Immunohistochemical demonstration of prostaglandins in various tissues of the rat.

To demonstrate the tissue localization of prostaglandin (PG) E2, PGF2 alpha and 6-keto-PGF1 alpha (a stable metabolite of PGI2) various tissues, including decalcified periodontal tissue of 7-week-old male Wistar strain rats, were immunohistochemically examined using a streptavidin-biotin complex method. Besides tissue macrophages and endothelial cells in various tissues, hepatocytes, renal tubular cells, and parietal and chief cells in the gastric mucosa showed a positive reaction for the various PGs examined. PGs were demonstrated in the cytoplasm or in association with the cell membrane. We generally observed no difference between the localization patterns of PGE2-, PGF2 alpha-, and 6-keto-PGF1 alpha-positive cells in these tissues. However, in the periodontal ligament and alveolar bone, 6-keto-PGF1 alpha was localized in the cytoplasm of osteocytes, osteoblasts, cementocytes, and cementoblasts, while no reaction for PGE2 or PGF2 alpha was revealed in these cells. We demonstrated the immunohistochemical localization of PGs in various rat tissues including decalcified periodontal tissue and discuss the important roles of PGs in the modulation of their normal functions in these tissues.

Release of immunoreactive arachidonate metabolites by equine endometrium in vitro.

The ability of equine endometrium to release prostaglandin (PG) F, PGE2, and leukotriene (LT) B4 was studied in vitro, using endometrial tissue from diestrous mares. Because of the high cross-reactivity of the PGF antiserum with PGF1 alpha and with PGF2 alpha, results were quoted as total immunoreactive PGF. Significant concentrations of these arachidonate metabolites were released into tissue culture medium between 1 and 24 hours of incubation. Significantly higher concentrations of PGE, but not of PGE2 or LTB4, were released from endometria of mares with chronic endometritis than from genitally normal mares. Prostaglandin F was released only in low concentrations from the endometrium of a mare with pyometra, but concentrations of PGE2 and LTB4 were similar to those of genitally normal mares.

[Comparative analysis of the levels of anti-prostaglandin F2 alpha antibodies in the normal state and in cardiovascular diseases]. / Sravnitel'nyi analiz soderzhaniia autoantitel protiv prostaglandina F2 alpha v norme i pri serdechno-sosudistykh zabolevaniiakh.

The ELISA test was performed on sera of normal donors and patients with myocardial infarction (MI) and essential hypertension (EH) to detect autoantibodies against prostaglandin F2 alpha (a-PGF2 alpha). The a-PGF2 alpha level was much higher after immune complexes dissociation. Baseline a-PGF2 alpha was elevated in EH patients with a sudden rise in blood pressure, and in MI patients without hypertension and unstable angina. MI patients with bradyarrhythmias also had elevated a-PGF2 alpha levels. Baseline a-PGF2 alpha was rather low in the sera of donors, chronic EH patients with hypertension and unstable angina, suggesting that a-PGF2 alpha has a physiological role to play.

Active immunization of heifers against prostaglandin F2 alpha: potential as a sterilization vaccine.

Four experiments were conducted with 210 heifers in an attempt to develop a sterilization vaccine by active immunization against prostaglandin F2 alpha (PGF2 alpha) and to evaluate feedlot performance following immunization against the combination of PGF2 alpha and estrogens. The objectives were: development of a PGF2 alpha-ovalbumin conjugate that would induce antibody formation when used with complete Freund's adjuvant (CFA); comparison of CFA with other adjuvants in relation to PGF2 alpha antibody binding and maintenance of the corpus luteum; examination of growth performance in immunized heifers against both PGF2 alpha and estradiol-17 beta and evaluation of sterilization in PGF2 alpha-immunized heifers maintained with bulls. A PGF2 alpha-ovalbumin conjugate was developed that resulted in antibody production against PGF2 alpha, although antibody binding was quite low. The antibody response in heifers was higher in Exp. I and II than in Exp. III and IV (P less than .05). Complete Freund's adjuvant was the best adjuvant in inducing antibody formation compared with all other adjuvants tested (P less than .01). Corpus luteum (CL) function was maintained for 2.5 mo and ovulation was apparently blocked in Exp. I. The results of Exp. II confirmed those of Exp. I. Fewer than half of the heifers in Exp. III and IV had prolonged estrous cycles. In Exp. IV, immunized heifers became sterile for at least 4 mo when kept with bulls, although the success rate was only 37%. The low levels of antibody titers to PGF2 alpha in Exp. III and IV may be the reason for the failure to maintain CL function in some heifers.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical demonstration of increase in prostaglandin F2-alpha after recirculation in global ischemic rat brains.

Immunohistochemical localization of prostaglandin F2 alpha (PG F2 alpha) was studied in 24 rats. In 21 rats, global brain ischemia was produced for 5 min by Pulsinelli's method. Prior to decapitation, 13 were recirculated for 5 min, while the remaining eight were not. Three recirculated rats were pretreated with indomethacin before the occlusion. Hypotension was induced during the occlusion to 40-50 mm Hg of mean arterial blood pressure in 11 rats including those unrecirculated, recirculated and pretreated with indomethacin. Three normal rats without occlusion of arteries served as control. The brains were snap frozen and 10-microns cryostat sections were incubated in rabbit anti-PG F2 alpha serum and stained by the indirect immunofluorescence method after fixation in carbodiimide and in Zamboni's solution. Positive staining for PG F2 alpha was noted mainly in pial vessels in normal and ischemic rats both with and without hypotension. The rats recirculated without hypotensive ischemia revealed a positive reaction in the walls of pial and parenchymal vessels. All rats recirculated after the hypotensive occlusion showed positive staining in blood vessels, in the cytoplasm of neurons (especially in hippocampi) and in the interfascicular oligodendrocytes. The above results indicate that recirculation after ischemia results in an increase in PG F2 alpha in parenchymal vessels, neurons and oligodendrocytes.

The six equilibrium binding parameters of two antibodies in competitive radioimmunoassays.

A procedure is described for measuring the six parameters that theoretically determine the equilibrium bindings of two ligands in two-antibody antisera used in competitive radioimmunoassays (RIA). Four intensity parameters and two quantities are identified. They are the dissociation constants of the complexes formed by the bindings of the tracer and the specific ligand of two antibodies and the two corresponding binding capacities of a certain volume of the antiserum. Four series of assays, runs, are required to give enough information. Two very different amounts of serum are used, either with various doses of the tracer only or with one tracer dose and suitable dose ranges of the specific ligand. The corresponding equations and their use are presented. The amount of information is sufficient to offer several controls of consistency of the parameters with the two-antibody model. The RIA antisera of two prostaglandins, PGF2 alpha and PGE2, with the heavily tritiated analogues as tracers were found to be very different with regard to the ratios of the binding capacities and the affinities of the two antibodies. The intensity parameters of the two sera were stable over the period of the present study, almost a year. To interpret assay results of a run of unknowns it is necessary to calibrate precisely the amount of antiserum used as well as the residual non-specifically bound tracer fraction and the tracer dose. The random analytical error of an assay is then also predictable when the amounts of tracer or antiserum vary between assays of the same run.

Implication of prostaglandin E2 in soluble factor-mediated immune suppression by murine decidual cells.

Cells from the uteri of pregnant mice mediate profound nonantigen-specific and nonmajor histocompatibility complex-restricted immune suppression in vitro. In part, those cells accomplish suppression by releasing soluble suppressor factors. The purpose of the present study was to initiate identification of uterine cell suppressor factors. Immune suppression was assayed by the effect of decidual cells or in vitro generated supernatants of decidual cells on mixed lymphocyte reactions (MLR). The following findings support the designation of prostaglandin E2 (PGE2) as a primary suppressor molecule originating with decidual cells: Suppression mediated by supernatants of decidual cells was relieved by removal of lipids but not proteins; indomethacin, an inhibitor of prostaglandin synthesis, produced partial relief of suppression mediated by uterine cells and totally inhibited soluble suppressor factor generation by those cells; decidual cells produced high levels of both PGE2 and PGF2a; the addition of exogenous PGE2 at levels comparable to those found in the decidual cell supernatants restored suppression by decidual cells and their supernatants whereas the addition of PGF2a had no effect; inhibition of the lipoxygenase pathway of arachidonate metabolism had no effect on cell or supernatant mediated suppression; nonspecific suppressor mechanisms, such as arginine depletion and peroxide generation, were excluded as possible mediators of MLR suppression by decidual cells and their supernatants. Fractionation of decidual cells revealed at least three indomethacin-sensitive cell types: small, lymphocyte-like cells, macrophages, and a third population of large decidual cells that was not identified by specific markers.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of arachidonic acid metabolites in mononuclear phagocytic cell interactions.

Numerous investigations support the theory that arachidonic acid metabolites play a critical role in dictating the progression of chronic immune reactions. With regard to macrophage-mediated inflammatory responses, enzymatic oxygenation of arachidonic acid via the lipoxygenase or cyclooxygenase pathway can result in the production of compounds that may potentiate or suppress the inflammatory lesion. We recently have presented data demonstrating that lipoxygenase derived leukotriene B4 and C4 can induce the release of IL-1 by macrophages, while PGE2 and PGI2 can suppress the production of IL-1. Macrophages are central to the induction of immune responses and the progression of chronic inflammatory reactions. Therefore, an understanding of the role that macrophage-derived arachidonic acid metabolites play in the initiation, maintenance, and resolution of chronic immune responses is essential. As shown in Figure 3, there are a number of chemical signals that occur between macrophages and lymphocytes that are critical for immune cell communication. The investigations described above have demonstrated that the macrophage may regulate the production and expression of any or all of these signals, such that the inflammatory response is potentiated, sustained, suppressed, or resolved. A better comprehension of the activity of these potent arachidonate derivates will undoubtedly aid in the therapeutic manipulation of inflammatory disease.

Effect of Rumex nepalensis extracts on histamine, acetylcholine, carbachol, bradykinin, and PGs evoked skin reactions in rabbits.

The antihistaminic, anticholinergic, antibradykinin, and/or antiprostaglandin activity of aqueous and the alcoholic leaf extracts of the plant Rumex nepalensis were tested on the prepared site on the back of rabbits, using a control and a test site. Aqueous extract was found to reduce the mean size of the wheal produced by histamine, acetylcholine, carbachol, and bradykinin. The alcoholic extract reduced the size of the wheal produced by histamine, acetylcholine and carbachol. The results indicate that Rumex nepalensis may have antihistaminic, anticholinergic, and/or antibradykinin activity.

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.

Macrophage function in the Schistosoma mansoni egg-induced pulmonary granuloma. Role of arachidonic acid metabolites in macrophage Ia antigen expression.

The ability of arachidonic acid (AA) metabolites to regulate I-region-associated (Ia) antigen expression on macrophages from schistosome-egg-induced pulmonary granulomas was examined. The prostaglandin (PG) analog 15-S-15-CH3-PGE1 (M-PGE1) and PGF2 alpha were found to modulate the kinetics of Ia expression when administered in vivo. Methyl-PGE1 significantly suppressed Ia antigen expression by hypersensitivity granuloma macrophages, while PGF2 alpha appeared to potentiate the expression. Lymphokine-induced Ia antigen expression by cultured granuloma macrophages was likewise dramatically inhibited by M-PGE1. Further analysis using systemically administered inhibitors of AA metabolism demonstrated that the cyclooxygenase inhibitor indomethacin caused augmentation of Ia expression. In contrast, lipoxygenase inhibitors significantly reduced both Ia expression and granuloma size. The role of AA metabolites in modulating chronic inflammation is discussed.

Peritoneal fluid prostanoids in unexplained infertility.

Elevated peritoneal fluid thromboxane B2 and 6-keto-prostaglandin F1 alpha have been reported in patients with biopsy-proved endometriosis. In this paper these peritoneal fluid prostanoids were measured in patients with unexplained infertility and a fertile control group matched for age and day of the menstrual cycle. A subgroup of the women with unexplained infertility demonstrated a marked elevation of both prostanoids (p less than 0.01). These elevations may serve to identify women in whom undetected endometriosis or some other peritoneal irritant is associated with infertility.

Radioimmunoassay for 13, 14-dihydro-15-ketoprostaglandin F2 alpha and its application in normo- and anovulatory women.

Highly specific antibodies to 13, 14-dihydro-15-ketoprostaglandin F2 alpha (PGFM) were raised in rabbits. The animals were immunized with PGFM-bovine serum albumin (BSA)-conjugates. Prior to the incubation procedure PGFM was extracted by a rapid method with dichloromethane followed by column chromatography. The antisera dilution was 1:10 000 amd the cross-reactivity towards prostaglandin A2, E2, F2 alpha, 13, 14-dihydro-15-ketoprostaglandin E2 and the 15-ketoprostaglandin E2 and F2 alpha 2 was less than 1%. The limit of detection was 1.9 +/- 0.6 pg/ml plasma over the standard range 1.9--250 pg. The intra- and inter-assay variations were 3.9 and 15%, respectively. PGFM was measured throughout the menstrual cycle in female volunteers. In normal ovulatory women (n = 3) plasma levels of PGFM varied between 65.6 to 107.1 pg/ml. No significant variations of plasma PGFM were seen during the cycle. In anovulatory women (n = 4) no difference of PGFM was found during the cycle. PGFM levels in hyperprolactinaemic but ovulating women tend to be higher than in anovulatory, and normoprolactinaemic subjects. These data strongly indicate that PGFM is not correlated with other hormonal parameters tested here in the normal and anovulatory cycles.

Prostaglandin F2 alpha is associated with alveolar bone cells: immunohistochemical evidence utilizing monoclonal antibodies.

Prostaglandin F2 alpha (PGF2 alpha) has been found in mineralized tissues and may be implicated in cellular remodeling activities. The objective of this study was to localize PGF2 alpha in cat jaw bone by immunohistochemistry. Monoclonal antibodies to PGF2 alpha were prepared by the hybridoma technique and used as the first step in the tissue section staining procedure, the rabbit anti-mouse IgG conjugated to horseradish peroxidase followed by diaminobenzidine (DAB) as the substrate. Periodontal cells and cementoblasts displayed dark DAB deposits over the cell periphery. Some alveolar bone osteoblasts and osteoclasts were stained for PGF2 alpha over the cytoplasm as well as the membrane region. In the maxillary-premaxillary suture, osteoblasts on the border of the maxilla were stained very intensely for PGF2 alpha, while those adjacent to the premaxilla were stained exceptionally lightly. These results demonstrate the ability to localize PGF2 alpha in bone cells in situ and to identify intracellular sites of prostaglandin localization. Our observation suggests that PGF2 alpha may play a role in physiologic activities of fibroblasts, osteoblasts, and osteoclasts.