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.

FQPD, a novel immunomodulatory drug, has significant in vitro activity in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy that claims thousands of lives each year and has considerable morbidity. The disease remains incurable despite recent advances in the understanding of the disease biology and the introduction of more effective drugs is needed. This study evaluated the anti-MM activity of 3-(7-fluoro-4H-quinazolin-3-yl)-piperidine-2,6-dione, hydrochloride (FQPD), a novel immunomodulatory drug. FQPD inhibited the proliferation of multiple MM cell lines, including those resistant to conventional treatments, such as dexamethasone. It induced apoptosis in MM cell lines, as well as freshly isolated patient MM cells, without cytotoxicity on normal human lymphocytes. Moreover, it induced apoptosis in MM cells adherent to bone marrow (BM) stromal cells or in the presence of cytokines, such as interleukin-6 and vascular endothelial growth factor, confirming its ability to overcome the protective effects of the BM milieu. Apoptosis in the MM cells was mediated via poly-ADP ribose polymerase cleavage as well as cleavage of caspase 8 and caspase 9. Our studies therefore demonstrated in vitro anti-MM activity of FQPD and provide the rationale for its in vivo evaluation in animal models and derived clinical trials.

Determination of prostacyclin in plasma through a bioluminescent immunoassay for 6-keto-prostaglandin F1alpha: implication of dosage in patients with primary pulmonary hypertension.

This work describes a solid-phase immunoassay for 6-keto-prostaglandin F1alpha, the stable hydrolysis product of prostacyclin (prostaglandin I2). Prostacyclin, a potent vasodilator with antiplatelet and antiproliferative properties is an effective treatment for primary pulmonary hypertension and pulmonary arterial hypertension associated with scleroderma and scleroderma-like syndrome. Levels of 6-keto-prostaglandin F1alpha can be directly correlated with levels of prostacyclin. Therefore, 6-keto-prostaglandin F1alpha, has become the indicator of choice to measure prostacyclin levels. The single-step immunoassay for 6-keto-prostaglandin F1alpha reported here was developed using the bioluminescent protein aequorin as a label. Analyte-label conjugates were constructed by linking the carboxyl group of 6-keto-prostaglandin F1alpha and lysine residues of aequorin by chemical conjugation methods. The binding properties of 6-keto-prostaglandin F1alpha toward its antibody and the bioluminescent properties of aequorin were retained in the conjugate, which was then used to generate a dose-response curve for the analyte in a convenient microtiter plate format. The concentration of 6-keto-prostaglandin F1alpha after extraction from plasma showed good correlation with the concentration of 6-ketoprostaglandin F1alpha obtained without prior extraction of the same plasma sample. This measurement demonstrated that the assay allows the measurement of 6-keto-prostaglandin F1alpha directly in plasma without any pretreatment of the samples, which results in a much simpler method with a faster assay time.

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.

Plasma iPGE2 and i6-keto PGF1 alpha in the course of liver cirrhosis.

Plasma iPGE2 and i6-keto PGF1 alpha were measured with an EIA assay in twenty patients with alcohol-related liver cirrhosis (ALC group) and 13 patients with hepatitis B virus as an etiologic factor of liver cirrhosis (HLC group). Significant increase of both prostanoids was observed irrespectively of the etiology of liver cirrhosis. Their levels increased depending on the degree of liver insufficiency with the highest values in patients classified as Child-Pugh C class. A significant, positive correlation with Child-Pugh score was found regarding PGE2 (r = 0.657; p < 0.001) as well as 6-keto PGF1 alpha (r = 0.736; p < 0.001). Correlation (r = 0.789; p < 0.001) was also observed between levels of both prostaglandins. In conclusion we have shown that plasma iPGE2 and i6-keto PGF1 alpha arise simultaneously with the degree of liver insufficiency, that can be a result of activation of non-parenchymal liver cells accompanying hepatic fibrosis.

Development of an enzyme-linked immunosorbent assay for 2,3-dinor-6-keto-prostaglandin F1 alpha in urine using a monoclonal antibody.

OBJECTIVES: To develop and validate an enzyme-linked immunosorbent assay (ELISA) for measurement of urinary 2,3-dinor-6-keto-prostaglandin F1 alpha (2,3D6KPGF1 alpha) using a monoclonal antibody and a horseradish peroxidase-linked antigen. DESIGN AND METHODS: Assay validation included optimization of the standard curve, antibody cross-reactivity, accuracy and imprecision studies together with preliminary measurement of clinical samples. RESULTS: Optimal conditions of the standard curve (0.078-10.0 micrograms/L) used 2 mg/L of antibody and 3 micrograms/L of peroxidase conjugate in each well, at pH 7.2. The coefficient of variation of various concentrations of the standard curve averaged 6.8%. Antibody cross-reactivity was < 0.01% for related prostanoids. Recovery of known amounts (0.1-5.0 micrograms/L) of 2,3D6KPGF1 alpha added to an urinary sample was 101.2 +/- 6.3%. Imprecision studies with non-pregnant (0.24 microgram/L) and pregnant (2.5 micrograms/L) samples displayed an intraassay variability of 8.9 and 9.9%, and an interassay variability of 9.6 and 10.0%, respectively. Urinary measurements in the non-pregnant and pregnant states were similar to those previously reported. An apparent decreased concentration was observed early in pregnancy in future preeclampsia. CONCLUSION: With similar precision and validity, our assay method is time- and cost-saving. Preliminary urinary measurements show that this analyte may be of interest as an early marker for preeclampsia.

Cross-reactivity of delta 17-6-keto-PGF1 alpha with 6-keto-PGF1 alpha antibodies.

The cross-reactivity of the PGI3 metabolite, delta 17-6-keto-PGF1 alpha, with antibodies against 6-keto-PGF1 alpha for radioimmunoassays (RIA) has been investigated. Delta 17-6-keto-PGF1 alpha was obtained either from commercial sources or after its purification from endothelial cells. In the latter case, primary cultured bovine aortic endothelial cells were incubated for 20 min at 37 degrees C with 10 microM eicosapentaenoic acid (EPA) in the presence of 2 microM 13-hydroperoxy-octadecadienoic acid, and activator of the EPA cyclooxygenation, and the 6-keto-PGF1 alpha and beta 17-6-keto-PGF1 alpha produced were separated by RP-HPLC. Then, cross-reactivities of the commercial and purified beta 17-6-keto-PGF1 alpha with 6-keto-PGF1 alpha antibodies were determined and found not to exceed 10%. In addition, the amounts of prostacyclin-related compounds detected by direct measurements in media of cells loaded with EPA were compared with those obtained after purification of 6-keto-PGF1 alpha. In accordance with the cross-reactivity data, we found that RIA in media mainly measured 6-keto-PGF1 alpha, the beta 17-6-keto-PGF1 alpha formed being undetected at 90%. It is concluded that 6-keto-PGF1 alpha antibodies generally used for RIA of 6-keto-PGF1 alpha are highly specific since they can discriminate a metabolite bearing an additional double band such as the PGI3 metabolite beta 17-6-keto-PGF1 alpha.

Application of an alpha-sidechain length-specific monoclonal antibody to immunoaffinity purification and enzyme immunoassay of 2,3-dinor-6-keto-prostaglandin F1 alpha from human urine.

2,3-Dinor-6-keto-prostaglandin F1 alpha (2,3-dinor-6-keto-PGF1 alpha) is a major urinary metabolite of PGI2 (prostacyclin) and one of the most reliable parameters of PGI2 production in vivo. A mouse was immunized with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha conjugated to bovine serum albumin for preparation of a monoclonal antibody which recognized the difference in the alpha-sidechain length of 6-keto-PGF1 alpha and its 2,3-dinor-metabolite. A sensitive and specific enzyme immunoassay was developed by the solid-phase competition method with 2,3-dinor-6-keto-20-carboxy-PGF1 alpha labeled with peroxidase protein. The detection range of the assay was 14-1200 fmol (IC50 = 120 fmol). The cross-reactivities of the antibody with 6-keto-PGF1 alpha, 6,15-diketo-13,14-dihydro-PGF1 alpha, and other arachidonate metabolites were less than 0.01%. An immunoaffinity column was prepared by coupling the anti-2,3-dinor-6-keto-PGF1 alpha antibody to BrCN-activated Sepharose 4B. Human urine was applied to an octadecylsilyl silica cartridge, and the extract was applied to the immunoaffinity column. This procedure allowed an efficient separation of 2,3-dinor-6-keto-PGF1 alpha from unidentified urinary substances which interfered with immunoassay. Validity of the results obtained by the enzyme immunoassay was confirmed by GC/MS employing selected ion monitoring for quantification.

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.

Differential effects of leukotriene C4 on endothelin-1 and prostacyclin release by cultured vascular cells.

Nanomolar concentrations of leukotriene C4 and phorbol 12-myristate acetate, a protein kinase C activator, stimulated endothelin-1 release by vascular endothelial but not smooth muscle cells. For both agonists, attenuation of this stimulatory effect was observed at higher concentrations, concomitant with but independent of enhanced prostacyclin biosynthesis.

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.

Age-related decline in prostacyclin synthesis by human aortic endothelial cells. Qualitative and quantitative analysis.

To investigate the functional alteration of human aortic endothelial cells with aging, prostacyclin synthesis was qualitatively and quantitatively examined. The endothelial cells of human aortas and umbilical veins or inferior vena cavae were immunohistochemically examined and found positive for prostacyclin, but the intensity of aortic endothelial cells from older subjects was low. In addition to the endothelial cells, smooth muscle cells in the thickened intima, not the media, of the aorta were also immunoreactive. Endothelial cells were successfully cultured from human aortas obtained from infants through aged subjects and were subdivided into three groups: young, middle, and old. Prostacyclin synthesis by endothelial cells from all types of blood vessels was extremely great at the primary culture, but decreased abruptly in the following subcultures. Among the aortic endothelial cells, the young group synthesized the largest amount of prostacyclin in a conventional culture condition, with synthesis progressively decreasing in the older groups. The in vitro prostacyclin biosynthesis was supported by the qualitative analysis on the tissue sections. These results indicate that prostacyclin synthesis of the aortic endothelial cells decreases with age, but intimal smooth muscle cells potentially have a back-up mechanism and substitute this synthesis to some extent. The decreased synthesis of prostacyclin with age may play an important role in the development and advancement of thrombosis and atherosclerosis.

An enzyme-linked immunosorbent assay for 6-keto PGF1 alpha.

An enzyme-linked immunosorbent assay for 6-keto prostaglandin F1 alpha, a stable metabolite of prostacyclin, has been developed. The assay allows quantitation of 6-keto PGF1 alpha in the range 1-200 pg/0.1 ml and shows very low cross reactivity to nine other prostaglandins. Dose dependent stimulation by thrombin of 6-keto PGF1 alpha formation in human endothelial cells in culture has been used to verify the assay. Quantitation by the enzyme linked immunosorbent assay agrees closely with determination by radioimmunoassay.

Monoclonal antibodies against E- and F-type prostaglandins. High specificity and sensitivity in conventional radioimmunoassays.

Polyclonal antisera against prostaglandins (PGs) are widely used for the assessment of the biological role of these mediators, but even the most specific contain antibodies against the major metabolites and degradation products of the haptens employed. To overcome this inherent problem we produced monoclonal antibodies (mAs) against PGE2, PGF2 alpha and 6-keto-PGF1 alpha using the somatic cell hybridization technique. The mAs against 6-keto-PGF1 alpha and PGF2 alpha proved to be highly specific, but allowed only for moderate detection limits (1-2 ng) in conventional fluid phase radioimmunoassays (RIAs). One of the mAs against PGE2 permitted a 100-fold improvement in the detection limit while being almost devoid of cross-reactivity with metabolites and other structurally related PGs. These results show that highly specific mAs against PGs can be produced to improve the available RIA technique for PG quantification.

Gastroduodenal ulceration in rabbits producing antibodies to prostaglandins.

The consistent occurrence of gastric and duodenal ulcers was observed in laboratory rabbits used for production of high-titer plasma antibody to 6-keto PGF1 alpha and PGE2. Perforations developed in 7 of 10 animals, usually just distal to the pyloroduodenal junction. The remaining rabbits showed gross and/or microscopic evidence of imperforate ulcers and erosions. These lesions appeared to be direct pathologic complications of an immune response directed against prostaglandins since animals immunized against met-enkephalin with similar methods had no ulcers.

[A radioimmunoassay of 6-keto PG F1 alpha using [125I]-6-keto PG F1 alpha-tyramide].

We established a radioimmunoassay of 6-keto PG F1 alpha, using 125I-6-keto PG F1 alpha-tyramide, which enabled us more easily to monitor the plasma levels of prostacyclin in clinical fields. Antibodies against 6-keto PG F1 alpha were obtained from rabbits immunized with 6-keto PG F1 alpha-BSA complex, which could be used for RIA at a final dilution of 1:648,000. The detection limit of 6-keto PG F1 alpha was 50-1000 pg/ml. The cross reactivities between PG I2 and PG F1 alpha were 26.6% and 1.62% respectively, but those between other prostaglandins were less than 1%. Using this radioimmunoassay with 125I-6-keto PG F1 alpha-tyramide, plasma levels of 6-keto PG F1 alpha were measured. The recovery rate by this assay was 92.9%, and the intra- and inter-assay coefficients of variation determined were 9.9% and 27.1%, respectively. The mean of plasma levels of 6-keto PG F1 alpha revealed 126.2 +/- 75.3 (mean +/- S.D.) pg/ml in 46 healthy Japanese males, 122.7 +/- 85.2 pg/ml in 60 females, and 124.2 +/- 81.3 pg/ml in 106 Japanese of both sexes.

Measurement of human venous plasma prostacyclin and metabolites by radioimmunoassay: a reappraisal.

Two radioimmunoassays, the first specific for 6-oxo-PGF1 alpha and the second for 6-oxo-PGF1 alpha, 13, 14-dihydro-6-oxo-PGF1 alpha, 13,14-dihydro-6, 15-dioxo-PGF1 alpha and 6-oxo-PGE1 are described. These radioimmunoassays were used to measure levels of immunoreactive 6-oxo-PGF1 alpha and alleged metabolites of prostacyclin in human venous plasma. Procedures for the direct measurement and extraction of plasma 6-oxo-PGF1 alpha are described and the limitations to which radioimmunoassay of plasma 6-oxo-PGF1 alpha is exposed are discussed. Direct measurement of plasma immunoreactive 6-oxo-PGF1 alpha gave maximal levels of 6.1 pg ml-1 and 4 pg ml-1 in the first and second radioimmunoassays respectively. The latter value reflected combined levels of less than 2 pg ml-1 of 6-oxo-PGF1 alpha and cross-reacting metabolites in venous blood. Extraction of human plasma gave 88.7 +/- 2.2% recovery (mean +/- S.E.M. n = 5 experiments) of [3H]-6-oxo-PGF1 alpha and 80 - 86% recovery of exogenous 6-oxo-PGF1 alpha as immunoreactive 6-oxo-PGF1 alpha. Basal plasma levels of extractable immunoreactive 6-oxo-PGF1 alpha were less than 2.5 pg ml-1. Prostacyclin incubated in vitro with blood was recovered in plasma as immunoreactive 6-oxo-PGF1 alpha and confirmed that conversion to metabolites that cross-reacted with the second antibody, in particular 6-oxo-PGE1, did not occur under experimental conditions. Extraction of [3H]-6-oxo-PGF1 alpha from acidified plasma with methanol resulted in formation of a prostanoid that had properties consistent with the methylated hemiketal isomer of 6-oxo-PGF1 alpha. Under radioimmunoassay conditions this prostanoid was less immunogenic than native [3H]-6-oxo-PGF1 alpha and [3H]-6-oxo-PGF1 alpha extracted from plasma using methyl formate. The low levels of plasma 6-oxo-PGF1 alpha which we report questions the validity of clinical studies that previously described altered levels of plasma 6-oxo-PGF1 alpha in pathophysiological conditions. These studies which were based upon measurement of plasma 6-oxo-PGF1 alpha by radioimmunoassay and GC/MS should now be re-evaluated. Preliminary results from this study have been reported elsewhere (1, 2).

A new technique for the measurement of urinary 6-keto-prostaglandin F1 alpha: normal values in adults.

A new sensitive and specific radioimmunoassay for measurement of urinary 6-keto-prostaglandin F1 alpha is described. In this technique, 6-keto-prostaglandin F1 alpha was first extracted and separated from prostaglandins F2 alpha, F1 alpha, E2, E1, A2 and A1 by using an original high performance liquid chromatographic method with an overall recovery of 74.4 +/- 0.9%. The radioimmunoassay used a highly specific antibody raised in the rabbit. The sensitivity of the method was 1.8 pg. Between-assay reproducibility was at 7.7%. As previously reported for prostaglandins E and F alpha, the urinary excretion of 6-keto-prostaglandin F1 alpha, measured in 28 healthy adult subjects, was found to be significantly (p less than 0.001) higher in men (319 +/- 21 ng/24 h) than in women (190 +/- 14 ng/24 h). The chromatographic separation method described here could also be applied to the radioimmunoassay of other primary prostaglandins.

Prostacyclin substitution for heparin in long-term hemodialysis.

We studied prostacyclin as a substitute for heparin in 12 patients who underwent maintenance hemodialysis. All subjects underwent initial hemodialysis with prostacyclin as the sole anticoagulant; 10 of the 12 were restudied during heparin hemodialysis. Few adverse reactions occurred during prostacyclin hemodialysis in the 10 patients in whom dialysis was performed against a bicarbonate-containing dialysate; however, significant hypotension developed in two subjects when an acetate bath was used. Platelet aggregation progressively decreased during prostacyclin hemodialysis (p less than 0.02), but not during heparin hemodialysis, and returned toward control values after hemodialysis. Platelet thromboxane release decreased during both prostacyclin and heparin hemodialysis. Intradialytic percent decrements in serum urea nitrogen and creatinine were greater during prostacyclin than heparin administration (42 +/- 2.9 percent versus 36 +/- 2.6 percent [p less than 0.05] and 33 +/- 2.6 percent versus 29 +/- 2.1 percent [0.05 less than p less than 0.1], respectively). The plasma concentrations of 6-keto-prostaglandin-F1 alpha, a prostacyclin metabolite, reached peak levels by 120 minutes of hemodialysis and declined biexponentially toward predialysis concentrations during 120 minutes after hemodialysis, thereby providing an index of cumulative prostacyclin dosage. We conclude that prostacyclin is not only a safe alternative to heparin anticoagulation during hemodialysis, but that prostacyclin might also increase the efficiency of hemodialysis.