown that their elastin synthesis is increased in vitro by retinoic acid (RA). We hypothesized that temporal changes in the endogenous RA content of LIF may correlate with changes in elastin synthesis by these cells. LIF were isolated from the lungs of rats at gestational day 19 and postnatal days 2, 4, 8, and 12 and their retinoid contents were quantitated. Retinyl esters were highest at gestational day 19 (2.9 +/- 0.6 pmol/10(6) cells, means +/- SE) and decreased to 1.6 +/- 0.2 pmol by postnatal day 2 (P < 0.05). This decrease in retinyl esters was accompanied by an increase in retinol from 0.4 +/- 0.1 to 2.0 +/- 0.6 pmol/10(6) cells (P < 0.05). RA increased in LIF from 0.07 +/- 0.04 pmol/10(6) cells at gestational day 19 to 0.29 +/- 0.05 pmol/10(6) cells at postnatal day 2 (P < 0.05) and increased in whole lung tissue from 0.07 +/- 0.04 to 0.29 +/- 0.05 nmol/g, over the same interval. The increase in RA content was accompanied by an increase in RA receptor (RAR)-beta and -gamma mRNAs. The steady-state mRNA level of cellular retinol binding protein (CRBP) was high in LIF, relative to whole lung tissue at day 2. Cellular RA binding protein (CRABP) mRNA rose fourfold from day 2 to day 8 and then fell by day 12. In summary, RA, RAR, and CRBP mRNA in LIF are highest before the period of maximal elastin synthesis, which occurs at postnatal days 8 and 12. These findings are consistent with the hypothesis that endogenous RA could contribute to the postnatal increase in elastin production by pulmonary fibroblasts.

Clinical isolates of Stenotrophomonas (Xanthomonas) maltophilia showed growth temperature-dependent variation in susceptibility (TDVS) to aminoglycoside antibiotics between 30 degrees C and 37 degrees C, but little or no TDVS effect for polymixin B, colistin, ceftazidime, chloramphenicol and piperacillin. When phenylethanol was added at sub-inhibitory concentrations, the TDVS effect was eliminated. Gas liquid chromatography showed that 13-methyl tetradecanoate (i-15;0), was the predominant fatty acid, and was present in lower proportions in cells grown at 30 degrees C than 37 degrees C, by contrast to the unsaturated acids, which were found in increased proportions in cells grown at 30 degrees C. However, the extent of these shifts in composition did not correlate with the extent of the TDVS effect in individual strains. Membrane analysis by spin label-electron spin resonance spectroscopy showed that strains exhibiting TDVS had significantly decreased membrane fluidity compared with susceptible strains at 30 degrees C. Furthermore, analysis of the outer and cytoplasmic membranes from the strains with TDVS revealed that in organisms grown at 30 degrees C, the outer membrane remained in a more rigid conformation than the cytoplasmic membrane. We conclude that resistance of S. maltophilia to aminoglycoside antibiotics at 30 degrees C correlates with changes in the conformation of the outer membrane so that binding and/or uptake of the antibiotic is inhibited.

We have studied the effects of bacterial endotoxin on the oxygen consumption of a variety of target cells, and found that the rate of utilization of oxygen by treated cells was decreased in a time- and dose-dependent manner. Precise EPR measurement of oxygen concentrations enabled us to demonstrate that this effect was linked to mitochondrial dysfunction and was particular to each cell type. Such detailed knowledge on oxygen utilization by viable whole cells and the varied effects of endotoxin are as yet undocumented. Oxygen consumption was shown to decrease quite markedly in CHO cells and kidney cells from the cortex region. Cells from the kidney medulla region had lower baseline consumption and were stimulated to increased levels of oxygen consumption on addition of similar doses of endotoxin. Macrophages exhibited a dual response in that in addition to inhibiting mitochondrial oxygen consumption, endotoxin pretreatment primed these cells to exhibit an enhanced oxidative burst on stimulation with Zymosan. These results show that endotoxin has a direct effect on normal cellular oxygen consumption and is an important parameter that must be considered when following the early effects on cells and tissues during the septic syndrome.

Aims: Animal studies have shown that antigens present within the gut play an important role in the development of acute graft versus host disease (GvHD) following allogeneic bone marrow transplantation (BMT). In previous studies, inert sugars have been found to penetrate the small bowel mucosa after conditioning therapy for BMT; endotoxaemia can also occur during acute GvHD. Data on absorption of antigenic proteins across the gut following BMT in humans have not been presented as yet., Methods: Six patients undergoing allogeneic BMT were studied to determine whether enteric ovalbumin absorption increased or endotoxaemia developed during acute GvHD., Results: Three patients had minimal antigenaemia and no detectable endotoxaemia before receiving conditioning therapy. At the onset of acute GvHD, however, much higher ovalbumin concentrations were detected in those patients with severe antigenaemia. Serum concentrations of specific antiovalbumin IgG and IgA, or antiendotoxin IgM or IgG had no bearing on detectable IgG or IgM ovalbumin or endotoxin concentrations. In five of six patients, small bowel permeability increased, as tested by the lactulose/mannitol sugar absorption test, but detectable ovalbumin absorption increased in only three of these and only two developed endotoxaemia., Conclusions: Antigens present within the gut can cross the mucosal epithelium during acute GvHD, probably resulting in an enhanced immune response.

Objectives of this case-control study were (1) to examine membrane conformational changes and dynamics in platelets of patients with stroke prior to and following short-term aspirin administration and (2) to investigate changes in platelet membrane fluidity which may be associated with the ageing process. The study population consisted of 10 patients with ischaemic stroke (age range 61-92, mean 76 years; 6 females and 4 males), 9 age- and sex-matched controls (age range 64-88, mean 73 years; 5 females and 4 males), and 8 healthy young controls (age range 22-39, mean 32 years; 4 females and 4 males). The patients were recruited from the medical and geriatric wards, whereas both young and old controls were recruited from hospital staff and the local community. Aspirin, at a dose of 150 mg/day, was administered to all three groups for 7 days. The order parameter S, which is an index of membrane fluidity and conformation, was measured by the techniques of spin labelling and electron spin resonance spectroscopy on platelets from venous blood samples from patients prior to (within 48 h of admission) and following 7 days of aspirin therapy and from venous blood samples from both young and old controls before and after 7 days of oral aspirin therapy. There was an age-related decrease in platelet membrane fluidity. There was no significant difference in the value of 'S' between patients with ischaemic stroke and old controls. Treatment with aspirin led to a significant decrease in membrane fluidity in both patients and old controls, but had no apparent effect on the platelet membrane fluidity of younger controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of this study was to investigate the effects of interferon-gamma and -beta (IFN-gamma, -beta), interleukin-4 and -10 (IL-4, -10) and Hpopolysaccharide (LPS) on the metabolism and composition of phospholipid fatty acids in macrophages. Murine J774.2 macrophages were incubated with radiolabelled fatty acids and the appropriate stimulus and the incorporation and composition of the phospholipid classes was determined. IFN-gamma and IL-4 specifically stimulated enhanced incorporation of [(14)C]-linoleic acid into the phosphatidytethanolamine fraction. IL-4 (in contrast to IFN-gamma and LPS) reduced incorporation of [(14)C]- arachidonic acid into phosphatidylinositol. Incubation of J774.2 cells with linoleic acid significantly increased TNFalpha and nitric oxide production; arachidonic acid enhanced TNFalpha production but reduced nitric oxide production. It is concluded that IFN-gamma, IL-4 and IL-10 may differentially regulate macrophage activation via effects on the metabolism of polyunsaturated fatty acids.

We have previously shown that interferon-gamma (IFN-gamma) increases the polyunsaturated fatty acid content of membrane phospholipids in cells that were sensitive to endotoxin. In this study, IFN-gamma was found to stimulate the binding of endotoxin to the murine macrophage cell line J774.2 and the human monocyte cell line U937. Interferon-gamma-activated J774.2 cells showed a 66% increase in fluoresceine isothiocyanate (FITC) labelled LPS binding (P < 0.0005 vs control cells) and a 49% increase in tritium labelled LPS binding (P < 0.0001 vs control cells). Interferon-gamma also induced a 35% increase in binding of FITC-LPS in U937 cells (P < 0.0001 vs control cells). In contrast, pretreatment of J774.2 cells with interferon-beta (IFN-beta) had no effect on binding of FITC-LPS. Preincubation with exogenously supplied polyunsaturated fatty acids, linoleic and arachidonic acids, resulted in increases of 74% and 69% in FITC-LPS binding, respectively (both P < 0.0005 vs control cells). On the other hand, pretreatment with the saturated fatty acid, palmitic acid, had no effect on FITC-LPS binding. We propose that IFN-gamma-induced changes in the membrane phospholipid fatty acid composition of macrophage-like cells influence the binding of endotoxin.

Interferon-gamma (IFN-gamma) specifically induced the uptake of the unsaturated fatty acid [14C]linoleic acid into membrane phospholipids of the murine macrophage-like P388D cell lineage, but did not alter the incorporation of the saturated fatty acid [14C]stearic acid. Spin label ESR spectroscopy was used to examine any effects of these IFN-gamma-induced changes on membrane fluidity and the results revealed significant increases in plasma membrane fluidity. This alteration in membrane fluidity may have important consequences in the dynamic properties of cellular physiochemical interactions and some of the stimulatory effects of IFN-gamma on macrophages might be attributed to its effects on the plasma membrane composition.

When P388D macrophage cells were exposed to [14C]linoleic acid, phosphatidylcholine (PC) phosphatidylethanolamine (PE), phosphatidylinositol (PI), and triacylglycerol (TAG) were major labelled lipid classes. PC was the major labelled phospholipid class at shorter incubation times with PE and PI becoming proportionally better labelled with time. Interferon-gamma (IFN-gamma) stimulated the labelling of all phospholipid classes at the expense of triacylglycerol (TAG) labelling whether it was added coincidently to the [14C]linoleic acid or after a prelabelling period. A similar pattern of labelling of all major phospholipid classes by interferon-gamma in J774.2 macrophage cells was also observed. Interferon-gamma also exerted a stimulatory effect on incorporation of [14C]arachidonic acid into the phosphatidylinositol fraction of the membrane phospholipids. However, uptake of [14C]stearic acid was not different in control compared with IFN-gamma-activated cells. Uptake of linoleic acid into the plasmalogen fraction of PE was also considerably enhanced in IFN-gamma-treated cells. The results suggest that IFN-gamma has a direct effect on the activity of enzymes controlling fatty acid turnover in phospholipids. This altered uptake and turnover of unsaturated fatty acids may have important consequences for the subsequent activation of macrophages by endotoxin.

Endotoxin was measured in over 1000 plasma samples from bone marrow transplant patients in a randomized trial of the IgM-enriched intravenous immunoglobulin (IVIG) Pentaglobin. Peak endotoxaemia was significantly reduced (P = 0.02) in patients receiving Pentaglobin and 70% of all pyrexias of unknown origin were associated with endotoxaemia. Gut mucosal damage, assessed by lactulose/mannitol ratios, was significantly associated (P = 0.02) with endotoxaemia. Specific IgM antibody to endotoxin core-glycolipid was significantly raised (P < 0.01) in patients receiving the IVIG, and the IgM fraction of Pentaglobin was found to contain most of the anti-endotoxin antibody activity of the IVIG. These results suggest a role for IgM-enriched IVIG as a prophylactic agent for the reduction of endotoxaemia and its consequences in bone marrow transplant patients.

Reference strains (2) and 29 isolates of Aeromonas spp. from clinical material and environmental specimens were characterised in traditional biochemical tests, and in pyrolysis mass spectrometry, which gives data reflecting whole-cell composition. Numerical taxonomic analyses of the data sets were compared with conventional identification at species level, and pathogenic potential, as inferred from the origin of the isolates. Clustering with conventional test reaction patterns showed, for each of the species represented, a clearly defined core group of 'typical' isolates, surrounded by a halo of aberrant strains. One further cluster comprised strains intermediate between A. caviae and A. hydrophila, and one strain was grossly atypical in both analyses. Clustering from pyrolysis data corresponded less well with species identification. Broadly, the biochemical division between core and halo strains was supported in pyrolysis for A. caviae and A. sobria, but the main group of A. hydrophila in pyrolysis comprised strains clustering in the core and halo groups of this species, and three strains intermediate between A. hydrophila and A. caviae in biochemical tests. Two further pyrolysis clusters comprised core and halo strains of A. hydrophila. However, pyrolysis clustering correlated well with inferred pathogenicity, showing four clusters of probable pathogens, six clusters of probable non-pathogens, and one two member cluster of doubtful status. Most strains that clustered in the species haloes, or in species-intermediate groups in biochemical tests, were non-human isolates, or were isolated in the absence of symptomatic infection. The correlation of inferred pathogenicity with biochemical clustering was poorer than that with pyrolysis clustering.

Incubation of murine macrophages or the macrophage-like cell line P388D with interferon-gamma in vitro induced a significant increase in the polyunsaturated fatty acid content of phosphatidylethanolamine. These increases were time and dose-dependent, being maximal at 12 hours and with 5000 U/ml interferon and were inhibited in the presence of anti-interferon-gamma monoclonal antibody. Interferon-gamma induced a significant increase in linoleate in peritoneal macrophages while in the cell line arachidonate was significantly increased. These results are of interest because such increases in the polyunsaturated fatty acid content of phosphatidylethanolamine were previously shown by us to be associated with increased sensitivity to endotoxin in mice in vivo. The implications for interferon-gamma sensitizing to endotoxin are discussed.

The molecular mechanisms of endotoxin action are poorly understood. A prerequisite to cellular activation by this agent must be interaction (binding) with the plasma membrane. In this study we have investigated the role of the polysaccharide region of endotoxin (LPS) in binding to macrophages and macrophage-like cell lines. The LPS molecules, from Escherichia coli O111.B4, J5 and the lipid-A, were spin labelled with 2,2,6,6-tetramethylpiperidine-N-oxyl] (Tempo) free radical in their sugar residues, and examined by electron spin resonance spectroscopy. This is the first report of the synthesis of spin-labelled endotoxins. Measurement of the rotational correlation times (Tc) indicated that the saccharide resides do not bind to membrane surface structures and suggests that the binding of LPS to macrophages is mediated by the lipid acyl chains. Anti-sera to LPS from E. coli O111.B4 was effective in binding to the polysaccharide of the same LPS bound to the cell surface.

The mechanisms modulating host susceptibility to endotoxin are unknown. Evidence suggests that endotoxin pathophysiology is mediated in part by oxidative reactions that lead to tissue damage and organ failure. The proposition is that conditions which favour oxidation sensitise the host to endotoxin. Central to this hypothesis is that an increase in the polyunsaturated fatty-acid composition of membrane phospholipids enhances susceptibility because such fatty acids are easily oxidised to produce mediators of the endotoxic crisis. Cytokines, such as tumour-necrosis factor and interferon-gamma, may be ultimately responsible for orchestrating these changes and thereby modify the host response to endotoxin.

Serum antibody to lipopolysaccharide core glycolipid was measured in normal children and in full term and premature (less than or equal to 1500 g) infants. Antilipopolysaccharide core glycolipid antibody was present in term infants and normal children, and reached adult titres by 15 years of age. The specific anti core glycolipid antibody was predominately of the IgG class. Preterm infants (less than 32 weeks' gestation) had significantly lower titres of antilipopolysaccharide core glycolipid than more mature preterm or term infants. The results suggest that administration of anticore glycolipid immunoglobulin may be beneficial in the treatment or prevention of Gram negative septicaemia in preterm and very young infants.

Circulating antibody to endotoxin core glycolipid and total serum immunoglobulin concentrations were measured in 86 children with cancer (54 with acute lymphoblastic leukaemia, four with acute myeloid leukaemia, and 28 with various solid tumours). Measurements were made before treatment in the group with acute lymphoblastic leukaemia as well as when patients were both on and off chemotherapy. In the other two groups measurements were made when patients were both on and off treatment. Significant reductions in endotoxin antibody and serum immunoglobulin concentrations were found only in patients with acute lymphoblastic leukaemia. In addition, there was a significant correlation between febrile episodes and the concentration of antibody to core glycolipid in the children with acute lymphoblastic leukaemia. These findings suggest that the use of prophylactic high titre endotoxin antibody may be of benefit to children with life threatening Gram negative infections who are receiving cytotoxic chemotherapy.

The effects of endotoxin on mouse liver phospholipid fatty acid composition have been investigated. Administration of endotoxin from Salmonella abortus equi led to a decrease in the polyunsaturated fatty acid content of livers from mice sensitized with Bacille Calmette Guérin (BCG). The content of arachidonic acid fell significantly in both the phosphatidylcholine and phosphatidylinositol fractions whereas in the phosphatidylethanolamine fraction the linoleic acid content was significantly reduced. The polyunsaturated fatty acids were replaced by increased amounts of oleic acid and palmitic acid, leading to a reduction in the polyunsaturated to saturated fatty acid ratio.

Changes in the lipids of tissues from mice infected with bacille Calmette-Guérin (BCG) have been detected by gas-liquid chromatography. Infection with BCG resulted in (1) an increase in the polyunsaturated to saturated fatty acid ratio of phospholipids and (2) a decrease in the total triacylglycerol fatty acid content of spleen, liver and peritoneal macrophages. The alteration in fatty acid composition was significant in the phosphatidylethanolamine fraction of the phospholipids. The relation of these findings to an increased sensitivity to bacterial endotoxins is discussed.

Macrophages and hepatocytes from normal and BCG-primed mice have been spin-labelled in their membranes with 5- and 16-doxyl stearic acid. Incubation of spin-labelled cells from BCG-primed animals with lipopolysaccharide from E. coli 0111.B4 produced a detectable and transient disturbance in the cell membranes as reflected by an increase in the order parameter measured from the electron spin resonance spectra of 5-doxyl-stearate. This membrane disturbance was maximal at 3-4 hours of incubation and was only detected with cells from mice primed with BCG. Spectra obtained from the 16-doxyl-stearate-labelled cells showed no change in order parameter on incubation with lipopolysaccharide.

The K-stimulated release of [3H]ACh from rat midbrain slices prelabeled by incubation with [3H]choline was dependent on extracellular Ca. Phenobarbital inhibited the K-stimulated [3H]ACh release and the IC50 was equal to that found for K-stimulated endogenous ACh release. These results support the suggestion that barbiturates primarily inhibit the Ca-dependent stimulated release of ACh and affect ACh synthesis only indirectly. K-Stimulated release of [3H]5-HT was also inhibited by removing Ca from the medium or by adding phenobarbital which further supports the effects of barbiturates on the depolarization-induced release process. Fluoxetine, an inhibitor of 5-HT uptake, increased the amount of [3H]5-HT found in the medium but did not fully block the uptake of [3H]5-HT in this slice preparation.

Cultures of 10-day rat embryos depleted a protein band with a molecular weight of approximately 125,000 from the rat serum medium. Delayed centrifuged serum differed from immediately centrifuged serum by a reduction in the staining intensity of the 125,000 molecular weight protein band and by the absence of two high molecular weight (greater than 200,000) protein bands.

The production of oxygen radicals by Bacille-Calmette-Guerin primed mouse macrophages stimulated with bacterial endotoxin has been investigated. Superoxide radicals were spin-trapped in this system with dimethylpyrroline-N-oxide after a lag period of 20-40 minutes. The electron spin resonance signals due to the superoxide radical adduct could be inhibited by superoxide dismutase but not by catalase.