Untargeted metabolomics reveals alterations in metabolites of lipid metabolism and immune pathways in the serum of rats after long-term oral administration of Amalaki rasayana.

Amalaki rasayana, a traditional preparation, is widely used by Ayurvedic physicians for the treatment of inflammatory conditions, cardiovascular diseases, and cancer. Metabolic alterations induced by Amalaki rasayana intervention are unknown. We investigated the modulations in serum metabolomic profiles in Wistar rats following long-term oral administration of Amalaki rasayana. Global metabolic profiling was performed of the serum of rats administered with either Amalaki rasayana (AR) or ghee + honey (GH) for 18 months and control animals which were left untreated. Amalaki rasayana components were confirmed from AR extract using HR-LCMS analysis. Significant reductions in prostaglandin J2, 11-dehydrothromboxane B2, and higher levels of reduced glutathione and glycitein metabolites were observed in the serum of AR administered rats compared to the control groups. Eleven different metabolites classified as phospholipids, glycerophospholipids, glucoside derivatives, organic acids, and glycosphingolipid were exclusively observed in the AR administered rats. Pathway analysis suggests that altered metabolites in AR administered rats are those associated with different biochemical pathways of arachidonic acid metabolism, fatty acid metabolism, leukotriene metabolism, G-protein mediated events, phospholipid metabolism, and the immune system. Targeted metabolomics confirmed the presence of gallic acid, ellagic acid, and arachidonic acid components in the AR extract. The known activities of these components can be correlated with the altered metabolic profile following long-term AR administration. AR also activates IGF1R-Akt-Foxo3 signaling axis in heart tissues of rats administered with AR. Our study identifies AR components that induce alterations in lipid metabolism and immune pathways in animals which consume AR for an extended period.

Microthrombotic Renal Vascular Lesions Are Associated to Increased Renal Inflammatory Infiltration in Murine Lupus Nephritis.

Background: Vascular microthrombotic lesions in lupus nephritis with or without antiphospholipid antibodies may relate to worse renal outcomes. Whether microthrombotic lesions are a consequence of renal inflammation or independently contribute to renal damage is unclear. Our aim was to investigate the relationship between microthrombotic renal vascular lesions and nephritis progression in MRL/lpr mice. Methods: MRL/lpr mice were analyzed for the presence of renal microvascular, glomerular and tubulointerstitial lesions and the effect of anti-aggregation (aspirin or clopidogrel) and dexamethasone on renal clinical and pathological manifestations was evaluated. Intravascular platelet aggregates (CD41), peri- (F4/80), and intraglomerular (Mac-2) macrophage infiltration, and C3 deposition were quantified by immunohistochemistry. Renal function was assessed by measuring proteinuria, and serum levels of creatinine and albumin. Anti-dsDNA and anti-cardiolipin antibodies, and thromboxane B2 levels were quantified by ELISA. Results: Frequency of microthrombotic renal lesions in MRL/lpr mice was high and was associated with immune-mediated renal damage. Proteinuria positively correlated with glomerular macrophage infiltration and was higher in mice with proliferative glomerular lesions. All mice had detectable anti-dsDNA and anti-cardiolipin IgG, regardless the presence of microthrombosis. Proteinuria and glomerular macrophage infiltration were significantly reduced in all treatment groups. Dexamethasone and platelet anti-aggregation similarly reduced glomerular damage and inflammation, but only platelet anti-aggregation significantly reduced anti-cardiolipin antibodies, renal complement deposition and thromboxane B2 levels. Conclusions: Platelet anti-aggregation reduced renal inflammatory damage, renal complement deposition, anti-cardiolipin antibodies, and thromboxane B2 levels and in MRL/lpr mice, suggesting that platelet activation has a pathogenic effect on immune-mediated nephritis. Our results point to MRL/lpr mice with lupus nephritis as an appropriate model to analyze the potential impact of anti-thrombotic intervention on renal inflammation.

Effects of carboxymethylpachymaran on signal molecules in chicken immunocytes.

The study was carried out to investigate the immunomodulation mechanism of carboxymethylpachymaran (CMP). Chicken splenic lymphocytes were cultured in medium alone or with CMP at the final concentration of 50mg/L, 100mg/L, 200mg/L or 400mg/L in vitro for 4h, 8h, 12h or 24h, respectively. The supernatants at different culture periods were analyzed for changes in levels of 6-keto-prostaglandin F1α (6-keto-PGF1α), thromboxane B2 (TXB2) and nitric oxide (NO). The cells were collected to determine contents of oxidized glutathione (GSSG), reduced glutathione (GSH), cyclic AMP (cAMP) and cyclic GMP (cGMP). The results showed that CMP increase the values of NO, 6-keto-PGF1α, TXB2, and the ratio of 6-keto-PGF1α to TXB2 in supernatants. The contents of intracellular GSH, cAMP, cGMP and the ratio of cAMP to cGMP were increased in the cells treated with CMP. The results suggested that CMP enhanced immune functions by increasing the contents of GSH and by regulating arachidonic acid signal transduction systems in chicken splenic lymphocytes. The signal pathway of NO-cGMP plays an important role in CMP-induced activation of chicken splenic lymphocytes.

Clinical and immunomodulating effects of ketamine in horses with experimental endotoxemia.

BACKGROUND: Ketamine has immunomodulating effects both in vitro and in vivo during experimental endotoxemia in humans, rodents, and dogs. HYPOTHESIS: Subanesthetic doses of ketamine will attenuate the clinical and immunologic responses to experimental endotoxemia in horses. ANIMALS: Nineteen healthy mares of various breeds. METHODS: Experimental study. Horses were randomized into 2 groups: ketamine-treated horses (KET; n = 9) and saline-treated horses (SAL; n = 10). Both groups received 30 ng/kg of lipopolysaccharide (LPS, Escherichia coli, O55:B5) 1 hour after the start of a continuous rate infusion (CRI) of racemic ketamine (KET) or physiologic saline (SAL). Clinical and hematological responses were documented and plasma concentrations of tumor necrosis factor-α (TNF-α) and thromboxane B(2) (TXB(2)) were quantified. RESULTS: All horses safely completed the study. The KET group exhibited transient excitation during the ketamine loading infusion (P < .05) and 1 hour after discontinuation of administration (P < .05). Neutrophilic leukocytosis was greater in the KET group 8 and 24 hours after administration of LPS (P < .05). Minor perturbations of plasma biochemistry results were considered clinically insignificant. Plasma TNF-α and TXB(2) production peaked 1.5 and 1 hours, respectively, after administration of LPS in both groups, but a significant difference between treatment groups was not demonstrated. CONCLUSIONS AND CLINICAL IMPORTANCE: A subanesthetic ketamine CRI is well tolerated by horses. A significant effect on the clinical or immunologic response to LPS administration, as assessed by clinical observation, hematological parameters, and TNF-α and TXB(2) production, was not identified in healthy horses with the subanesthetic dose of racemic ketamine utilized in this study.

Effects of selective and non-selective COX inhibitors on antigen-induced release of prostanoid mediators and bronchoconstriction in the isolated perfused and ventilated guinea pig lung.

The contribution of cycloxygenase (COX)-1 and COX-2 in antigen-induced release of mediators and ensuing bronchoconstriction was investigated in the isolated perfused guinea pig lung (IPL). Antigen challenge with ovalbumin (OVA) of lungs from actively sensitised animals induced release of thromboxane (TX)A(2), prostaglandin (PG)D(2), PGF(2)(alpha), PGI(2) and PGE(2), measured in the lung effluent as immunoreactive TXB(2), PGD(2)-MOX, PGF(2)(alpha), 6-keto PGF(1)(alpha) and PGE(2), respectively. This release was abolished by the non-selective COX inhibitor flurbiprofen (10 microM). In contrast, neither the selective COX-1 inhibitor FR122047 nor the selective COX-2 inhibitor celecoxib (10 microM each) significantly inhibited the OVA-induced bronchoconstriction or release of COX products, except for PGD(2). Another non-selective COX inhibitor, diclofenac (10 microM) also significantly inhibited antigen-induced bronchoconstriction. The data suggest that both COX isoenzymes, COX-1 and COX-2 contribute to the immediate antigen-induced generation of prostanoids in IPL and that the COX-1 and COX-2 activities are not associated with different profiles of prostanoid end products.

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells.

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.

Determination of the eicosanoid response to inflammatory stimuli in whole blood and its pharmacological modulation ex vivo.

Recognition of pathogens by immune cells initiates the release of numerous signaling molecules, including cytokines and eicosanoids. Here, we describe a simple procedure by which eicosanoids such as prostaglandin E(2) (PGE(2)), leukotriene B(4) (LTB(4)) and thromboxane B(2) (TxB(2)) can be measured using commercial enzyme immunoassays (EIAs) in the supernatant of whole blood stimulated with inflammatory stimuli. This is illustrated for numerous stimuli. The kinetics by which lipopolysaccharide (LPS) induces cyclooxygenase (COX)-2 expression in this setup were determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). The eicosanoid response of the blood of 160 healthy volunteers to 1 microg/ml LPS was measured. To determine whether the action of a drug in vivo is represented ex vivo in the eicosanoid response of blood, one volunteer took a standard dose of a number of commercially available cyclooxygenase inhibitors on different days and the eicosanoid response of his blood to LPS was determined before ingestion as well as 2 and 6 h afterwards. The efficacy of the different pharmaceuticals on cyclooxygenase but not lipoxygenase products or cytokines could be monitored ex vivo. Similarly, ex vivo eicosanoid release was measured in blood from 10 volunteers who had taken 50 mg flurbiprofen. The method described extends approaches for studying whole blood cytokine release to the lipid mediators formed from arachidonic acid. These important signaling molecules represent targets for pharmacological intervention, which can now be monitored in vitro, as well as ex vivo employing the same model. Furthermore, the assay could be used to characterize the immune status of patient groups or to monitor the course of disease.

Inhibition of mediator release from dispersed nasal polyp cells by cyclosporin A.

The mechanisms of action of cyclosporin A require further elucidation since this drug includes anti-inflammatory properties unrelated to its previously documented effect of T cells. A study was performed using enzymatically dispersed cells from nasal polyps of 7 subjects to examine the effects of cyclosporin A on the release of histamine, leukotriene C4/D4 (LTC4/D4) and thromboxane (TxB2) following stimulation by anti-IgE. Cells were resuspended and preincubated with cyclosporin A (0.1, 1 and 10 microM) or 0.1% DMSO (the vehicle used to dissolve cyclosporin A) for 20 min prior to challenge with 10 microgram/ml epsilon-chain-specific anti-IgE for 45 min at 37 degrees C. Histamine, LTC4/D4 and TxB2 were measured using EIA. Cyclosporin A significantly inhibited the release of histamine, LTC4/D4, and TxB2 in a concentration-dependent manner. IC30 values, histamine (5.1 microM), LTC4/D4 (7.8 microM) and TxB2 (6.2 microM), were determined. These data demonstrate new antiallergic properties of cyclosporin A using a novel in vitro model which mimics more closely allergic inflammation.

Urinary leukotriene E4 and 11-dehydrothromboxane B2 in patients with aspirin-sensitive asthma.

The objective of this study was to define the participation of cysteinyl leukotrienes (LTs) or thromboxane A2 in the pathogenesis of aspirin-sensitive asthma (ASA). Leukotriene E4 (LTE4) and 11-dehydrothromboxane B2 (11DTXB2) values in spot urine were measured in 22 asthmatics with a history of aspirin sensitivity and in 17 without such a history (non-aspirin-sensitive asthma [NASA]) in the outpatient clinic. The urinary LTE4 value was significantly higher in ASA patients than in NASA (340 +/- 47 vs 65 +/- 15 pg/mg.cr, P < 0.001), but there was no significant difference in urinary 11DTXB2 between the two groups (891 +/- 77 vs 657 +/- 90 pg/mg.cr). A high value of LTE4 was not associated with type of asthma, severity of disease, oral prednisolone treatment, sex, or age. A higher value of 11DTXB2 was observed in the atopic type than the nonatopic type in ASA (1086 +/- 111 vs 697 +/- 147 pg/mg.cr, P < 0.05). No correlation was observed between urinary LTE4 and 11DTXB2 in either ASA or NASA. In conclusion, LTs may play an important role in the pathogenesis of ASA, and TXA2 in the pathogenesis of the atopic type in ASA.

Effect of TYB-2285 on lung anaphylaxis in actively sensitized rats.

1. We examined the effect of TYB-2285 on the acute phase and the late phase of lung anaphylaxis in rats. 2. TYB-2285 (3-30 mg/kg PO) inhibited antigen-induced bronchoconstriction and TxB2 production during the acute phase of lung anaphylaxis in a dose-dependent manner. 3. Ketotifen fumarate (30 mg/kg p.o.) inhibited bronchoconstriction and TxB2 production less potently than TYB-2285. 4. TYB-2285 (30 mg/kg p.o.) inhibited the accumulation of neutrophils during the late phase of lung anaphylaxis significantly without a significant change in total cells. 5. Hydrocortisone acetate (100 mg/kg p.o.) inhibited the accumulation of total cells as potent as neutrophils.

Release of calcitonin gene-related peptide in cardiac anaphylaxis.

We have investigated the antigen-stimulated release of calcitonin gene-related peptide (CGRP) from ovalbumin-sensitized guinea-pig isolated hearts and the interaction with other mediators of anaphylaxis released concomitantly. It was found that antigen challenge caused a significant increase of CGRP release (from basal 31.2 +/- 2.9 to 51.6 +/- 4.9 fmol/5 min). Anaphylactic CGRP release was significantly attenuated in the presence of the cyclooxygenase inhibitor indomethacin while the 5-lipoxygenase inhibitor Bay-X1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) had no significant effect. Combined treatment with the histamine receptor (H1,H2) antagonists mepyramine and cimetidine also significantly attenuated anaphylactic release of CGRP. Under control conditions antigen injection increased release of cysteinyl-leukotrienes (LT), thromboxane (TXB2) and 6-keto-prostaglandin (PG)F1 alpha from basal values of 0.96 +/- 0.09, 2.7 +/- 0.7 and 3.4 +/- 0.28 ng/5 min respectively, to 5.9 +/- 0.9, 48.4 +/- 3.4 and 6.9 +/- 1.4 ng/5 min. Indomethacin abolished the release of cyclooxygenase products of arachidonate metabolism and simultaneously increased cysteinyl-LT release significantly (8.8 +/- 1.4 ng/5 min). Conversely Bay-X1005 completely abolished cysteinyl-LT release and had no significant effect on anaphylactic release of TXB2 and 6-keto-PGF1 alpha. Simultaneous blockade of H1 and H2 receptors abolished release of 6-keto-PGF1 alpha, while release of TXB2 and cysteinyl-LT was not significantly affected. The results indicate that CGRP is not a primary mediator of the immediate hypersensitivity reaction of the heart, but is in turn released by arachidonic acid metabolites of the cyclooxygenase pathway and histamine. In contrast, LT obviously do not contribute to anaphylactic CGRP release. CGRP is a potent coronary vasodilator and could act as endogenous functional antagonist of vasoconstrictor mediators also released during cardiac anaphylaxis such as cysteinyl-LT, platelet activating factor and TXA2.

Thromboxane receptor stimulation/inhibition and perfusion redistribution after acute lung injury.

Perfusion redistribution (PR) after acute oleic acid (OA) lung injury may be the result of changes in the tissue concentration ratio of thromboxane (Tx) and prostacyclin (A. H. Stephenson et al. J. Appl. Physiol. 73: 2126-2134, 1992). We tested this hypothesis by determining whether the Tx mimetic U-46619 would mimic PR caused by cyclooxygenase inhibition with meclofenamate and whether the Tx receptor antagonist ONO-3708 would inhibit PR even in the presence of meclofenamate. Measurements of regional pulmonary blood flow (PBF) and lung water concentration were made with the nuclear medicine imaging technique of positron emission tomography. Measurements were made at baseline and 2 h after OA. At baseline, the spatial distribution of PBF was similar in all experimental groups. Two hours after OA, fractional PBF was reduced to the edematous lung in all groups given OA, but the magnitude of change was greater in those groups receiving meclofenamate or U-46619 compared with the change in the group given OA only. Thus, although the Tx mimetic produced the same amount of PR as meclofenamate, Tx inhibition did not prevent PR after meclofenamate. Therefore, the ratio of Tx to prostacyclin per se is not the critical determinant of PR.

Thromboxane contributes to pulmonary hypertension in ischemia-reperfusion lung injury.

Exposure of isolated perfused rabbit lungs (IPL) to ischemia-reperfusion causes a transient increase in pulmonary arterial (PA) pressure at the onset of reperfusion. Because thromboxane A2 (TxA2) is a potent vasoconstrictor, we hypothesized that it may contribute to the ischemia-reperfusion-induced pressor response. To evaluate this hypothesis, we exposed IPL perfused with a cell-free solution to 40 min of warm ischemia followed by reperfusion and measured perfusate immunoreactive thromboxane B2 (iTxB2) and 6-ketoprostaglandin F1 alpha (i6-keto-PGF1 alpha). We observed that ischemia-reperfusion IPL compared with controls had an increase in PA pressure (40.2 +/- 4.8 vs. 9.3 +/- 0.3 mmHg, P < 0.05), lung edema (29.3 +/- 6.3 vs. -0.2 +/- 0.2 g, P < 0.05), iTxB2 perfusate levels (155 +/- 22 vs. < 50 pg/ml, P < 0.05), and i6-keto-PGF1 alpha (436 +/- 33 vs. 61 +/- 16 pg/ml, P < 0.05). In ischemia-reperfusion IPL, infusion of SQ 29548 (10(-6) M), a specific TxA2/prostaglandin H2 receptor antagonist, attenuated the PA pressor response and the degree of edema. We conclude that pulmonary hypertension associated with ischemia-reperfusion results in part from pulmonary release of TxA2. Furthermore, TxA2 directly through membrane effects or indirectly through hydrostatic mechanisms increases the severity of ischemia-reperfusion-induced lung edema.

The differences in 12-hydroxyeicosatetraenoic acid and thromboxane B2 release from guinea pig platelets.

Anti-12(S)-hydroxyeicosatetraenoic acid (12-HETE)-antibody and anti-thromboxane B2 (TXB2)-antibody were generated and applied to the radioimmunoassay. The detection limit for 12-HETE was 16 pg. The cross-reactivities of anti-12-HETE-antibody were 4.6% for 15-HETE, 0.18% for 5-HETE and below 0.15% for leukotrienes and prostaglandins (PGs). 12-HETE and TXB2 released from guinea pig platelets were measured by radioimmunoassay. Platelet activating factor (PAF) at 10(-9) M induced the aggregation of platelets, the releases of immunoreactive-12-HETE (1.8 +/- 1.2 ng/10(8) platelets, mean +/- S.D.) and immunoreactive-TXB2 (18.5 +/- 17.3 ng/10(8) platelets). Collagen at 1 microgram/ml also evoked platelet aggregation, the releases of immunoreactive-12-HETE (2.7 +/- 1.1 ng/10(8) platelets) and immunoreactive-TXB2 (11.8 +/- 4.6 ng/10(8) platelets). By the stimulation with these compounds, TXB2 was produced in a greater amount than 12-HETE from guinea pig platelets. Although 10(-7) M and 10(-6) M U46619, a TXA2 mimetic, caused platelet aggregation, arachidonic acid metabolites were not released. These data suggest the presence of different mechanisms of platelet activation depending on each stimulus.

Novel derivatization and immunoextraction to improve microanalysis of 11-dehydrothromboxane B2 in human urine.

A new, highly selective procedure for the determination of 11-dehydrothromboxane B2 (11-dehydro-TXB2) in human urine is described. Following the addition of [19,19,20,20-2H4]11-dehydro-TXB2 as an internal standard, samples were extracted with an affinity column of anti-11-dehydro-TXB2. Conversion of the immunoextracted 11-dehydro-TXB2 into its 1-methyl ester-11-n-propylamide-9,12,15-tris-dimethylisopropylsilyl ether derivative was followed by gas chromatography-selected-ion monitoring. The mass spectrum of the 11-dehydro-TXB2 derivative was dominated by the base peak ion of [M-C3H7]+ at m/z 698, which accounted for more than 10% of the total ion current. A typical result showed that the immunoaffinity purification procedures provided an extremely clean alternative to more conventional methods of chromatographic fractionation, and that interfering substances from the urine matrix were almost entirely eliminated during the microanalysis.

Purification and characterization of an NAD(+)-dependent dehydrogenase that catalyzes the oxidation of thromboxane B2 at C-11 from porcine liver. Development and application of 11-dehydro-thromboxane B2 radioimmunoassay to enzyme assay.

11-Dehydro-thromboxane B2 has been identified as a major metabolite of infused as well as endogenous thromboxane B2 in mammalian plasma and urine. This metabolite is derived from thromboxane B2 by enzymatic oxidation at C-11 catalyzed by 11-hydroxythromboxane B2 dehydrogenase. A radioimmunoassay for 11-dehydro-thromboxane B2 has been developed and used for enzyme assay, purification and characterization. Antibodies were generated against 11-dehydro-thromboxane B2 conjugated to bovine thyroglobulin. Labeled marker was prepared by radioiodinating 11-dehydro-thromboxane B2-tyrosine methyl ester conjugate. A sensitive radioimmunoassay capable of detecting 10 pg of 11-dehydro-thromboxane B2 per assay tube was developed. The antibodies showed minimal crossreaction with thromboxane B2 (0.03%), prostaglandin D2 (2.76%) and other eicosanoids (less than 0.03%). The enzyme activity was determined by assaying NAD(+)-dependent formation of immunoreactive 11-dehydro-thromboxane B2 from thromboxane B2. The enzyme was found to be enriched in liver although significant activity was also detected in gastrointestinal tract and kidney in pig. The enzyme was purified from porcine liver cytosol to apparent homogeneity using conventional and affinity chromatography. The purified enzyme exhibited coenzyme specificity for NAD+ and used thromboxane B2 as a substrate. The enzyme also catalyzes NADH-dependent reduction of 11-dehydro-thromboxane B2 to thromboxane B2 indicating the reversibility of the enzyme catalyzed reaction. The apparent Km values for thromboxane B2, 11-dehydro-thromboxane B2 and NAD+ are 8.1, 8.0 and 23 microM, respectively. Subunit Mr was shown to be 55,000, whereas the native enzyme Mr was found to be 110,000 indicating that the enzyme is a dimer. The enzyme is sensitive to sulfhydryl inhibitions suggesting cysteine residues are essential to enzyme activity. The availability of a homogeneous enzyme preparation should allow further studies on the substrate specificity and the structure and function of the enzyme.

The booster injection of antigen during active sensitization of guinea-pig modifies the anti-anaphylactic activity of the PAF antagonist WEB 2086.

1. Serum IgG levels of sensitized guinea-pigs bled at various times after the booster injection were evaluated and its capacity to sensitize passively lung strips from normal guinea-pigs assessed. Following the booster injection, both serum IgG and the ability to sensitize passively lung strips increased during the first week and decreased slowly thereafter. 2. The PAF antagonist WEB 2086 (3 mg kg-1, i.v.) blocked the anaphylactic bronchoconstriction induced by intravenous administration of ovalbumin (1 mg kg-1) when guinea-pigs were challenged 2 and 4 days after the booster injection, but became ineffective when tested in guinea-pigs challenged 7, 28 and 56 days after the booster injection. 3. The ability of WEB 2086 to reduce anaphylactic bronchoconstriction of guinea-pigs challenged 2 and 4 days after the booster injection was unrelated to either the selective involvement of one type of immunoglobulin, low IgG titres in sera or a reduced sensitizing capacity. 4. The booster injection, which accounts for the loss of efficacy of WEB 2086 from the fourth day thereafter, probably operates as a PAF-independent inflammatory challenge. 5. The protocol for immunisation and the day of experiment after the booster injection determines the sensitivity of the anaphylactic bronchoconstriction to inhibition of PAF antagonists.

Development of an enzyme-linked immunosorbent assay of thromboxane B2 using a monoclonal antibody.

An inhibition enzyme-linked immunosorbent assay (ELISA) was developed using a monoclonal antibody against thromboxane B2 (TXB2). As a specific antigen, the bovine serum albumin conjugate of TXB2 was adsorbed onto polystyrene microtiter plates. The sensitivity of the monoclonal antibody was compared by means of three different enzyme conjugates, all commercially available. The detection limit with immunoglobulin conjugates of alkaline phosphatase and horseradish peroxidase was 0.04 ng of TXB2 per sample. The use of horseradish peroxidase coupled with an avidin-biotin complex allowed a tenfold increase in sensitivity to 0.0045 ng of TXB2 per sample. The suitability of the assay was checked with TXB2-containing human serum and urine samples, which yielded unchanged standard curves. Recovery experiments had an accuracy of r = 0.960 and r = 0.987. Validity was confirmed by a good correlation between radioimmunoassay and ELISA (r = 0.949). Results of an inhibition experiment with platelet-rich plasma in the presence and absence of ibuprofen demonstrated the practical applicability of this method.

A monoclonal anti-thromboxane B2 antibody.

A monoclonal antibody against thromboxane B2 which may be used in standard fluid phase radioimmunoassays with a detection limit of around 40 pg and a binding affinity of 1.98 X 10(9) M-1 is described. Limited crossreactivity could be observed only with structurally closely related compounds such as 2,3-dinor-thromboxane B2 (8.9%), thromboxane B1 (15.7%) and thromboxane B3 (39.7%). Detectable crossreactivity with 11-dehydro-thromboxane B2, omega-carboxy-thromboxane B2, omega-hydroxy-thromboxane B2, prostaglandins of the D-, E- and F-type as well as metabolites of prostacyclin was lacking. The monoclonal anti-thromboxane B2 antibody proved well suited for measuring the thromboxane B2 content in tissue culture supernatants as well as in human serum.

The complement membrane attack complex stimulates the prostanoid production of cultured glomerular epithelial cells.

Incubation of cultured rat glomerular epithelial cells (GEC) with sublytic amounts of the purified complement components C5b6, C7, C8 and C9 greatly stimulated the release of the prostanoids prostaglandin E (PGE) and thromboxane B2. Incubation of GEC with C5b-8 was also stimulatory, whereas omission of C7 abolished the enhanced prostanoid production. These effects were dose-dependent. The increased release of PGE was biphasic with peaks at 5 min and 24 h of incubation. The second peak could be prevented by treatment with cycloheximide, suggesting its dependence on protein synthesis. The observations on cultured GEC provide evidence that terminal complement components alter the metabolism of glomerular cells, resulting in increased production of prostanoids. The results are consistent with the concept that deposition of nonlytic amounts of complement in the glomerular capillary wall may affect the GEC in vivo and may indirectly contribute to abnormalities of the glomerular filter as it is seen in glomerular disease.