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Cholangiocarcinoma (CCA), consisting of intrahepatic (IHCCA), perihilar (PHCCA), and distal (DCCA) CCA, is a type of highly aggressive malignancy with a very dismal prognosis. Potential biomarkers and drug targets of CCA are urgently needed. As a new member of the Annexin (ANXA) family, the role of ANXA10 in the progression and prognosis of CCA is unknown. In our study, ANXA10 upregulation was revealed by exome and transcription sequencing in 5 pairs of PHCCA and adjacent tissues. The clinical significance and prognostic value of ANXA10 was evaluated by analyzing its correlation with clinicopathological variables and survival rates. As a result, ANXA10 expression was upregulated in PHCCA and DCCA but not in IHCCA. High ANXA10 expression was significantly associated with poor tumor differentiation and unfavorable prognosis. With in vitro and in vivo experiments, we demonstrated that ANXA10 promoted the epithelial-mesenchymal transition (EMT), proliferation, migration and invasion of the PHCCA cell line QBC939. The key molecule in ANXA10-induced CCA progression was screened by mRNA sequencing. Consequently, PLA2G4A expression was regulated by ANXA10 and it was required in ANXA10-induced metastasis. High PLA2G4A expression also predicted poor prognosis in PHCCA and DCCA. With multiple in vivo experiments, we showed that ANXA10 facilitated EMT and promoted metastasis by upregulating PLA2G4A expression, thus increasing PGE2 levels and activating STAT3. In conclusion, ANXA10 was an independent prognostic biomarker of PHCCA and DCCA but not IHCCA. ANXA10 promoted the proliferation, migration and invasion of PHCCA and facilitated metastasis by promoting the EMT process via the PLA2G4A/PGE2/STAT3 pathway. Our results suggested that ANXA10, PLA2G4A and their downstream molecules, such as COX2 and PGE2, may be promising drug targets of PHCCA and DCCA. Funding Statement: Our study was supported by National Natural Science Foundation of China (Grant no. 81601668), Shandong Province Major Research and Design Program (Grant No. 2018GSF118169), Jinan City Science and Technology Development Program(Grant No. 201805017, 201805013), and Hengrui Hepatobiliary and Pancreatic Foundation (Grant No.Y-2017-144). Declaration of Interests: The authors declare no conflicts of interests. Ethics Approval Statement: All experiments were approved by the Ethics Committee of Qilu Hospital of Shandong University.

Background Defective clearance of apoptotic cells (ACs) has been suggested to be involved in the pathogenesis of systemic lupus erythematosus (SLE). Mesenchymal stem cells (MSCs) exhibit promising therapeutic effects on SLE, but whether MSCs phagocytose ACs and contributes to the underlying mechanism in the treatment of SLE remain unknown. Methods Human umbilical cord (UC) MSCs were co-cultured with ACs, and the engulfment of ACs by MSCs was either detected by flow cytometry or observed under confocal laser scanning microscope. Peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) were cultured in MSC conditioned medium (MCM) or MSC exposed to ACs (AC-MSC) conditioned medium (ACMCM), and then CD4+ T cell proliferation was detected. Soluble factors including prostaglandin (PG)E2 in the supernatants of MSCs and AC-MSCs, as well as in the mouse peritoneal lavage fluids (PLF) were determined by enzyme-linked immunosorbent assay (ELISA). Cyclooxygenase (COX)2 inhibitors and siRNA transfection were utilized to determine the function of COX2/PGE2 in AC-MSC-mediated immunosuppression. PGE2 metabolites (PGEM) in the plasma of SLE patients were measured before and 24 h after MSC transplantation respectively. Findings Human UC MSCs possessed the ability to engulf ACs. AC-MSCs increased MSC-mediated suppression of CD4+ T cell proliferation compared to MSCs alone. Mechanistically, ACs stimulated MSCs to express COX2 and consequently produced PGE2 that inhibited T cell responses. NF-κB signalling pathway mediated the activation of COX2/PGE2 in AC-MSCs. Importantly, in patients with SLE, the plasma PGEM levels increased significantly in those with reduced apoptotic mononuclear cells in peripheral blood after MSC transplantation. Interpretation Clearance of ACs by MSCs contributes to immunosuppressive function via increasing PGE2 production. These findings reveal a previously unrecognized role of MSC-mediated phagocytosis of ACs in MSC-based immunotherapy. Fund This study was supported by grants from the Chinese Major International (Regional) Joint Research Project (No. 81720108020), the Jiangsu Province Major Research and Development Program (No. BE2015602) and the Jiangsu Province 333 Talent Grant (BRA2016001). WJ. Chen was supported by the Intramural Research Program of NIH, NIDCR.

This work presents a reliable analytical procedure combining micro-extraction by packed sorbent (MEPS) and ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry to determine 8-iso prostaglandin F2α, 8-iso prostaglandin E2 and prostaglandin E2 in dried blood spots (DBSs). To reach this goal, we optimized a fast semi-automated MEPS procedure for the clean-up and pre-concentration of the analytes extracted from a single DBS (50 μL) by a 70:30 v/v methanol:water mixture. Limits of detection of about 20 pg mL−1, satisfactory recoveries (90–110%) and very good intra- and inter-day precisions (RSD ≤10%) were obtained for all the analytes. The innovative addition of internal standards on the filter paper before DBS sampling allowed to compensate changes in the amount of analyte during storage. Since prostanoids and isoprostanoids are biomarkers involved in the pathogenesis and progression of many diseases (e.g. ductal patency, diabetic nephropathy, and acute lung injury), our analytical method offers interesting diagnostic and prognostic opportunities in the medical field. The present method is currently used for the analysis of such biomarkers in DBSs from preterm newborns collected in the clinical setting.

Many studies seek to explore the impact of extrinsic soluble factors present in serum, interstitial fluids or cell-conditioned media on cells in vitro. A convenient approach to elucidate the effects of a particular factor is its selective neutralization. However, intrinsic production of soluble factors such as cytokines by the cultured cells is common and can have an impact via autocrine mechanisms. The addition of cytokine-specific neutralizing antibodies leads to neutralization of the targeted factors irrespective of their source and affects paracrine and autocrine effects alike. Thus, neutralization assays are not suitable to irrevocably demonstrate that the examined factors exert their effect via a paracrine mechanism. We were interested in investigating the impact of immunosuppressive factors present in ovarian carcinoma-associated ascites by dissecting paracrine versus autocrine effects of interleukin 10 (IL-10) and prostaglandin E2 (PGE2) on the activation of monocyte-derived dendritic cells (DC). We explored several methods of depletion based on introduction of the neutralizing antibodies bound to beads. Here we describe the pitfalls of the investigated depletion approaches and show the importance of monitoring the presence of residual neutralizing antibodies in the sample upon depletion, which impacts on the suitability of the approach to distinguish paracrine from autocrine effects. Only one of three investigated approaches showed no dislocation of neutralizing antibody from the beads into the sample. This method, which is based on covalently linking antibody to magnetic beads harbouring a reactive group allowed for the complete removal of the investigated factors from ascites and represents an elegant tool to elucidate immunoregulatory or -stimulatory cytokine networks in considerably more depth than the use of neutralizing antibodies in cell cultures alone can contribute.

Aspiration of gastric acid commonly injures airway epithelium and, if severe, can lead to respiratory failure from acute respiratory distress syndrome. Recently, we identified cyclooxygenase-2 (COX-2)-derived prostaglandin E(2) (PGE(2)) and lipoxin A(4) (LXA(4)) as pivotal mediators in vivo for resolution of acid-initiated acute lung injury. To examine protective mechanisms for these mediators in the airway, we developed an in vitro model of acid injury by transiently exposing well-differentiated normal human bronchial epithelial cells to hydrochloric acid. Transmission electron microscopy revealed selective injury to superficial epithelial cells with disruption of cell attachments and cell shedding. The morphological features of injury were substantially resolved within 6 hours. Acid triggered and early marked increases in COX-2 expression and PGE(2) production, and acid-induced PGE(2) significantly increased epithelial LXA(4) receptor (ALX) expression. LXA(4) is generated in vivo during acute lung injury, and we observed that nanomolar quantities increased basal epithelial cell proliferation and potently blocked acid-triggered interleukin-6 release and neutrophil transmigration across well-differentiated normal human bronchial epithelial cells. Expression of recombinant human ALX in A549 airway epithelial cells uncovered ALX-dependent inhibition of cytokine release by LXA(4). Together, these findings indicate that injured bronchial epithelial cells up-regulate ALX in a COX-2-dependent manner to promote LXA(4)-mediated resolution of airway inflammation.

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.

Background The paper is aimed at uncovering the mechanism of miR-204-5p in regulating inflammatory responses of human osteoarthritic synovial fibroblasts (SFs). Methods IL-1β-induced osteoarthritic SFs were established as an osteoarthritis (OA) cell model. The osteoarthritic SFs were accordingly transfected with mimics-miR-204-5p, inhibitors-miR-204-5 or FOXC1 siRNA. MTT tested the vitality of osteoarthritic SFs by analyzing the cell optical density. The expressions of miR-204-5p, FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 in osteoarthritic SFs were measured by qRT-PCR, Western blotting and/or ELISA. The binding of miR-204-5p to FOXC1 was verified through luciferase reporter assay. The regulatory effect of miR-204-5p on FOXC1 was also tested in normal SFs. Results miR-204-5p was under-expressed and FOXC1 was over-expressed in osteoarthritic SFs. The expressions of FOXC1, TNF-α, IL-6, PGE2, MMP-1, MMP-13 and COX-2 were up-regulated in IL-1β-treated SFs. Up-regulation of miR-204-5p or down-regulation of FOXC1 suppressed the inflammatory responses of osteoarthritic SFs. miR-204-5p negatively regulated FOXC1 by being a sponge in osteoarthritic SFs as well as in normal SFs. Conclusion miR-204-5p down-regulates FOXC1 to ameliorate inflammation of SFs in OA.

In a previous paper we gave evidence that chronic oral defibrotide antagonizes the noxious effect of developing atherosclerosis in the cardiovascular system. In the present paper we give evidence that defibrotide is still capable of exerting beneficial effects on cardiovascular function once atherosclerosis is established. In fact, there was statistically significant amelioration by defibrotide infusion in the following, all of which were hampered by established atherosclerosis: in rabbit aorta relaxation to acetylcholine, prostaglandin E2, and 6-keto-prostaglandin F1alpha generation from rabbit aortas, rabbit heart left ventricular end-diastolic pressure, coronary perfusion pressure, and left ventricular developed pressure, vasopressor activity of acetylcholine and endothelin-1 on coronary perfusion pressure, and 6-keto-prostaglandin F1alpha generation from the rabbit heart. Since prostacyclin takes part in NO generation, is cellular protective, and inhibits 5-lipoxygenase product synthesis, its increase, caused by defibrotide, could explain defibrotide cardioprotective activity. Prostacyclin activity could be backed by prostaglandin E2, another cardioprotective prostaglandin.

Prostaglandin (PG) synthesizing capability of the rat uterus in early pseudopregnancy and in early pregnancy was determined in the above paper by incubation of uterine homogenates. It was shown that 6-keto-PGFr, was the major prostaglandin produced in both reproductive states and that 6keto-PGFt,, PGF,, and PGE;! peaked on day 5 of pseudopregnancy, while 6-keto-PGFr, and, PGE2 peaked on day 5 of pregnancy (day of implantation). Testing of the data given in this paper for the values of PG production by pregnant uteri compared to pseudopregnant uteri showed that 6-keto-PGFr, production was significantly greater on days 4, 5, 6; PGEz production was significantly higher on day 4; and, while PGFk production was significantly higher on day 4, it was significantly lower on day 5. The lowered production of PGF2, on day 5 by the pregnant uterus compared to that by the pseudopregnant uterus supported the view that the presence of the embryo affected uterine PG synthesis in the rat. A similar decrease in PGF production has recently been reported for endometrium from pregnant mares incubated in the presence of a horse embryo (1). As the various prostanoids show differences in modulating myometrial contractility and may bring about different vascular changes, it is considered appropriate in terms of attempting to relate levels of PG production to uterine functioning to consider the ratios (expressing the balances between PGs) rather than the absolute levels of PGs produced (2, 3). The data presented in the paper by Phillips and Poyser for PG production capacity of the uteri of pseudopregnant rats (days 4-6) and pregnant rats (days 4-7) have been examined in this way and the

It is still an open question as to whether or not aseptic injuries affect the generation of fever due to exogenous pyrogens including bacterial products. Therefore, in the present paper we have investigated the course of endotoxin fever in rats induced with lipopolysaccharide (LPS; given intraperitoneally in a dose of 50 μg/kg) 48 h after subcutaneous administration of turpentine oil (TRP; 0.1 mL per rat) that causes aseptic necrosis of tissues. We found that febrile response was significantly augmented in the animals pre-treated with turpentine compared to control rats (pre-treated with saline), and that observed excessive elevation of body temperature (Tb) was accompanied by enhanced release of fever mediators: interleukin-6 (IL-6) and prostaglandin E2 (PGE2) into plasma. Moreover, we found that sensitization to pyrogenic effects of lipopolysaccharide was associated with the increase in plasma level of high mobility group box 1 protein (HMGB1), one of the best-known damage-associated molecular patterns (DAMP), which was recently discovered as inflammatory mediator. Since the injection of anti-HMGB1 antibodies weakened observed hyperpyrexia in the animals pre-treated with turpentine, we conclude that HMGB1 is a plasma-derived factor released in the course of aseptic injury that enhances pyrogenic effects of LPS.

Prostaglandin E2 (PGE2) is an eicosanoid derived from cyclooxygenase, an enzyme responsible for the cyclisation and oxygenation of arachidonic acid. In response to bacterial infection, PGE2 binds to EP3 receptors on a population of GABAergic neurons in the pre-optic area. Activation of the EP3 receptor decreases the intracellular cyclic adenosine monophosphate (cAMP) concentrations of these neurons, and the resulting dis-inhibition activates spinal motor outputs responsible for shivering thermogenesis, tachycardia, and brown adipose tissue activation. These involuntary responses increase core body temperature to varying degrees depending on the magnitude of infection; an immune response which is crucial for the survival of the host. However, evidence in animal and human models, primarily through the use of cyclooxygenase inhibitors (which block the production of PGE2), suggests that PGE2 may also be an important molecule for the defence of core temperature against body cooling and cold stress (in the absence of fever). In this paper, evidence within human and animal models is discussed which supports the hypothesis that the eicosanoid PGE2 has a role in maintaining human core temperature during environmental cooling. Given that over-the-counter PGE2 inhibiting drugs [i.e. Non-Steroidal Anti Inflammatory Drugs (NSAIDS)] are frequently used worldwide, it is possible that the use of such medication during environmental cooling could impair one's ability to thermoregulate. Support for such findings could have major implications in the pathology of hypothermia, thus, we suggest that future researchers investigate this specific hypothesis in vivo, using healthy human models. Suggestions for the implementation of such experiments are provided in the present work.

Our previous papers indicate that peritoneal exudate macrophages (PM) of newborn mice are strongly suppressive for the tumor cell growth in comparison with those of adult mice. Neither newborn PM nor adult PM are cytolytic, but they both can be activated to be cytolytic by being cultured overnight either with a high concentration of bacterial lipopolysaccharide (LPS) or with a low concentration of LPS plus lymphokine. This paper shows that the activation of adult PM was inhibited by prostaglandin E2 (PGE2) added simultaneously with LPS, though that of newborn PM was not affected by PGE2. Adult PM, however, acquired the resistance to PGE2 by the preculture with LPS and/or lymphokine for 4 hr so as to be activated by LPS in the presence of PGE2. These results indicate that newborn PM are more activated than adult PM to a state which adult PM attain after moderate activation with LPS or lymphokine.

Gangliosides, sialic acid-containing glycosphingolipids, have been considered to be involved in the development, differentiation, and function of nervous systems in vertebrates. However, the mechanisms for anti-inflammation caused by gangliosides are not clear. In this paper, we investigated the anti-inflammation effects of ganglioside GD1a by using RAW264.7 macrophages. Our data demonstrated that treatment of macrophages with lipopolysaccharide significantly increased the production of NO and pro-inflammatory cytokines. GD1a suppressed the induction of iNOS and COX-2 mRNA and protein expression and secretory pro-inflammatory cytokines in culture medium, such as TNFα, IL-1α and IL-1β. In addition, LPS-induced phosphorylation of mitogen-activating protein kinases and IκBα degradation followed by translocation of the NF-κB from the cytoplasm to the nucleus were attenuated after GD1a treatment. Furthermore, GD1a probably inhibited LPS binding to macrophages and LPS-induced accumulation between TLR4 and MyD88. Taken together, the results demonstrated that ganglioside GD1a inhibited LPS-induced inflammation in RAW 264.7 macrophages by suppressing phosphorylation of mitogen-activating protein kinases and activation of NF-κB through repressing the Toll-like receptor 4 signaling pathway.

The recent proposal of using Zn-based alloys for biodegradable implants was not supported with sufficient toxicity data. This work, for the first time, presents a thorough cytotoxicity evaluation of Zn-3Mg alloy for biodegradable bone implants. Normal human osteoblast cells were exposed to the alloy's extract and three main cell-material interaction parameters: cell health, functionality and inflammatory response, were evaluated. Results showed that at the concentration of 0.75mg/ml alloy extract, cell viability was reduced by ~50% through an induction of apoptosis at day 1; however, cells were able to recover at days 3 and 7. Cytoskeletal changes were observed but without any significant DNA damage. The downregulation of alkaline phosphatase protein levels did not significantly affect the mineralization process of the cells. Significant differences of cyclooxygenase-2 and prostaglandin E2 inflammatory biomarkers were noticed, but not interleukin 1-beta, indicating that the cells underwent a healing process after exposure to the alloy. Detailed analysis on the cell-material interaction is further discussed in this paper.

Root canal contents are potent stimuli for proinflammatory cytokines involved in apical periodontitis. This study investigated target gram-negative bacterial species and endotoxins in primary endodontic infection with apical periodontitis, determined their antigenicity against macrophages through the levels of PGE(2), and evaluated their relationship with clinical findings.Samples were taken from 21 root canals with primary infection and apical periodontitis by using paper points. Polymerase chain reaction (16S rDNA) was used for bacterial detection and limulus amebocyte lysate assay for endotoxin measurement. Levels of prostaglandin E2 (PGE(2)) were measured by enzyme-linked immunosorbent assay (Duoset Kit; RD, Minneapolis, MN).Prevotella nigrescens (13/21), Fusobacterium nucleatum (6/21), and Porphyromonas endodontalis (6/21) were the most frequently observed species. A positive association was found between F. nucleatum and P. endodontalis (P.05). A correlation was found between the number of gram-negative bacterial species and the levels of endotoxins, such as PGE(2) (P.05). Higher levels of endotoxin were detected in teeth with exudation, whereas elevated levels of PGE(2) were found in teeth with tenderness to percussion and pain on palpation.Our findings imply an additive effect between the number of gram-negative bacterial species involved in endodontic infection regarding the induction of proinflammatory cytokine by macrophage cells. Moreover, teeth with clinical symptomatology were related to higher levels of endotoxins and PGE(2) secretion.

The study aim was to determine whether prostaglandin E(2) (PGE(2)) in gingival crevicular fluid (GCF) could serve as a risk factor for periodontitis in human immunodeficiency virus-positive (HIV(+)) patients. Clinical measurements, including gingival index (GI), plaque index, bleeding index, probing depth (PD), attachment loss (AL) and GCF samples were taken from two healthy sites (including sites with gingival recession, GI=0; PDor =3 mm; ALor =2 mm), three gingivitis sites (GI0; PDor =3 mm; AL=0) and three periodontitis sites (GI0; PDor =5 mm; ALor =3 mm) of each of the 30 patients at baseline and 6-month visits. GCF samples were also taken by means of paper strips. GCF PGE(2) levels were determined by a sandwich ELISA. The progressing site was defined as a site which had 2 mm or more attachment loss during the 6-month study period. The mean amounts of PGE(2) were significantly higher in gingivitis and periodontitis sites than in healthy sites (p0.0001). GCF levels of PGE(2) were significantly correlated with probing depth, attachment loss, CD4(+) cells, viral load, age and smoking pack-years at baseline and 6-month visits (0.0001p0.05). Repeated measures analysis of 19 active sites versus 221 inactive sites indicated that PGE(2) levels were significantly higher in active sites than in inactive sites (p0.0001). It is likely that the compromised immune system contributes to the pathogenesis of periodontitis in HIV(+) patients. It is well known that the activated inflammatory cells produce inflammatory mediators which stimulate the production of PGE(2). Longitudinal evaluation of GCF PGE(2) with respect to the progression of untreated periodontitis sites in HIV(+) subjects will contribute to the understanding of the pathogenesis of periodontitis in HIV(+) patients. These data indicate that sites with high GCF levels of PGE(2) in HIV(+) patients are at significantly greater risk for progression of periodontitis.

Free radicals are associated with the appearance of disorders such as tumours, CNS alterations and inflammatory pathologies. Their levels are known to be increased in inflammatory diseases due to the activity of prostaglandins, which are related to protein secretion including enzymes. Peroxidase is an oral enzyme that is implicated in the defence of the oral cavity. In this paper, investigations of the effect and mechanism of the activity of prostaglandin E2 (PGE2) on peroxidase secretion of female rat submandibular glands are reported. Results showed that PGE2 significantly increased the secretion of submandibular peroxidase and that this effect was mediated by an increase of intracellular cAMP and nitric oxide synthase activation. This could imply that prostaglandins play a modulatory role in diseases where free radicals are involved.

In this paper we have determined the different signal pathways involved in M(1) and M(3) muscarinic acetylcholine receptor (mAChR) dependent stimulation of cyclo-oxygenase 1 (cox-1) mRNA gene expression and PGE(2) production on rat cerebral frontal cortex. Carbachol stimulation of M(1) and M(3) mAChR exerts an increase in cox-1 mRNA gene expression without affecting cox-2 mRNA expression and increased PGE(2) generation. Besides, increased phosphoinositide (PI) turnover and stimulation of nitric oxide synthase (NOS) and cyclic GMP (cGMP) production. Inhibitors of phospholipase A(2) (PLA(2)), COX and phospholipase C (PLC), calcium/calmodulin (CaM), NOS and soluble guanylate cyclase prevent the carbachol effect. These results suggest that carbachol-activation of M(1) and M(3) mAChR increased PGE(2) release associated with an increased expression of cox-1 and NO-cGMP production. The mechanism appears to occur directly to PLC stimulation and indirectly to PLA(2) activation. These results may contribute to understand the effects and side effect of non-steroidal anti-inflammatory drugs in patients with cerebral degenerative diseases.

The superfamily of phospholipase A 2 (PLA 2 α) enzymes implicated in arachidonic acid release has grown in number with members having different structural characteristics, substrate specificity, calcium dependency and cellular localization. Despite this growing complexity and multiple attacks on its position as the reigning monarch of this superfamily, the 85 kDa cytosolic PLA 2 α continues to rule supreme, having a primary role in many pathophysiological conditions. The most recent addition to the list of accolades of this enzyme is reflected in two important papers in which the use of the cytosolic cPLA 2 α ( cPLA 2 α )-null mouse demonstrates crucial roles of this enzyme in collagen-induced arthritis and prostaglandin E 2 (PGE 2 )-mediated bone resorption associated with inflammation.

Recently, we demonstrated that bradykinin (BK) counteracts the stimulatory effect of Ang-(1-7) on the Na(+)-ATPase activity from basolateral membrane of the proximal tubule through B2 receptor. In the present paper, the signaling pathway involved in the inhibitory response of the Na(+)-ATPase activity to BK was investigated. The following results indicate that the phospholipase A2 (PLA2)/COX/prostaglandin E (PGE2) pathway is implicated in this process: (1) The inhibitory effect of BK on Ang-(1-7)-stimulated enzyme is abolished in a dose-dependent manner by quinacrine (10(-9)-10(-6)M), a nonspecific PLA2 inhibitor, and by PACOCF3 (10(-7)M), an inhibitor of a Ca(2+)-independent PLA2. However, AACOCF3 (2 x 10(-4) M), an inhibitor of the cytosolic PLA2, does not modify the inhibitory effect of BK. (2) The inhibitory effect of BK on the Ang-(1-7)-stimulated enzyme is reversed by cyclooxygenase (COX) inhibitors diclofenac (10(-12) M) and indomethacin (10(-12) M). (3) PGE2 (10(-12)-10(-5) M) inhibits the Na(+)-ATPase activity in a dose dependent manner. (4)The inhibitory effects of PGE2 and BK on the Na(+)-ATPase activity are not cumulative. (5) PGE2 (10(-12)-10(-8) M) counteracts the stimulatory effect of Ang-(1-7) on the enzyme activity in a dose-dependent manner.

Several lines of evidence suggest that non-steroidal antiinflammatory drugs (NSAIDs) have anticarcinogenic effects. The causal relationship linking the preventive effect of NSAIDs on colon cancer and the inhibition of prostaglandin synthesis is questioned by the contrasting results obtained by many laboratories. The experiments reported in this paper demonstrate that prostaglandin E 2 (PGE 2 ) did not stimulate the proliferation in HT-29 human colon adenocarcinoma cells under several experimental conditions. Moreover, PGE 2 and 17-phenyl trinor prostaglandin E 2 (a specific agonist of EP1 receptors) did not increase intracellular Ca 2+ concentration. Finally, PGE 2 did not affect the intracellular cAMP and did not reduce the isoproterenol dependent increase in cAMP. These results indicate that in HT-29 cells: (1) proliferation is not directly sensitive to PGE 2 ; and (2) PGE 2 does not stimulate a signal transduction pathway leading to intracellular increase in cAMP or Ca 2+ mobilization. Therefore, other cell lines should be used to assess the direct role played by prostanoids in promoting cell proliferation in colon cancer.

An immune mechanism has been suggested in the pathogenesis of periodontal disease. Actinobacillus actinomycetemcomitants (Aa) has been implicated as one of the etiological agents that induces the major immune response together with a dense infiltrate of inflammatory cells. But the exact role of these immune cells in periodontal disease has not yet been clarified. In this study the T lymphocyte (TL) proliferative response was evaluated after having being exposed to free cell supernatant (SN) from Aa. Aa SN increased TL proliferation. This mitogenic effect of Aa SN was attenuated by pretreating TL with indomethacin (INDO) or acetylsalicylic acid (ASA) but not by polymyxin B. The inhibitory effect of INDO on cell proliferation was reversed by the addition of prostaglandin E2 (PGE2) to the culture assay. Moreover, when immune cells were exposed to Aa SN they were able to generate PGE2 at the same time as intracellular levels of cAMP decreased. Both, PGE2 release and decrease accumulation of cAMP in TL were blunted by treated lymphocytes with INDO. In this paper we demonstrate that cell free SN from Aa induces a mitogenic effect on murine lymphocytes. The mechanism involves the host's immunecompetent cells and the release of PGE2 and appears not to be induced by capsular-like polysaccharide antigen. Results show a paradoxical mitogenic effect of Aa SN accompanied by increased generation of PGE2 and decreased production of cAMP by lymphocytes.

In a previous paper we presented evidence for a negative regulation of adenylyl cyclase activity by tyrosine protein kinase(s) in the human leukemic T cell line Jurkat. In order to examine this point in non malignant cells, we conducted the present study in human peripheral blood mononuclear cells (PBMC). In these cells, staurosporine, a broad spectrum protein kinase inhibitor, enhanced not only the receptor-mediated, induced by prostaglandin E2 (PGE2), but also the direct (forskolin-induced) stimulation of adenylyl cyclase activity. Herbimycin A, a specific protein tyrosine kinase inhibitor, reproduced only in part the effect of staurosporine, whereas bisindolylmaleimide, the most specific protein kinase C (PKC) inhibitor known at present time, was ineffective. All these observations were made both in the absence and presence of isobutylmethylxanthine, a phosphodiesterase inhibitor, indicating that the effects of staurosporine and herbimycin A on cAMP accumulation were not due to phosphodiesterase inhibition. The calcium ionophore A 23187 also enhanced the PGE2-induced cAMP accumulation, and this effect was not additive to that of staurosporine, but additive to that of herbimycin A. These results confirm and extend those obtained in Jurkat cells. Taken together, they indicate that in human PBMC the adenylyl cyclase activity is negatively regulated by tyrosine kinase(s) and not by PKC, and positively regulated by Ca2+. They also suggest that the major enhancement by staurosporine of the PGE2-induced cAMP accumulation, although chiefly mediated by protein tyrosine kinase inhibition, also depends on another, presently undetermined, effect of the drug simulating that of Ca2+.

Prostaglandins (PGs) are well known for their role in reproductive processes. At the time of pregnancy recognition, PGF2alpha is luteolytic and PGE2 may be antiluteolytic and luteotropic. During the preimplantation period, interferon-tau (IFN-tau) is produced by the conceptus and plays a crucial role in maternal recognition of pregnancy in domestic ruminants. We have demonstrated previously that recombinant bovine and ovine interferon-tau (rbIFN-tau and roIFN-tau) stimulate PGE2 production in epithelial cells, changing the primary PG produced by these cells from F2alpha to E2. In stromal cells, where PGE2 is the major PG produced, roIFN-tau induced an increase of both types of PGs. The aim of this paper is to identify the possible involvement of cyclooxygenases (COXs) in the modulation of PG production by trophoblastic interferons. Epithelial and stromal cells cultured in vitro were isolated from bovine endometrium and stimulated with increasing doses (1, 10 and 20 microg/ml) of roIFN-tau. PG levels in the culture media were measured by enzyme immunoassays (EIA) and total RNA was extracted from the cells. Northern blot analysis was performed to quantify cyclooxygenase COX-1 (constitutive), COX-2 (inducible) and phospholipase A2 (PLA2) messenger RNA (mRNA) production in response to treatment. The results indicate that roIFN-tau treatment did not affect COX-1 and PLA2 mRNA production in either cell type, whereas COX-2 expression was upregulated in both. The up-regulation of COX-2 transcript was greater in stromal than in epithelial cells. The increase in COX-2 mRNA levels was concurrent with increased production of PGE2 and PGF2alpha in stromal cells and principally PGE2 in epithelial cells. Furthermore, addition of indomethacin (1 microM) and a specific COX-2 inhibitor (NS-398, 1 microM) blocked the roIFN-tau-stimulation of PG production in both cell types. The mechanism whereby elevated COX-2 expression results in a selective increase of PGE2 in epithelial cells remains to be elucidated. In stromal cells, an increase in COX-2 mRNA levels may explain increased PG production. The overall effect of roIFN-tau in the two cell types is a net increase in PGE2 output.

In this study the relationship of prostaglandin E2 (PGE2) concentrations in periapical exudates with clinical findings of teeth with periapical periodontitis is discussed. Periapical exudate samples were obtained from root canals of 77 endodontically involved teeth during routine root canal treatment by the quantitative sampling method using paper points. PGE2 concentrations in periapical exudates (PE-PGE2) were determined by radioimmunoassay. Significantly higher levels of PGE2 were found in periapical exudates from teeth with radiolucent areas than from teeth without radiolucent areas (236.8 +/- 521.3 pg/microliter vs. 14.9 +/- 23.1 pg/microliter, respectively). The elevated PE-PGE2 levels were associated with the presence of clinical symptoms that reflected an acute inflammation in the periapical lesion. In contrast, a significantly negative association of decreased PE-PGE2 levels with increasing size of radiolucent areas was demonstrated. These results suggested that PGE2 was produced locally in periapical lesions and that the PGE2 concentration in periapical exudate could reflect the state of the disease activity in periapical periodontitis.

In this paper, we demonstrated that the production of PGE 2 by CD8 + T lymphocytes through muscarinic cholinergic receptor (mAChR) activation of lymphocytes from mice acutely infected with nonlethal Trypanosoma cruzi CA-1 strain could enhance resistance to infection. Treatment in vivo with either atropine or cyclooxygenase inhibitors enhanced mortality rates and parasitemia of mice infected with T. cruzi CA-1 strain. The mechanism by which CD8 + T lymphocytes released PGE 2 appears to involve the activation of the cells by circulating IgG present in mice infected with T. cruzi CA-1 strain. Binding of these antibodies to mAChR on CD8 + T lymphocytes triggered the release of large amounts of PGE 2 . The results point to a role of serum antibodies against mAChR in the protection of T. cruzi infection. The prostanoid acting as an immunomodulator contributed to the maintenance of the chronic course of experimental Chagas disease.

The activation and proliferation of a prostaglandin E 2 (PGE 2 ) producing monocyte/macrophage may play a major role in down-regulating immune function after injury. But the mechanism by which monocyte or macrophage activation occurs in injury is unknown. Endothelin is a 21-amino acid peptide produced by vascular endothelium in response to ischemia, injury, or endotoxin. Prior work from our laboratory has shown that endothelin increases intracellular calcium in monocytes and causes production of interleukins-6 and -8. In the data reported in this paper, 10 -9 M endothelin stimulated human monocytes to produce 1050 ± 63 pg/ml of PGE 2 at 6 hr and 1328 ± 47 pg/ml at 24 hr. This was nearly as much PGE 2 production as that by bacterial lipopolysaccharide (endotoxin) stimulation (1295 ± 47 pg/ml at 6 hr and 1506 ± 94 at 24 hr). Endothelin had no effect on production of leukotriene B4. Endothelin may play an important initiating role in post-traumatic immunosuppression.

An essential role for an intact vagal nerve has been proven in the development of gastric mucosal cyto- and general protection. On the other hand, chemically-induced (ethanol, HCl, indomethacin) gastric mucosal damage is enhanced after acute surgical vagotomy. The aims of this paper were to study the possible mechanisms of the vagal nerve in the development of gastric mucosal defense. The following questions were addressed: 1) effect of surgical vagotomy on the development of ethanol- (ETOH), HCl-, and indomethacin (IND)-induced gastric mucosal damage: 2) changes in the gastric mucosal defense by scavengers, prostacyclin and other compounds (small doses of atropine and cimetidine; 3) changes in the gastric mucosal vascular permeability due to chemicals; 4) effect of indomethacin in the ETOH and HCl models with and without surgical vagotomy; 5) changes in the gastric mucosal content of prostacyclin and PGE2 in the ETOH and HCl models after surgical vagotomy; and 6) changes in the role of SH-groups in gastric mucosal defense after surgical vagotomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent double blind studies have shown some benefit of borage oil in treatment of rheumatoid arthritis. Tumor necrosis factor-alpha has been shown to be a central mediator of inflammatory and joint destructive processes in rheumatoid arthritis. In this paper, evidence from published research is reviewed that indicates gamma linolenic acid component of borage oil increases prostaglandin E levels that increase cAMP levels that in turn suppress tumor necrosis factor-alpha synthesis. If this biochemical path of borage oil is correct then (1) concomitant non-steroidal anti-inflammatory drug use would tend to undermine borage oil effects, and (2) borage oil would be contraindicated in pregnancy given the teratogenic and labor inducing effects of prostaglandin E agonists.

Hydrolysis of phosphatidyl inositol-4,5-biphosphate, leading to generation of inositol phosphates (IPs), occurs after crosslinking CD3 antigens on the surface of murine and human T-cell lines and clones, and is thought to represent a basic mechanism of signal transduction after antigen receptor binding. Previous investigators have had difficulty demonstrating this phenomenon using human peripheral blood T-cells. In this paper we demonstrate significant IP generation after anti-CD3 stimulation of human peripheral blood lymphocytes and T-cells. The amount of IP generation is less and considerably more variable than what is obtained using a T-cell line. Also, exposure to ammonium chloride in the E-rosetting procedure totally inhibits IP generation, perhaps explaining previous unsuccessful attempts. Finally, prostaglandin E2 and other agents that raise cyclic AMP inhibit IP generation by anti-CD3 antibodies in human T-cells and a T-cell line.

13 ,14 -Dihydro-15-keto-PGF2 alpha (PGFM) serum levels were determined by radioimmunoassay in 101 postpartum women who were treated with 200 micrograms methergin, 5 I.U. oxytocin and 500 micrograms sulprostone, respectively, 30 min after expulsion of placenta. All patients had normal deliveries. The present radioimmunoassay system did not show cross-reactivity with sulprostone. In addition, radioimmunoassayable sulprostone serum levels were monitored. Covariance analysis of area under PGFM serum levels between time zero and 180 min after application of oxytocics was performed. A higher but statistically not significantly PGFM serum level was maintained in subjects treated with sulprostone. Sulprostone serum levels are rapidly attained after application. Decrease of radioimmunoassayable sulprostone indicates a half-life of 75 min. These data corroborate clinical findings of an accompanying paper and combine to suggest that sulprostone may be a useful alternative therapy in high-risk patients with severe postpartum atony and hemorrhage in whom prior preventive measures have failed.

This paper reports the conformation energy (CE) calculations on PGE2, PGE2 alpha and 15-(s)-methyl PGE2 alpha on the basis of empirical potential energy functions for the simultaneous rotations around C7-C8 (psi), C12-C13 (theta) and C14-C15 (phi) bonds. The variation of the minimum conformation energy E for each isoenergy map in the psi theta plane with respect to phi gives two minima around 90 degrees and 240 degrees in PGE2, 60 degrees and 245 degrees in PGF2 alpha, and 60 degrees and 150 degrees in 15-(s)-methyl PGF2 alpha. The latter two forms also have a small dip at 270 degrees. The pattern of allowed low energy conformations of PGF2 alpha and 15-(s)-methyl PGF2 alpha is quite similar and is characterized by the existence of six low energy regions.

The profiles of circulating metabolites of prostaglandin F2 alpha were investigated in a number of species, viz. rat, rabbit, guinea pig, cattle and sheep. The aim of the study was to identify in each animal major plasma metabolites that outlast the initially formed, short-lived 15-keto-13, 14-dihydroprostaglandin F2 alpha and might thus serve as better parameters for monitoring prostaglandin production in vivo. Tritium-labeled prostaglandin F2 alpha was injected intravenously and frequent blood samples were collected. The metabolic profiles at different stages were visualized using two-dimensional thin-layer chromatography and autoradiography. Identification of circulating products was achieved by comparison with reference compounds using several chromatographic methods, and by gas chromatography-mass spectrometry in cases where larger amounts of the prostaglandin had been administered. In the rabbit a similar study was also done with tritium-labeled prostaglandin E2. Certain species differences were seen in the removal of labeled compounds from the circulation, the elimination being most efficient in the guinea pig. Further differences were seen in the profiles of circulating prostaglandin metabolites. The first appearing major prostaglandin F2 alpha metabolite was always 15-ketodihydroprostaglandin F2 alpha. However, this compound was later replaced in the circulation by a number of more degraded products, the profiles of which were relatively typical for each species. Thus, in cattle, rat and guinea pig, the earliest-formed metabolites, 15-ketodihydroprostaglandin F2 alpha and its tetranor counterpart, 5 alpha, 7 alpha-dihydroxy-11-ketotetranorprostanoic acid, remained comparatively prominent plasma products, whereas highly polar dicarboxylic acids rapidly dominated the metabolite spectrum in the ovine and lapine circulation. These differences were further supported by separate kinetic experiments, using unlabeled prostaglandin F2 alpha and radioimmunological determination of formed products. These latter experiments also demonstrated further pronounced species differences in the rat of elimination of the different prostaglandin metabolites. A considerable interconversion between prostaglandin E and F compounds was also demonstrated in some species. In conclusion, the traditional prostaglandin parameters in plasma, the 15-ketodihydrometabolites, were found not to be the best parameters in all species. It is suggested that species differences in prostaglandin metabolism are taken ito consideration when the optimal analytical protocol is sought for future biological studies. Some alternatives are suggested in the present paper.

Summary Pirenzepine (P) exerts cytoprotective action throught a mechanism so far poorly understood. The aim of the present work was to evaluate the protective effect of P and by comparison PGE 2 , in different cytoprotective models: NaOH 0.2 N; antral ulcer induced by indomethacin; and in an “in vivo” model using ethanol (50% 1ml/rat p.o.) as mucosal barrier damaging agent. In this last group of experiments gastric mucosal potential difference (DP), Na + concentrations and pH were measured. These parameters provided a clear measure of the mucosal barrier recovery from damaging agents. P., like PGs, significantly protect mucosa against NaOH 0.2 N induced ulcers with an ED 50 = 21.94 mg/kg os, a similar activity was found for the antral ulcers induced by indomethacin (ED 50 22.06 mg/kg os). Moreover P. significantly antagonized the PD reduction induced by ethanol in rat. PGE 2 was strikingly active in this model. P. and PGE 2 significantly decreased also Na + ion concentration altered by ethanol. Although this paper shows a positive effect of P on the gastric mucosal barrier, further studies are necessary to clarify the mechanism of gastric cytoprotection of P.

Nonsteroidal antiinflammatory drugs (NSAID) have been thought to act by inhibiting the production of prostaglandins. Until recently, prostaglandins have been considered to be mainly proinflammatory compounds, responsible for pain, increased blood flow, and edema. It has more lately been realized that prostaglandins. particularly PGE 2 , have important immunoregulatory properties. 1 Thus NSAID, by inhibiting PGE 2 production, modulate immune responses in vitro and in vivo. One or more of these immunomodulating effects of NSAID may play a role in their efficacy as arthritis medications. The purpose of this paper is to review the immunologic role of PGE 2 and the immunologic effects of NSAID.

We have recently shown that the novel neuropeptide calcitonin gene-related peptide, CGRP, is a potent vasodilator. In this paper we report a detailed study of the effects of CGRP in human skin. CGRP induces a clearly defined, long-lasting erythema. We have measured the effect of CGRP on blood flow in human skin using a laser Doppler technique and have demonstrated increased local blood flow that persists for a number of hours. We compared the response of CGRP with other known vasodilators [histamine, prostaglandin (PG) E2, PGI2, substance P, and vasoactive intestinal peptide (VIP)] in the skin, and in all subjects the erythema induced by CGRP was more persistent than that induced by the other mediators tested. Except at high doses the local vasodilatation induced by CGRP was not associated with a wheal and flare as seen with histamine, substance P, and VIP. CGRP is an extremely potent vasodilator and if released into the circulation, or locally from peripheral nerve endings, it could have a role in the regulation of blood flow in both physiologic and pathologic conditions; CGRP may be the endogenous mediator of the flare in the triple response. A deficiency in CGRP secretion or action could be an important component of peripheral vascular disease. Sonic flushing reactions (e.g., those associated with medullary thyroid carcinoma) may result from circulating CGRP.

Prostaglandins (PG) are endogenous compounds that have been extensively studied by various techniques. In this paper, the syntheses of methyl esters of PGD2 and PGE2, labelled with 11C for PET investigations, are reported. The prostaglandin was esterified via its carboxylate anion, with [11C]methyl iodide as alkylating reagent, in a dimethyl sulphoxide-dimethylformamide mixture. To minimize the base-catalysed degradations, the carboxylate anion was generated in situ by use of tetramethylpiperidine. The radio-chemical yield, including purification, was 70%, with a total synthesis time of 20–25 min counted from the end of the [11C]methyl iodide synthesis.