Infection of a knee prosthesis with Tsukamurella species.

A 69-year-old woman with a history of multiple infections of a postoperative wound from a knee replacement was diagnosed with an infection with Tsukamurella sp. The infection was treated with a course of vancomycin and pipercillin/tazobactam, followed by a course of clarithromycin, ciprofloxacin, and ethambutol. The patient responded well. This represents the first report of a Tsukamurella infection of an artificial joint.

Modulation of endothelin-1 production by a pulmonary epithelial cell line. I. Regulation by glucocorticoids.

Endothelin-1 (ET-1) is one of the most potent bronchoconstrictor agents yet described. Bronchial epithelial cells of asthmatic patients in vivo express preproET-1 and in vitro release high amounts of ET-1. Healthy and chronic bronchitic controls do not express preproET-1 or release ET-1. Interleukin-2 (IL-2) and other cytokines up-regulate the in vitro ET-1 release in guinea pig airway epithelial cells. We explored whether two glucocorticoids, dexamethasone (Dex) and triamcinolone acetonide (TA), inhibit the synthesis and release of ET-1 by A549 cells, a transformed human pulmonary epithelial cell line, since ET-1 may have a basic role in the pathogenesis of asthma. Cells were grown to confluence in RPMI 1640 plus 10% fetal bovine serum (FBS). Cells were then cultured for 3 days without serum to obtain ET-1 basal levels. The effects of 10% FBS, IL-2 (10 U/mL), Dex, TA or mifepristone, a steroid antagonist (1, 10 or 100 nM), were evaluated on ET-1 as measured by radioimmunoassay (RIA). ET-1 production increased from 57.6 +/- 5 pg/mg cell protein at 6 hr to 170 +/- 9 pg/mg cell protein at 72 hr in control cultures. Ten percent FBS increased ET-1 production from 58.7 +/- 9.6 to 399 +/- 14.5 pg/mg cell protein. IL-2 significantly increased ET-1 from 100.7 +/- 6.1 to 144 +/- 6.7 at 24 hr and from 170 +/- 9 to 207.7 +/- 24 at 72 hr. Dex and TA (10 and 100 nM) at 24-72 hr decreased ET-1 under basal conditions. Both drugs (only at 100 nM) decreased ET-1 production in 10% FBS- and IL-2-stimulated cells. Mifepristone (10 and 100 nM) reversed the decreased production of ET-1 induced by Dex (100 nM) at 24-72 hr. Northern blot analysis showed that Dex (100 nM) decreased the expression of ET-1 mRNA at 6 and 24 hr, but that mifepristone (100 nM) reversed this effect in cells cultured with Dex. In conclusion, Dex and TA down-regulate the synthesis and production of ET-1 by this human pulmonary epithelial cell line under basal or stimulated conditions, and these effects are reversed by mifepristone. These findings suggest a novel mechanism of glucocorticoid effect during the treatment of asthma.

Multiple high-affinity cAMP-phosphodiesterases in human T-lymphocytes.

Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that inactivate intracellular cyclic AMP (cAMP). Because the functions of T-lymphocytes are modulated by cAMP levels, the isozymes of PDE in these cells are potential targets for new drugs designed to modify the body's immunity through selective alteration of T-lymphocyte PDE activity. Cyclic GMP and 3(2H)-pyridazinone-4,5- dihydro-6-[4-(1H-imidazol-1-yl)phenyl]-5-methyl-monohydrochloride (CI-930) selectively inhibit the catalytic activity of one of the two high affinity cAMP-PDE isozyme families known to occur in mammals, whereas d,l-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone (Ro 20-1724) selectively inhibits the other. The objectives of this investigation were: (1) to determine whether human T-lymphocytes contain one or both of these pharmacologically distinguishable high-affinity cAMP-PDEs, and (2) to determine the effects of selective inhibitors of these PDEs on lymphocyte blastogenesis. High-affinity cAMP-PDE was found in both the soluble and particulate fractions of T-lymphocyte sonicates. Cyclic GMP and CI-930 inhibited PDE in the particulate fraction better than in the soluble fraction, but the converse was found for Ro 20-1724. CI-930 or Ro 20-1724, used alone, attenuated T-lymphocyte blastogenesis, but neither suppressed it completely. In combination, the same PDE inhibitors caused greater suppression of blastogenesis than either produced alone. The results indicate that human T-lymphocytes contain both CI-930- and Ro 20-1724-inhibitable isozymes. Either of the isozymes can modulate human T-lymphocyte blastogenesis, but inhibition of both isozymes produces synergistic antiblastogenic effects.

High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes.

The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low Km cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,1-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC50 = 17 microM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes.

Beta-adrenergic receptors in nasal tissue with reference to their role in asthma and atopy.

Thirty-three samples of nasal tissue (turbinates and polyps) were collected and assayed for beta-adrenergic receptor (BAR) sites (radio-ligand assay). Patients were divided into groups: 1) nasal turbinates, 2) nasal polyps, both under the aspects of atopy and bronchial hyperreactivity. Different radiobinding was found in both groups: Patients A with high BAR and patients B with low BAR numbers. There was no correlation between BAR numbers--clinical symptoms and bronchial hyperreactivity. Generally the BAR numbers were higher in turbinates (p less than 0.01) than in polyps and in nonatopic compared to atopic patients (p less than 0.005). "Affinity state 1" BAR are equal in all groups; however, "affinity state 2"BAR are significantly less in polyps (p less than 0.05). In 50% of the examined cases there was a combination of polyps, asthma and atopy putting foreward a common line of pathogenesis in this entity. It is discussed that the underlying inflammation (allergic and non-allergic) is the cause for reduced BAR numbers in the effected tissue. So far nasal tissue seems to be like a mirror compared to the results from lower respiratory tract tissue with respect to beta-adrenergic receptors.

Effects of ketotifen on the responsiveness of peripheral blood lymphocyte beta-adrenergic receptors.

The effects of ketotifen therapy on the responsiveness of lymphocyte beta-adrenergic receptors was evaluated by measuring cyclic AMP elevations caused by isoproterenol in cells isolated from patients treated with ketotifen for more than 1 year. Binding of 3H-dihydroalprenolol to beta-receptors was also evaluated. The isoproterenol-induced rise in cyclic AMP relative to each individual's baseline level was greater in patients on current ketotifen therapy than in other asthmatic patients or non-asthmatic subjects. Ketotifen therapy increased the apparent equilibrium dissociation constant for specific 3H-dihydroalprenolol binding to the receptors. Receptor numbers in symptomatic asthma patients on standard drug therapy were decreased. The results indicate that long term ketotifen therapy is associated with increased responsiveness of beta-receptors to stimulation by catecholamines and that this alteration may involve changes in the receptors themselves, their membrane environment, adenylate cyclase or components of the adenylate cyclase coupling system.

Interferon induction by endotoxin-derived nontoxic polysaccharides.

Polysaccharide-rich preparations from Serratia marcescens-derived endotoxin and components thereof, including Lipid A, were studied in terms of their ability to induce interferon (IFN) activity in murine spleen cell cultures in vitro. Although the polysaccharide-rich derivatives, similar to intact endotoxin, were only weak stimulators for IFN induction, pretreatment of splenocyte cultures with recombinant interleukin-2 (IL-2) significantly increased IFN-inducing activity. Both an endotoxin-derived polysaccharide and a "White type" polysaccharide prepared from intact Serratia had similar ability to induce IFN in vitro, but only when spleen cells were first treated with IL-2. The polysaccharide preparations were nontoxic as compared with the high degree of toxicity of the intact endotoxin, yet induced similar IFN levels as whole endotoxin. Much of the IFN induced by these preparations was of the gamma type, since activity was either not neutralized or only incompletely neutralized by treatment with anti-alpha/beta interferon antibody.

Effects of adenosine and theophylline on canine tracheal smooth muscle tone.

The postulated mechanisms by which theophylline induces relaxation of airway smooth muscle include, among others, inhibition of cyclic nucleotide phosphodiesterase(s) and antagonism of adenosine-induced contraction. This latter possibility was examined by investigation of the interaction of theophylline and adenosine in canine tracheal smooth muscle preparations. Adenosine did not alter basal tone i.e. there is no evidence of a contractile response. However, when contraction was induced with methacholine, adenosine caused relaxation. It appears that this relaxation occurred as a consequence of the combination of adenosine with a site within the smooth muscle cell. The prior addition of theophylline (10(-6)-10(-3) M) did not alter adenosine-induced relaxation and in the reverse experiment, the prior addition of adenosine (10(-6)-10(-3) M) did not alter the relaxation produced by theophylline. It is concluded that adenosine relaxes canine tracheal smooth muscle by combination with an intracellular site, rather than a receptor on the cell surface. The hypothesis that theophylline relaxes airways smooth muscle by antagonism of adenosine or that adenosine antagonizes theophylline was not supported by our data.

The mechanism of ergonovine-induced airway smooth muscle contraction.

There have been three articles in the clinical literature of ergonovine maleate-induced bronchospasm. The effect of the alkaloid on isolated canine tracheal smooth muscle was analyzed to investigate the mechanism of ergonovine-induced airway smooth muscle contraction. Both ergonovine and 5-hydroxytryptamine (5HT, serotonin) contracted the smooth muscle preparations with EC50s of 1.35 X 10(-8) mol/L and 5.06 X 10(-7) mol/L, respectively. The maximal contractile response observed with ergonovine was approximately 30% less than that observed with 5HT. Methysergide competitively blocked both ergonovine and 5HT responses with similar calculated pKB values (8.33 against ergonovine and 8.46 against 5HT) and also similar pA2 values determined by Schild plots (8.50 and 8.45, respectively). The relative affinity and efficacy of ergonovine versus 5HT were determined by use of a concentration of the irreversible antagonist, phenoxybenzamine, which partially blocked receptor sites. The calculated affinity of ergonovine was about 16 times higher than that of 5HT. The relative efficacy at EC100 for ergonovine was 0.2, but at EC10 it was 41.9 (5HT efficacy = 1). Ergonovine 10(-9) or 10(-8) mol/L shifted the 5HT dose-response curve to the right without reducing the maximal response, but the shift was nonparallel. Blockade of muscarinic (atropine), alpha 1-adrenergic (prazosin), beta-adrenergic (propranolol), H1 (pyrilamine), or H2 (cimetidine) receptors did not alter ergonovine-induced contraction. These data indicate that ergonovine directly contracts canine tracheal smooth muscle as a result of its combination with 5HT receptors. This effect may result in precipitation of an asthmatic attack in susceptible individuals.

The mechanism of Ptychodiscus brevis toxin-induced rat vas deferens contraction.

Ptychodiscus brevis, which causes Florida red tide, produces Ptychodiscus brevis toxin (PBTX) known to contain neurotoxins and to induce rhinorrhea, tearing, and cough in normal humans and wheezing in asthmatic subjects. It was previously reported (J Allergy Clin Immunol 69:418, 1982; 73:824, 1984) that PBTX causes canine tracheal smooth muscle contraction via stimulation of sodium channels in the axons of parasympathetic postganglionic nerves and the release of acetylcholine from these nerve endings. This was postulated to be an asthma-triggering mechanism. In this article the toxins were evaluated to determine if they also stimulate sodium channels on adrenergic nerve endings and release norepinephrine. Rat vas deferens was selected as the experimental tissue. Both PBTX and norepinephrine contracted rat vas deferens. Prazosin 10(-6) mol/L blocked the response to PBTX (3 micrograms/ml) (88.3% to 27.3% contraction [n = 6; p less than 0.001]) and the response to norepinephrine (EC50 was shifted from 1.67 X 10(-6) mol/L to 1.25 X 10(-4) mol/L in the presence of prazosin 10(-6) mol/L [n = 6; p less than 0.001]). Phentolamine 10(-6) mol/L also blocked both PBTX and norepinephrine. Tetrodotoxin 10(-7) mol/L, a sodium channel blocker, completely blocked the response to PBTX but not to norepinephrine. The response to PBTX was significantly reduced from 1.53 gm of tension in controls to 0.29 gm of tension (n = 6; p = 0.002) in tissues obtained from rats pretreated with reserpine (2 mg/kg per day for 2 days, injected intraperitoneally). Verapamil 10(-5) mol/L blocked the PBTX response, and PBTX caused no contraction in calcium-free media.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between inhibition of a cyclic GMP phosphodiesterase and relaxation of canine tracheal smooth muscle.

Inhibition of partially purified cyclic nucleotide phosphodiesterase activity as well as pharmacologically induced relaxation of respiratory airways smooth muscle was examined to determine whether any correlation between these two effects could be found. The phosphodiesterase in extracts of canine tracheal smooth muscle was chromatographed on a DEAE Bio-Gel A column and eluted with a sodium chloride gradient. The peak I activity hydrolyzed cGMP at a higher rate than cAMP although the apparent Km values for these two cyclic nucleotides were relatively close. Comparison of the Ki values for alkylxanthine inhibitors of peak I activity correlated remarkably well with the EC50 values of the same compounds as relaxants of canine tracheal smooth muscle strips. It is concluded that inhibition of the peak I enzyme may cause accumulation of an intracellular pool of cyclic nucleotide and thus produce or contribute to the muscle relaxant effects that were observed.

The site of action of Ptychodiscus brevis toxin within the parasympathetic axonal sodium channel h gate in airway smooth muscle.

The red tide toxin produced by Ptychodiscus brevis ( PBTX ) may cause cough, sneezing, and asthma. Previous in vitro studies with isolated canine tracheal smooth muscle demonstrated that PBTX stimulates sodium channels of parasympathetic nerve endings and thus causes a contractile response. The present study investigated the mechanism of the PBTX effect on canine tracheal smooth muscle. Repeated exposure of the muscle strip to PBTX (final concentration 46 micrograms/ml) followed by washout of the toxin resulted in reestablishment of baseline tension but a failure of contraction on further addition of PBTX . However, veratridine and scorpion toxin (SCT), which are voltage-sensitive sodium channel activators, still induced contraction. Furthermore, the contraction caused by veratridine was enhanced by a high dose of PBTX , whereas contraction caused by SCT was not. Responses to veratridine and SCT as well as PBTX (previously reported) were blocked by tetrodotoxin (a sodium channel blocker), while acetylcholine responsiveness remained intact. These results indicate that PBTX receptors in parasympathetic nerves influence Na+ flux at the h gate, that these receptors differ from the veratridine and SCT receptors, and that the conformational change in the receptors induced by PBTX affects the tissue response to veratridine.

Investigation of adrenergic and prostaglandin influences in the endotoxin alteration of hepatic heme oxygenase, microsomal mixed-function oxidase, and glucocorticoid-induced tryptophan oxygenase activities.

The possible role for adrenergic influences or prostaglandins in the effects of endotoxin to inhibit the glucocorticoid induction of hepatic tryptophan oxygenase (TO) activity, to decrease the hepatic microsomal cytochrome P--450-dependent drug-metabolizing activity, and to induce heme oxygenase activity was examined. Administration of the alpha-adrenergic locking agents phenoxybenzamine or phentolamine attenuated the inhibitory effect of the bacterial lipopolysaccharide on the induction of TO activity by dexamethasone. Injection of a beta-adrenergic blocker, propranolol, or of indomethacin, an inhibitor of prostaglandin biosynthesis, accentuated the effect of endotoxin to inhibit TO induction. Neither phenoxybenzamine, propranolol, nor indomethacin altered the effect of endotoxin to decrease aniline hydroxylase activity, ethylmorphine N-demethylase activity, or the levels of cytochrome P--450. Also, dexamethasone administration did not significantly protect against the effects of endotoxin on the hepatic microsomal drug metabolizing enzyme system, and none of the pharmacological agents diminished the effects of endotoxin to induce hepatic heme oxygenase activity. Endotoxin administration was also shown to diminish, but not prevent, the induction of cytochrome P--450 and ethylmorphine N-demethylase activity produced by phenobarbital. The results indicate that alpha-adrenergic mechanisms are involved in the endotoxic inhibition of the glucocorticoid induction of TO activity and suggest that neither adrenergic influences nor prostaglandins play a significant role in the effect of endotoxin to decrease hepatic mixed-function oxidase activity.

The effect of prostaglandin E2 on histamine-stimulated calcium mobilization as a possible explanation for histamine tachyphylaxis in canine tracheal smooth muscle.

Isolated strips of canine tracheal smooth muscle rapidly lost their responsiveness to histamine when placed in a zero calcium Krebs buffer. Responsiveness to acetylcholine, however, was not rapidly lost, and following 120 min of incubation in zero calcium buffer with frequent washes, 10% of the contractile response still remained. The kinetics of each loss of response suggest that primarily a loosely bound source of calcium is mobilized by histamine and a more tightly bound source is mobilized by acetylcholine. Consistent with these data were the effects of the calcium antagonist verapamil. In normal calcium Krebs solution, dose-response curves to histamine were markedly reduced by verapamil while acetylcholine responses were relatively unaffected. In calcium depleted tracheal strips, indomethacin potentiated the calcium dose-response curve, determined by incremental readdition of calcium in the presence of histamine (10(-4) M), with comparatively little effect on the calcium dose-response curve in the presence of acetylcholine (10(-6) M). Also, in indomethacin pretreated tracheal strips, a reduction in the histamine-calcium dose-response curve could be produced by exogenous addition of 2.8 X 10(-9) M and 2.8 X 10(-8) M PGE2. In the acetylcholine-calcium responses there was a significant reduction only at 2.8 X 10(-8) M PGE2. These data suggest that histamine mobilizes primarily a loosely bound, possibly extracellular source of calcium necessary for contraction, and this histamine-stimulated calcium mobilization is sensitive to the effects of PGE2.

Inhibition of a high affinity cyclic AMP phosphodiesterase and relaxation of canine tracheal smooth muscle.

The cyclic nucleotide phosphodiesterase (PDE) activity of canine tracheal smooth muscle (CTS,) was examined. Column chromatography of soluble CTSM-PDE revealed five peaks of activity. One of these peaks (V) was examined further in this study and showed a high affinity for adenosine 3',5'-cyclic monophosphate (Km = 0.63 microM). Seven pharmacological PDE inhibitors were tested for their abilities to inhibit the peak V enzyme and also for their abilities to cause mechanical relaxation of CTSM strips in isolated tissue baths. A strong correlation (P greater than 0.001) between peak V PDE inhibition (-log Ki) and airway muscle relaxation (-log ED50) was found.

Effects of toxin of red tide, Ptychodiscus brevis, on canine tracheal smooth muscle: a possible new asthma-triggering mechanism.

The red tide toxin produced by Ptychodiscus brevis becomes airborne by the thrashing action of the surf and wind and induces cough, rhinorrhea, watery eyes, and sneezing in normal humans and wheezing in asthmatic patients. The mechanism of the contractile response induced by P. brevis toxin (PBTX) was investigated with isolated canine tracheal smooth muscle. Tetrodotoxin and atropine blocked the contractile effect of PBTX, and neostigmine potentiated the contraction. Mepyramine, phentolamine, methysergide, and chlorisondamine did not inhibit the effect of PBTX. This is the first description of a naturally occurring airborne substance that causes smooth muscle contraction by stimulating the axon sodium channels, resulting in the release of acetylcholine at postganglionic parasympathetic efferent nerve endings. The in vitro effect of PBTX on canine tracheal smooth muscle indicates that PBTX is capable of causing respiratory irritation and thus may precipitate an asthmatic attack. It is possible, however, that the mechanism is vivo may also include stimulation of a cough receptor reflex and/or stimulation of sodium channels of afferent vagus nerve fibers. In vitro evidence suggests that isoproterenol, atropine, and verapamil may be used to eliminate or prevent the respiratory symptoms that follow exposure to airborne red tide toxin. The use of high-pressure liquid chromatography separated fractions indicates that the neurotoxic component, not the hemolytic component, is responsible for contractions.