Resolvin D2 promotes host defense in a 2 - hit model of sepsis with secondary lung infection.

In the development of sepsis, there is early, massive inflammation which can lead to multiple organ failure. Later there is an immunosuppressed phase where the host is susceptible to secondary infections or is unable to clear existing infection. Specialized Pro-resolving Mediators (SPMs) are endogenously produced lipids which resolve infection by decreasing bacteria load and reducing systemic inflammatory response. There has been little work studying if SPMs given late, can promote host defense. We examined if an SPM, Resolvin D2 (RvD2) could promote host defense in a 2-hit mouse model of cecal ligation and puncture (CLP) sepsis and secondary Pseudomonas aeruginosa lung infection. RvD2 given 48 h after mild CLP (1st hit), increased gene expression of Toll-like receptor-2 (TLR-2) and alveolar macrophage/monocyte phagocytic ability compared to CLP mice given saline vehicle. In this model, RvD2 did not affect plasma IL-6 or IL-10. These effects induced by RvD2, lowered lung bacterial load and decreased mortality after the secondary infection of Pseudomonas aeruginosa (2nd hit). Splenic T-cell numbers were also increased in RvD2 treated mice compared to saline vehicle treated animals. The results suggest that RvD2 promoted mechanisms of host defense in a 2-hit model sepsis and secondary lung infection.

Lipoxin A4 promotes reduction and antibiotic efficacy against Pseudomonas aeruginosa biofilm.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium commonly found in wound infections and airways of cystic fibrosis patients. P. aeruginosa readily forms biofilms which can reduce the efficacy of antibiotics used to eradicate the pathogen. We have previously shown that a Specialized Pro-resolving Mediator (SPM), Lipoxin A4 (LxA4) is a quorum sensing inhibitor which can reduce P. aeruginosa virulence. In this study, we examined the direct actions of LxA4 and RvD2 on P. aeruginosa biofilm formation and virulence gene expression. The influence of LxA4 on antibiotic efficacy and the combined effects on biofilm formation were also investigated. LxA4 and RvD2 reduced P. aeruginosa biofilm formation and virulence gene expression. LxA4 increased ciprofloxacin inhibition on biofilm formation but did not affect ciprofloxacin's action on non-adherent bacteria. On the other hand, LxA4 increased bacterial killing action of imipenem but did not affect imipenem's action on biofilm. We also found that LxA4 can increase ciprofloxacin's bacterial killing ability in established biofilm. Together these results suggest that LxA4 has direct effects on P. aeruginosa biofilm formation and can increase antibiotic efficacy directly.

Sign of lipid peroxidation as measured in the urine of patients with probable Alzheimer's disease.

Free radical-induced oxidative damage may be involved in the neurodegenerative process associated with Alzheimer's disease (AD). 8-Isoprostaglandin F(2alpha) (iPF(2alpha)-III) is an isoprostane derived from free radical-induced non-enzymatic oxidation of arachidonic acid. It is formed in vivo and is an indicator of lipid peroxidation. Measurements were made of iPF(2alpha)-III in the urine of patients with mild to moderate dementia associated with probable AD and compared to those in the urine of non-demented subjects, who were similar in age and gender. 2,3-Dinor thromboxane B(2) (dinor TXB(2)), a urinary metabolite of TXB(2) was also measured, and served as an indicator of the enzymatic transformation of a product of arachidonic acid. Enzyme linked immunoassays were used to measure iPF(2alpha)-III and dinor TXB(2) in the urine. The concentration of iPF(2alpha)-III was significantly elevated in urine of patients assessed to have mild to moderate dementia as compared to non-demented patients. The concentration of urinary dinor TXB(2) was also significantly elevated in the patients with dementia and probable AD as compared to the non-demented subjects. There was considerable overlap of values obtained for demented and non-demented patients for iPF(2alpha)-III and dinor TXB(2), respectively. The observed elevation of iPF(2alpha)-III suggests that patients with mild to moderate dementia associated with probable AD are experiencing significant oxidative stress. This finding is consistent with current data suggesting that oxidative stress may be occurring in patients with dementia and probable AD. The increase of dinor TXB(2) may indicate that enzymatic processes related to the metabolism of arachidonic acid-derived products are also increased in demented patients with probable AD.

The percutaneous penetration of prostaglandin E1 and its alkyl esters.

The percutaneous delivery of PGE1 and its alkyl esters in alcoholic saline solution through hairless mouse skin was compared. The quantification of alkyl esters was based on the same principle as that for PGE1, which was converted to PGB1 to enhance the sensitivity and minimize the interference. Results showed that it was PGE1 that appeared in the receiver compartment for all alkyl esters examined. The flux of all alkyl esters of PGE1 in the same concentration was higher than PGE1 itself at most of saline vehicle with various fractions of alcohol. The maximal flux for a fixed concentration of each alkyl ester appeared at different fractions of alcohol. When the fractions of alcohol was kept constant, the alkyl ester that showed the maximal flux at this concentration appeared to have a longer chain length with increasing the fraction of alcohol. But isopropyl ester deviated from this order. It was concluded that the alkyl ester derivatives promoted the penetration of PGE1 mainly as a result of enhancing the drug partitioning into the stratum corneum. The alcohol fraction that needed to achieve the maximal flux at the same concentration increased with the increase of alkyl chain length, which resulted in the decrease of solubility parameter. It is necessary to optimize the fraction of alcohol in the saline solution in order to achieve the maximal flux at a fixed concentration for these alkyl esters with different alkyl chain length.

Comparison between oral and vaginal administration of misoprostol on uterine contractility.

OBJECTIVE: To compare the degree of absorption and the effect on uterine contractility of the prostaglandin E1 analogue misoprostol after vaginal and oral administration. METHODS: Thirty women with a normal intrauterine pregnancy between 8 and 11 weeks' gestation who requested termination of pregnancy were given either 0.2 mg (orally n = 5; vaginally n = 6) or 0.4 mg (orally n = 10; vaginally n = 9) of misoprostol. Intrauterine pressure was recorded using a Grass polygraph connected to a pressure transducer 30 minutes before misoprostol was given and for 4 hours thereafter. At the end of the recording, suction curettage was performed. Blood samples were obtained at 0, 0.5, 1, 2, 4, and 6 hours for measurement of misoprostol, which was assayed by high-pressure liquid chromatography-mass spectrometry. RESULTS: In all patients, the first effect was an increase in uterine tonus. After 0.4 mg of misoprostol administered orally, uterine tonus started to increase after a mean (+/- standard deviation) time of 7.8+/-3.0 minutes and reached its maximum after 25.5+/-5.0 minutes. The corresponding times after vaginal administration were 20.9+/-5.3 minutes and 46.3+/-20.7 minutes, respectively. The initial increase in tonus was also more pronounced after oral than after vaginal administration. After vaginal administration, all patients developed uterine contractions; the activity, measured in Montevideo units, increased continuously during the observation period. This was not the case after oral administration. Plasma levels of misoprostol were measured in 18 patients. The highest levels were found 30 minutes after oral treatment and 1-2 hours after vaginal administration. CONCLUSION: The long-lasting and continuously increasing uterine contractility after vaginal administration can be explained only in part by a direct effect of misoprostol. The longer period of elevated plasma levels of misoprostol may also have initiated the prolonged events leading to increased uterine contractility.

Investigation of the percutaneous penetration of prostaglandin E1 and its ethyl ester.

The optimization of percutaneous delivery of PGE1 and its ethyl ester in alcoholic buffer solution through hairless mouse skin was investigated. A reversed-phase HPLC system with a photodiode array detector was used to differentiate the UV spectra of the penetration products. By comparison of the UV spectrum for each chromatographic peak, the conversion of PGE1 ethyl ester to PGE1 by enzymatic hydrolysis was found to be the predominant degradation pathway during the in vitro penetration. The quantification of ethyl ester was developed based on the same principle as that for PGE1. It was then applied to monitor the penetration of prostaglandins through hairless mouse skin from the vehicles containing various fractions of alcohol. Results demonstrated that the alkyl group promoted the penetration mainly as a result of enhancing the drug partitioning into the stratum corneum at its maximal thermodynamic activity. The alcohol fraction around 20% seemed to be optimal for the percutaneous delivery of the ethyl ester. The use of collagen gel to carry PGE1 ethyl ester for percutaneous application was included for comparison. The effect of adding alcohol in the collagen gel on the penetration of PGE1 ethyl ester was found to be slightly lower than that from the same vehicle without collagen.

Stimulation of protein kinase C redistribution and inhibition of leukotriene B4-induced inositol 1,4,5-trisphosphate generation in human neutrophils by lipoxin A4.

1. To test the hypothesis that protein kinase C (PKC) is involved in the inhibitory actions of lipoxin A4 (LXA4) on second messenger generation, we studied the effects of LXA4 on PKC in human neutrophils and on leukotriene B4 (LTB4)-stimulated inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) generation. 2. LXA4, 1 microM, caused a fall in cytosolic PKC-dependent histone phosphorylating activity to 23.5% of basal levels. 3. LXA4, caused an increase in particulate PKC-dependent histone phosphorylating activity with a bell-shaped dose-response fashion; maximal stimulation was observed at 10 nM LXA4. 4. Western blot analysis with affinity-purified antibodies to alpha- and beta-PKC showed that only the beta-PKC isotype was translocated by LXA4. 5. LXA4 inhibited LTB4-stimulated Ins(1,4,5)P3 generation in a bell-shaped fashion with maximal inhibition at 1 nM LXA4. The observed inhibition was dose-dependently removed by pre-incubation with a PKC inhibitor (Ro-31-8220). 6. These results show that LXA4 activates PKC in whole cells and supports a role for PKC activation in the inhibitory action of LXA4 on LTB4-induced Ins(1,4,5)P3 generation. 7. LXA4 (1-1000 nM) pre-incubation did not affect specific binding of [3H]-LTB4 to neutrophils. Thus, the inhibitory effect of LXA4 on LTB4-stimulated Ins(1,4,5)P3 generation could not be attributed to an effect on LTB4 receptors.

Antithrombotic effects of peroxynitrite: inhibition and reversal of aggregation in human platelets.

The inhibition of platelet aggregation by peroxynitrite, a reactive oxygen species derived from the interaction of nitric oxide (NO) and superoxide, was examined in platelet-rich plasma. In this report, we have used a preparation of peroxynitrite that was free of H2O2 and MnO2. As such, peroxynitrite dose-dependently (50-200 microM) inhibited aggregation of human platelets stimulated by ADP (5 microM), collagen (0.5 microgram), thrombin (0.5U/microL) and U46619 (1 microM). In addition, peroxynitrite reversed platelet aggregation induced by collagen, ADP, and thrombin. Peroxynitrite, preincubated with platelet-poor plasma or albumin (7%) for 30 min, did not alter the inhibition of platelet aggregation. This suggested that the inhibitory action of peroxynitrite may be due to nitrosylation of proteins, which by themselves possess activity, rather than conversion to NO or NO donors. Furthermore, we show that peroxynitrite increased the cGMP level only at 200 microM concentrations, further suggesting that the action of peroxynitrite was not completely due to its conversion to NO or NO donors.

Effects of lipoxin A4 on chemotaxis and degranulation of human eosinophils stimulated by platelet-activating factor and N-formyl-L-methionyl-L-leucyl-L-phenylalanine.

Lipoxins are trihydroxytetraene metabolites derived through a double lipoxygenation of arachidonic acid. Lipoxin A4 (LXA4) was prepared by total chemical synthesis, and its capacity to modulate eosinophil migration has been evaluated. LXA4 is a weak and partial chemotactic agent; at 10(-6) M, it achieved about 20% of the response of 10(-6) M platelet-activating factor (PAF). Preincubation of eosinophils with increasing doses of LXA4 (10(-10)-10(-5) M) resulted in a concentration-dependent inhibition of cell migration induced by 10(-6) M formyl-methionyl-leucyl-phenylalanine (FMLP) and 10(-6) M PAF. The concentration of LXA4 which produced 50% inhibition (IC50) of eosinophil migration was approximately 10(-6) M. LXA4 (10(-10)-10(-6) M) did not elicit ECP release or modulate ECP release induced by 10(-6) M FMLP. LXA4 may have antiallergic properties in preventing eosinophilic migration.

Structure/activity relationship of leukotriene B4 and its structural analogues in chemotactic, lysosomal-enzyme release and receptor-binding assays.

The biological activities of chemically synthesized leukotriene B4 and eight structural analogues have been studied using chemotaxis, lysosomal-enzyme release and receptor-binding assays on human neutrophils. The results show that increasing the number of double bonds between C14 and C20, having triple bonds at C6 or C14, substitution of the primary carboxyl group at C1, changing the geometry of the double bond at C6 from the cis to trans configuration and changing the chirality of the hydroxyl group at C12 from the R to the S configuration result in substantial loss of both biological activity and the capacity to bind to the LTB4 recognition site in parallel. We suggest that the functional epitopes of 5S,12R-dihydroxy-6,14-cis-8,10-trans-icosatetraenoic acid (LTB4) are either the same, or reside in the same domain as the binding site for the LTB4 receptor. Development of LTB4 antagonists to the high-affinity LTB4 receptor, based on the structure of LTB4, is unlikely to be successful.

Airway responsiveness to leukotriene C4 (LTC4), leukotriene E4 (LTE4) and histamine in aspirin-sensitive asthmatic subjects.

We wanted to determine whether the airway response to inhaled leukotriene C4 (LTC4) is similar to inhaled leukotriene E4 (LTE4) in aspirin-sensitive asthma and, therefore, determined airway responsiveness to histamine, LTC4 and LTE4 in seven aspirin-sensitive subjects and 13 control asthmatic subjects, who were tolerant of aspirin. The concentration of inhaled lysine-aspirin which produced a 15% fall in forced expiratory volume in one second (FEV1) (PC15) was determined in aspirin-sensitive asthmatic subjects. The dose of histamine, LTC4 and LTE4 which produced a 35% fall in specific airways conductance (PD35sGaw) was determined by linear interpolation from the log dose response curve. There was no correlation between the PC15 for lysine-aspirin and the airway reactivity to inhaled LTC4 or LTE4. There was no difference in airway response to histamine and LTC4 between any of the groups of asthmatic subjects. There was a rank order of potency LTC4 > LTE4 > histamine in both groups, with LTC4 approximately 1,000 fold more potent than histamine in both groups. Aspirin-sensitive asthmatic subjects were significantly more responsive to LTE4 (p = 0.02) than aspirin-tolerant asthmatic subjects. The relative responsiveness of LTE4 to histamine (PD35 histamine/PD35 LTE4) was significantly greater in aspirin-sensitive asthmatic subjects compared to aspirin-tolerant asthmatic subjects (p = 0.05). There was no difference in relative responsiveness of LTC4 to histamine between aspirin-sensitive or aspirin-tolerant asthmatic subjects. We conclude that the airways of aspirin-sensitive asthmatic subjects demonstrate a selective hyperresponsiveness to LTE4, which is not observed for LTC4.

Leukotriene E4 and granulocytic infiltration into asthmatic airways.

Sulphidopeptide leukotrienes are potent bronchoconstrictors and increase bronchial hyperreactivity, one of the hallmarks of asthma. We have demonstrated that leukotriene LTE4, the most stable of the sulphidopeptide leukotrienes, elicited an increase in the numbers of eosinophils and neutrophils in the lamina propria of the airway mucosa 4 h after inhalation in 4 asthmatic subjects. The numbers of eosinophils were, on average, 10-fold greater than those of neutrophils. There was no significant change in numbers of lymphocytes, plasma cells, mast cells, or macrophages. Since LTE4 recruits granulocytes, the potential of antisulphidopeptide leukotriene drugs as anti-inflammatory and "steroid-sparing" agents should be tested.

Effect of indomethacin on leukotriene4-induced histamine hyperresponsiveness in asthmatic subjects.

The effect of indomethacin on the capacity of LTE4 to enhance airway histamine responsiveness was evaluated in eight mild asthmatic subjects. Subjects attended the laboratory on three separate pairs of study days when inhalation challenges with methacholine or LTE4 were performed and the airway responses to histamine were measured 4 and 7 h later. An open pair of study days was followed by a pair of study days during ingestion of either placebo or indomethacin capsules. The dose of agonist that produced a 35% fall in specific airways conductance (PD35 SGaw) was obtained by linear interpolation from the logarithmic dose-response curve. Indomethacin treatment did not affect baseline SGaw or methacholine airway responsiveness. However, indomethacin significantly inhibited LTE4-induced histamine hyperresponsiveness. Maximum enhancement of histamine responsiveness by LTE4 on the open and placebo study days was 4.1 +/- 0.9- (mean +/- SEM) and 5.7 +/- 1.2-fold, respectively (p = 0.36). Maximal enhancement on the indomethacin day was 1.68 +/- 0.46, and this was significantly decreased compared with that on the placebo day (p = 0.02). This suggests that LTE4-induced enhanced responsiveness to histamine is mediated in part by cyclooxygenase pathway-derived products.

The effect of epithelium removal on leukotriene E4-induced histamine hyperresponsiveness in guinea-pig tracheal smooth muscle.

1. Removal of the epithelium resulted in a threefold increase in guinea-pig tracheal sensitivity to histamine without increasing the maximal response. 2. Preincubation of epithelially-denuded guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased the subsequent maximal response of the tissues to histamine. The sensitivity of the tissues to histamine was unaffected by LTE4 pretreatment. 3. Pretreatment of the epithelially-denuded tissues with the LTE4-analogue, 20-COOH LTE4, did not affect the maximal response to histamine. 4. LTE4 pretreatment increased the maximal response of the epithelially-denuded tissues to substance P (SP) but did not affect the maximal response to carbachol, KCl nor to the beta-adrenoceptor agonist, isoprenaline. 5. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM) and atropine (1 microM). 6. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 7. It is proposed that LTE4 induces an increased maximal response of epithelially-denuded guinea-pig airway smooth muscle to both histamine and substance P via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. Further, it is suggested that the increased maximal response of the epithelially-intact tissues to both histamine and substance P, after LTE4 pretreatment, may be suppressed by an epithelially-derived factor.

The effects of lipoxin A4 on airway responses in asthmatic subjects.

This study was performed to determine whether lipoxin A4 (LXA4) inhalation in asthmatic subjects has an effect on airways response. Eight subjects (six asthmatic, two normal) attended for bronchial inhalation challenge with LXA4. In three of these subjects (two asthmatics, one normal) blood pressure, pulse, and symptoms before and after challenge were recorded. Subsequently five male patients with mild asthma (22 to 34 yr of age) reattended for bronchial inhalation challenge with either leukotriene C4 (LTC4) or the combination of LTC4 and 1 x 10(-4) M LXA4. After inhalation of each dose of agonist SGaw and V25 were measured. Airway responsiveness was determined by the concentration of agonist in the nebulizer required to induce a 35% fall in SGaw (PC35). There was no effect of LXA4 inhalation on SGaw, V25, blood pressure, pulse, or symptoms. There was a significant shift of the SGaw and V25 dose-response curve to the right after inhalation challenge with LTC4 combined with 1 x 10(-4) M LXA4 as compared with that after inhalation challenge with LTC4 alone (p less than 0.01 and p less than 0.025, respectively). Thus, LXA4 may modulate LTC4-induced airway obstruction and may act as an endogenous sulfidopeptide leukotriene receptor antagonist.

The mechanism of LTE4-induced histamine hyperresponsiveness in guinea-pig tracheal and human bronchial smooth muscle, in vitro.

1. Preincubation of guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased its subsequent responsiveness to histamine. 2. LTE4 pretreatment of guinea-pig tracheal strips did not affect the subsequent responsiveness to either the contractile agents, carbachol and KCl, or to the relaxant beta-adrenoceptor agonist, isoprenaline. 3. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM), atropine (1 microM) and tetrodotoxin (1 microM). 4. U46619, a stable thromboxane A2-analogue, at a non-contractile concentration of 4 nM, increased tracheal smooth muscle sensitivity to histamine. 5. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 6. Preincubation of human bronchial spirals with LTE4 in vitro increased its subsequent responsiveness to histamine. 7. LTE4-induced histamine hyperresponsiveness of human bronchus was inhibited by GR32191 (3 microM) and atropine (1 microM). 8. It is proposed that LTE4 induces guinea-pig airway smooth muscle hyperresponsiveness to histamine via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. In addition, it is suggested that LTE4 may induce histamine hyperresponsiveness of human bronchus in vitro by a similar mechanism as to that seen in guinea-pig central airway smooth muscle.

Leukotrienes C4, D4, and E4 enhance histamine responsiveness in asthmatic airways.

The effects of prior inhalation of each of the sulfidopeptide leukotrienes (LT), LTC4, LTD4, and LTE4 on airway responsiveness to histamine have been compared in seven asthmatic and six normal subjects. Each subject underwent histamine inhalation challenge at 1, 4, and 7 h after inhalation of phosphate-buffered saline and bronchoconstricting doses of LTC4, LTD4, LTE4, and methacholine, which produced a greater than 30% fall in specific airway conductance. In asthmatic subjects, prior inhalation of LTC4, LTD4, and LTE4 enhanced airway responsiveness to histamine when compared with saline inhalation, on average by a maximum of 3.9-, 2.8-, and 3.1-fold, respectively, at 4 h after inhalation. Methacholine inhalation did not significantly after histamine responsiveness throughout the time course studied. In normal subjects, inhalation of LTC4, LTD4, LTE4, and methacholine did not change airway responsiveness to histamine. Thus, LTC4 and LTD4 were similar to LTE4 in their capacity to enhance airway responsiveness to histamine in asthmatic subjects, and, in common with LTE4, they failed to elicit a change in airway responsiveness to histamine in normal subjects.

Inhibition of leukotriene B4-induced neutrophil migration by lipoxin A4: structure-function relationships.

Lipoxins are trihydroxytetraene metabolites which are derived from arachidonic acid through an interaction between different lipoxygenase pathways. Previous work has shown that lipoxin A4 (LXA4) inhibits the chemotactic responsiveness of neutrophils (PMN) to leukotriene B4. We have now assessed the structural determinants of the lipoxin A4 molecule which are necessary for its inhibitory activity, using structural analogs of LXA4 prepared by chemical synthesis. Our results indicate the importance of two adjacent free hydroxyl groups in either the R or the S configuration; one hydroxyl group has to be in the C-6 position, but the other hydroxyl group can be in either the C-5 or the C-7 position for the conferment of inhibitory activity.