Malva sylvestris L. extract suppresses desferrioxamine-induced PGE2 and PGD2 release in differentiated U937 cells: the development and validation of an LC-MS/MS method for prostaglandin quantification.

Malva sylvestris is a species used worldwide as an alternative to anti-inflammatory therapies; however, its mechanism of action remains unknown. In this paper, the anti-inflammatory effects of M. sylvestris alcoholic extracts were evaluated by measuring the pro-inflammatory mediators PGE2 and PGD2 in desferrioxamine-stimulated phorbol 12-myristate 13-acetate-differentiated U937 cells. An HPLC-DAD fingerprint of the M. sylvestris extract was performed and caffeic acid, ferulic acid and scopoletin were identified and quantified. An HPLC-MS/MS method was developed and validated to separate and measure the prostaglandins. The lower limits of detection (~0.5 ng/mL for PGE2 and PGD2) and quantification (1.0 ng/mL for PGE2 and PGD2) indicated that the method is highly sensitive. The calibration curves showed excellent coefficients of correlation (r > 0.99) over the range of 1.0-500.0 ng/mL, and at different levels, the accuracy ranged from 96.4 to 106.4% with an RSD < 10.0% for the precision study. This method was successfully applied using U937-d cells. A significant dose-dependent reduction of PGE2 and PGD2 levels occurred using 10 µg/mL (10.74 ± 2.86 and 9.60 ± 6.89%) and 50 µg/mL of extract (48.37 ± 3.24 and 53.06 ± 6.15%), suggesting that the anti-inflammatory mechanisms evoked by M. sylvestris may be related to modulation of these mediators.

Prostaglandin D2 regulates joint inflammation and destruction in murine collagen-induced arthritis.

OBJECTIVE: Prostaglandin D2 (PGD2) may exert proinflammatory or antiinflammatory effects in different biologic systems. Although this prostanoid and the enzymes responsible for its synthesis are up-regulated by interleukin-1ß (IL-1ß) in human chondrocytes in vitro, the role of PGD2 in arthritis remains unclear. This study was undertaken to investigate the role of PGD2 in the inflammatory response and in joint destruction during the development of collagen-induced arthritis (CIA) in mice. METHODS: PGD2 and cytokine levels in mice with CIA were determined by enzyme-linked immunosorbent assay. Expression of hematopoietic PGD synthase (h-PGDS), lipocalin-type PGD synthase (l-PGDS), and DP1 and DP2 receptors was analyzed by immunohistochemical methods. PGE2 levels were determined by radioimmunoassay. RESULTS: The arthritic process up-regulated the expression of h-PGDS, l-PGDS, DP1, and DP2 in articular tissue. PGD2 was produced in the joint during the early phase of arthritis, and serum PGD2 levels increased progressively throughout the arthritic process, reaching a maximum during the late stages of CIA. Treatment of arthritic mice with the DP1 antagonist MK0524 soon after the onset of disease increased the incidence and severity of CIA as well as the local levels of IL-1ß, CXCL-1, and PGE2, whereas IL-10 levels were reduced. The administration of the DP2 antagonist CAY10595 did not modify the severity of arthritis. The injection of PGD2 into the paw, as well as the administration of the DP1 agonist BW245C, significantly lowered the incidence of CIA, the inflammatory response, and joint damage. CONCLUSION: Our findings indicate that PGD2 is produced in articular tissue during the development of CIA and plays an antiinflammatory role, acting through the DP1 receptor.

Mast cell phenotype, location, and activation in severe asthma. Data from the Severe Asthma Research Program.

RATIONALE: Severe asthma (SA) remains poorly understood. Mast cells (MC) are implicated in asthma pathogenesis, but it remains unknown how their phenotype, location, and activation relate to asthma severity. OBJECTIVES: To compare MC-related markers measured in bronchoscopically obtained samples with clinically relevant parameters between normal subjects and subjects with asthma to clarify their pathobiologic importance. METHODS: Endobronchial biopsies, epithelial brushings, and bronchoalveolar lavage were obtained from subjects with asthma and normal subjects from the Severe Asthma Research Program (N = 199). Tryptase, chymase, and carboxypeptidase A (CPA)3 were used to identify total MC (MC(Tot)) and the MC(TC) subset (MCs positive for both tryptase and chymase) using immunostaining and quantitative real-time polymerase chain reaction. Lavage was analyzed for tryptase and prostaglandin D2 (PGD2) by ELISA. MEASUREMENTS AND MAIN RESULTS: Submucosal MC(Tot) (tryptase-positive by immunostaining) numbers were highest in "mild asthma/no inhaled corticosteroid (ICS) therapy" subjects and decreased with greater asthma severity (P = 0.002). In contrast, MC(TC) (chymase-positive by immunostaining) were the predominant (MC(TC)/MC(Tot) > 50%) MC phenotype in SA (overall P = 0.005). Epithelial MC(Tot) were also highest in mild asthma/no ICS, but were not lower in SA. Instead, they persisted and were predominantly MC(TC). Epithelial CPA3 and tryptase mRNA supported the immunostaining data (overall P = 0.008 and P = 0.02, respectively). Lavage PGD2 was higher in SA than in other steroid-treated groups (overall P = 0.02), whereas tryptase did not differentiate the groups. In statistical models, PGD2 and MC(TC)/MC(Tot) predicted SA. CONCLUSIONS: Severe asthma is associated with a predominance of MC(TC) in the airway submucosa and epithelium. Activation of those MC(TC) may contribute to the increases in PGD2 levels. The data suggest an altered and active MC population contributes to SA pathology.

Identification of novel oxidized levuglandin D2 in marine red alga and mouse tissue.

In animals, the product of cyclooxygenase reacting with arachidonic acid, prostaglandin(PG)H(2), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form levuglandin(LG)E(2) and LGD(2). These LGs and their isomers are highly reactive γ-ketoaldehydes that form covalent adducts with proteins, DNA, and phosphatidylethanolamine in cells. Here, we isolated a novel oxidized LGD(2) (ox-LGD(2)) from the red alga Gracilaria edulis and determined its planar structure. Additionally, ox-LGD(2) was identified in some tissues of mice and in the lysate of phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells incubated with arachidonic acid using LC-MS/MS. These results suggest that ox-LGD(2) is a common oxidized metabolite of LGD(2). In the planar structure of ox-LGD(2), H8 and H12 of LGD(2) were dehydrogenated and the C9 aldehyde was oxidized to a carboxylic acid, which formed a lactone ring with the hydrated ketone at C11. These structural differences imply that ox-LGD(2) is less reactive with amines than LGs. Therefore, ox-LGD(2) might be considered a detoxification metabolite of LGD(2).

Development of enzyme-linked immunosorbent assay for Δ12-prostaglandin J2 and its application to the measurement of the endogenous product generated by cultured adipocytes during the maturation phase.

Peroxisome proliferator-activated receptor (PPAR)γ is a well-known master regulator for the differentiation and maturation of adipocytes. Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to J(2) series of PGs including 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2), which serve as pro-adipogenic prostanoids through the activation of PPARγ. However, the quantitative determination of Δ(12)-PGJ(2) has not been attempted during the life stage of adipocytes. In this study, we developed an enzyme-linked immunosorbent assay using mouse antiserum specific for Δ(12)-PGJ(2). According to the standard curve, the amount of Δ(12)-PGJ(2) can be measured from 0.5 pg to 14.4 ng in an assay. Our antiserum did not recognize most other prostanoids including 15d-PGJ(2), while it only showed the cross-reaction of 28% with unstable PGJ(2). This immunological assay was applied to the determination of the endogenous formation of Δ(12)-PGJ(2) in cultured 3T3-L1 adipocytes during the maturation phase. The ability of cultured adipocytes to form endogenous Δ(12)-PGJ(2) increased gradually at an earlier stage of the maturation phase and detectable at higher levels than 15d-PGJ(2). Treatment of cultured cells with either aspirin or indomethacin, a general cyclooxygenase inhibitor, significantly reduced the production of endogenous Δ(12)-PGJ(2) in the maturation medium as expected. Furthermore, we evaluated individually the exogenous effects of PGJ(2) series at various doses on adipogenesis during the maturation phase. Although Δ(12)-PGJ(2) was slightly less potent than 15d-PGJ(2), each of these PGJ(2) series rescued effectively both the accumulation of fats and the gene expression of typical adipocyte-markers that were attenuated in the presence of aspirin. Taken together, our findings indicate that endogenous Δ(12)-PGJ(2) contributes substantially to the up-regulation of adipogenesis program through the activation of PPARγ together with 15d-PGJ(2) during the maturation phase of cultured adipocytes.

Simultaneous determination of histamine and prostaglandin D2 using an LC-ESI-MS/MS method with positive/negative ion-switching ionization modes: application to the study of anti-allergic flavonoids on the degranulation of KU812 cells.

A new method for simultaneous determination of histamine and prostaglandin D(2) (PGD(2)) by liquid chromatography-electrospray ionization tandem mass spectrometry operated in positive and negative ionization switching modes was developed and validated without a previous derivatization step. This method was used to measure histamine and PGD(2) release following degranulation of KU812 human basophilic cells, using pyrazol and d(4)-PGD(2) as internal standards. Analyses were performed on a liquid chromatography system employing a Cosmosil 5C(18) PAQ column and an isocratic elution with mixed solution of methanol-water (7:3, v/v) with 0.0015% trifluoroacetic acid at a flow rate of 0.2 mL/min. A triple-quadrupole mass spectrometer operating in selected reaction monitoring mode simultaneously monitored using the following transitions: positive m/z 112/95 for histamine and negative m/z 351/271 for PGD(2). The retention times of histamine and pyrazol were 4.2 and 5.0 min, respectively. PGD(2) and d(4)-PGD(2) had retention times of 8.5 min. The limits of detection were 0.3 and 0.5 ng/mL for histamine and PGD(2), respectively. The relative standard deviations of the retention time and peak area for histamine were between 1.6% and 7.7%, and were 1.2% and 7.8% for PGD(2). This method was used to evaluate the anti-allergic effects of 26 flavonoids and sodium cromoglicate which are first-line anti-allergic drugs. Of these compounds, baicalein and morin were the most potent inhibitors.

Antigen-induced generation of lyso-phospholipids in human airways.

The goal of the current study was to examine the formation of phospholipids, 1-radyl-2-lysosn-glycero-phospholipids (lyso-PL) and 2-acetylated phospholipids (such as PAF) as well as mechanisms responsible for generating these phospholipids in bronchoalveolar lavage fluid (BAI.F) from allergic subjects challenged with antigen. Bronchoalveolar lavage was performed in normal and allergic subjects before, 5-30 min, 6 h, and 20 h after segmental antigen challenge via a wedged bronchoscope. Levels of 1-hexadecyl-2-lyso-phospholipids and 1-hexadecyl-2-acetyl-phospholipids were initially determined by negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS). Antigen dramatically elevated quantities of 1-hexadecyl-2-lyso-phospholipids in allergic subjects 20 h after challenge when compared to non-allergic controls. In contrast, there was not a significant increase in levels of 1-hexadecyl-2-acetyl-phospholipids after antigen challenge. Closer examination of 1-radyl-2-lyso-sn-glycero-3-phosphocholine (GPC) revealed that 1-palmitoyl-2-lyso-GPC, 1-myristoyl-2-lyso-GPC and 1-hexadecyl-2-lyso-GPC were three major molecular species produced after antigen challenge. 1-palmitoyl-2-lyso-GPC increased sevenfold to levels of 222 +/- 75 ng/ml of BALF 20 h after antigen challenge. The elevated levels of lyso-PL correlated with levels of albumin used to assess plasma exudation induced by allergen challenge. In contrast, the time course of prostaglandin D2 (PGD2) or 9 alpha, 11 beta PGF2 (11 beta PGF2) formation did not correlate with lyso-PL generation. To examine the mechanism leading to lyso-phospholipid formation in antigen-challenged allergic subjects, secretory phospholipase A2 (PI.A2) and acetyl hydrolase activities were measured. There was a significant increase in PLA2 activity found in BALF of allergic subjects challenged with antigen when compared to saline controls. This activity was neutralized by an antibody directed against low molecular mass, (14 kD) human synovial PLA2 and dithiothreitol. Acetyl hydrolase activity also markedly increased in BALF obtained after antigen challenge. This study indicates that high levels of lyso-PLs are present in airways of allergic subjects challenged with antigen and provides evidence for two distinct mechanisms that could induce lyso-PL formation. Future studies will be necessary to determine the ramifications of these high levels of lyso-phospholipids on airway function.

Prostaglandin expression profile in hypoxic osteoblastic cells.

Conditions such as fracture and unloading have been shown to be associated with tissue and cellular hypoxia in bone. The effects of hypoxia on bone cell physiology and ultimately its impact on bone tissue repair and remodeling are not well understood. In this study, we investigated the role of hypoxia on prostaglandin release from osteoblastic cells cultured in 2% (hypoxia), 5% (potentially cellular normoxia), and 21% (normoxia for standard cell culture conditions) oxygen for up to 24 h. We quantified the effects of reduced oxygen tension on the release of prostaglandin (PG)E(2), PGF(2alpha), PGD(2), and PGI(2). The mechanism by which hypoxia increases PG production was investigated by examining the various regulatory components of the PG biosynthetic pathway. Our data show that PGE(2) levels alone are significantly elevated under hypoxic conditions. Also, we show that cyclooxygenase (COX)-1 and COX-2 play an important role in hypoxia-induced PGE(2) production, possibly via a mechanism involving changes in their respective activity levels under low oxygen conditions. The effect of hypoxia on PGE(2) levels was mimicked by dimethyloxaloglycine, a known activator of the HIF pathway. In addition, we confirmed that HIF-1alpha was stabilized in osteoblastic cells under hypoxia. Taken together these data suggest a role for the HIF pathway in regulation of PGE(2) levels under hypoxic conditions. Previous studies have detected release of prostaglandins from areas of damaged bone, such as a fracture site, and our data may contribute to an understanding of how this release is regulated.

Increased cyclooxygenase activity impairs apoptosis of inflammatory neutrophils in mice lacking gelatinase B/matrix metalloproteinase-9.

Matrix metalloproteinase-9 (MMP-9)/gelatinase B plays an important role in neutrophil infiltration during inflammation and cyclooxygenases (COX-1 and COX-2) and their products are important regulators of inflammation. Recently, we reported that a genetic lack of MMP-9 impairs neutrophil infiltration during early zymosan-induced peritonitis but at later stages (> 24 hr) neutrophils persist in the peritoneal cavity. Here we show that this is the result of impaired apoptosis of MMP-9(-/-)-derived leucocytes. As enhanced COX-1 expression was reported in MMP-9(-/-) mice, we evaluated the hypothesis that altered COX expression induced the above phenomenon as COX-dependent prostaglandins can act either anti-apoptotically (PGE(2)) or pro-apoptotically (PGD(2)). The current data demonstrate that messenger RNA and protein expression of both COX isoforms and their activities are increased in MMP-9(-/-) mice during late peritonitis. Application of selective COX inhibitors revealed enhanced COX-1-dependent PGE(2) production and impaired COX-2-dependent PGD(2) synthesis in MMP-9(-/-) mice. Most importantly, inhibition of COX-1 abolished prolonged neutrophil accumulation in the peritoneal cavity of MMP-9(-/-) mice and increased apoptosis of inflammatory leucocytes. Similarly, weaker apoptosis of MMP-9(-/-) bone marrow neutrophils treated in vitro with zymosan was reversed by COX-1 inhibition. In conclusion, enhanced COX-1 expression is responsible for persistent neutrophil presence in the peritoneum of MMP-9(-/-) mice because of increased synthesis of anti-apoptotic PGE(2). In non-transgenic mice, however, inflammatory leucocytes die apoptotically in the late stages of peritonitis as a result of COX-2-dependent PGD(2) activity. Overall, we show a dependence of COX expression on the presence of MMP-9.

Lipid metabolite levels of prostaglandin D2 and eicosapentaenoic acid recovered from bronchoalveolar lavage fluid correlate with lung function of chronic obstructive pulmonary disease patients and controls.

One-step global profiling of analyte (mRNA, protein, metabolite) biomarkers may soon replace conventional blood and histological/biopsy diagnostics technologies. It is important to establish whether the numerous blood and other body fluid-derived potential novel diagnostics will be sufficiently efficacious and precise to replace, for example, imaging and functional diagnostic tests. Currently, imaging technologies and spirometry are indispensable for the diagnosis and management of chronic obstructive pulmonary disease (COPD). To validate the concept of using body fluid biomarkers in COPD and to address the question of whether biomarker levels correlate with lung function, we measured the level of a number of biologically relevant lipids and metabolites in the bronchoalveolar lavage (BAL) fluid of COPD and control subjects and examined whether these correlate with numeric parameters of lung function. Both the diagnosis and management of COPD rely on costly and labor intensive lung function tests. Thus, there is an imminent need to replace the current diagnostic approaches with simpler clinical assays. As a first step, we demonstrate proof of principle; the correlation of lipid biomarkers as measured by LC-MS with lung function. In the apparently BAL-accessible fluid compartment, the total recovered lipid metabolite amount, particularly prostaglandin D(2) and eicosapentaenoic acid show a remarkable linear correlation with lung function (R(2)>0.7). The study outcome is encouraging for the continuation of the work toward the measurement of lipid metabolite levels in more easily obtainable biological fluids such as sputum, exhaled air condensate, urine and plasma.

Quantitative characterization of prostaglandins in the uterus of early pregnant cattle.

Prostaglandins (PGs) are important regulators of reproductive processes including early embryonic development. We analyzed the most relevant PG in bovine uteri at different preimplantation pregnancy stages when compared with non-pregnant controls. Additionally, endometrium and trophoblast tissues were examined regarding specific enzymes and receptors involved in PG generation and function. Simmental heifers were artificially inseminated or received seminal plasma only. At days 12, 15, or 18, post-estrus uteri were flushed for PG determination by liquid chromatography-tandem mass spectrometry. Endometrium and trophoblast tissues were sampled for RNA extraction and quantitative real-time PCR analysis. At all days and points of time examined, the concentration of 6-keto PGF(1alpha) (stable metabolite of PGI(2)) was predominant followed by PGF(2alpha)>PGE(2)>PGD(2) approximately TXB(2) (stable metabolite of TXA(2)). At days 15 and 18, PG increased from overall low levels at day 12, with a much more pronounced increase during pregnancy. The PGF(2alpha)/PGE(2) ratio was not influenced by status. The highest PG concentration was measured at day 15 with 6-keto PGF(1alpha) (6.4 ng/ml) followed by PGF(2alpha) (1.1 ng/ml) and PGE(2) (0.3 ng/ml). Minor changes in endometrial PG biosynthesis enzymes occurred due to pregnancy. Trophoblasts revealed high transcript abundance of general and specific PG synthases contributing to uterine PG. As PGI(2) and PGF(2alpha) receptors were abundantly expressed by the trophoblast, abundant amounts of PGI(2) and PGF(2alpha) in the uterine lumen point towards an essential role of PG for the developing embryo. High amounts of PG other than PGE(2) in the preimplantation uterus may be essential rather than detrimental for successful reproduction.

Statins activate peroxisome proliferator-activated receptor gamma through extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase-dependent cyclooxygenase-2 expression in macrophages.

Both statins and peroxisome proliferator-activated receptor (PPAR)gamma ligands have been reported to protect against the progression of atherosclerosis. In the present study, we investigated the effects of statins on PPARgamma activation in macrophages. Statins increased PPARgamma activity, which was inhibited by mevalonate, farnesylpyrophosphate, or geranylgeranylpyrophosphate. Furthermore, a farnesyl transferase inhibitor and a geranylgeranyl transferase inhibitor mimicked the effects of statins. Statins inhibited the membrane translocations of Ras, RhoA, Rac, and Cdc42, and overexpression of dominant-negative mutants of RhoA (DN-RhoA) and Cdc42 (DN-Cdc42), but not of Ras or Rac, increased PPARgamma activity. Statins induced extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. However, DN-RhoA and DN-Cdc42 activated p38 MAPK, but not ERK1/2. ERK1/2- or p38 MAPK-specific inhibitors abrogated statin-induced PPARgamma activation. Statins induced cyclooxygenase (COX)-2 expression and increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) levels through ERK1/2- and p38 MAPK-dependent pathways, and inhibitors or small interfering RNA of COX-2 inhibited statin-induced PPARgamma activation. Statins also activate PPARalpha via COX-2-dependent increases in 15d-PGJ(2) levels. We further demonstrated that statins inhibited lipopolysaccharide-induced tumor necrosis factor alpha or monocyte chemoattractant protein-1 mRNA expression, and these effects by statins were abrogated by the PPARgamma antagonist T0070907 or by small interfering RNA of PPARgamma or PPARalpha. Statins also induced ATP-binding cassette protein A1 or CD36 mRNA expression, and these effects were suppressed by small interfering RNAs of PPARgamma or PPARalpha. In conclusion, statins induce COX-2-dependent increase in 15d-PGJ(2) level through a RhoA- and Cdc42-dependent p38 MAPK pathway and a RhoA- and Cdc42-independent ERK1/2 pathway, thereby activating PPARgamma. Statins also activate PPARalpha via COX-2-dependent pathway. These effects of statins may explain their antiatherogenic actions.

Prostaglandin D2 expression is prognostic in high­grade serous ovarian cancer.

To identify biomarkers that could predict response or lack of response to conventional chemotherapy at the time of diagnosis of high­grade serous ovarian carcinoma (HGSOC), the present study compared large­scale gene expression from patients with short or long disease­free survival times, according to the last cycle of chemotherapy, and validated these findings using reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and conventional immunohistochemical (IHC) analysis. Samples were selected for microarray evaluation, at the time of diagnosis, using the following criteria: Identical debulking primary surgery, International Federation of Gynaecology and Obstetrics staging, histological subtype and grade. These were divided into 2 groups, regarding the outcome after 2 years of follow-up. Prostaglandin D2 synthase 21 kDa (brain) (PTGDS) was found to be expressed at a significantly higher level in the tumours of patients with a short disease­free survival time, and this was validated by RT­qPCR in all samples. Furthermore, the study evaluated PGD2, the protein product of the PTGDS gene, in a large cohort of 114 HGSOC patients using the Ventana Benchmark automated platform, and IHC positivity was correlated with clinicopathological data and outcome. The global gene expression analysis identified 1,149 genes that were differentially expressed in microarray data, according to the patient outcome. Further analysis RT­qPCR validated PTGDS gene expression in the same samples (r=0.945; P<0.001). IHC analysis showed an inverse profile, with positivity for PGD2 strongly associated with an increase in disease­free survival (P=0.009), the absence of relapse (P=0.039) and sensitivity to platinum­based therapy (P=0.016). Multiple Cox regression showed that IHC evaluation of PGD2 was also a prognostic marker associated with relapse (hazard ratio, 0.37; P=0.002). Overall, the results showed that IHC evaluation of PGD2 is an independent marker of good prognosis in HGSOC. This finding contributes to our understanding of the mechanism of tumour regulation and to investigations into biomarkers that predict response to chemotherapy.

7beta-Hydroxy-epiandrosterone-mediated regulation of the prostaglandin synthesis pathway in human peripheral blood monocytes.

7alpha-Hydroxy-DHEA, 7beta-hydroxy-DHEA and 7beta-hydroxy-EpiA are native metabolites of dehydroepiandrosterone (DHEA) and epiandrosterone (EpiA). Since numerous steroids are reported to interfere with inflammatory and immune processes, our objective was to test the effects of these hydroxysteroids on prostaglandin (PG) production and related enzyme gene expression. Human peripheral blood monocytes were cultured for 4 and 24 h in the presence of each of the steroids (1-100 nM), with and without addition of TNF-alpha (10 ng/mL). Levels of PGE(2), PGD(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) were measured in the incubation medium, and cell content of cyclooxygenase (COX-2), and PGE and PGD synthases (m-PGES1, H-PGDS, L-PGDS), and peroxisome proliferator activated receptor (PPAR-gamma) was assessed by quantitative RT-PCR and Western blots. Addition of TNF-alpha resulted in elevated PG production and increased COX-2 and m-PGES1 levels. Among the three steroids tested, only 7beta-hydroxy-EpiA decreased COX-2, m-PGES1 and PPAR-gamma expression while markedly decreasing PGE(2) and increasing 15d-PGJ(2) production. These results suggest that 7beta-hydroxy-EpiA is a native trigger of cellular protection through simultaneous activation of 15d-PGJ(2) and depression of PGE(2) synthesis, and that these effects may be mediated by activation of a putative receptor, specific for 7beta-hydroxy-EpiA.

Clinical and biochemical effects of a combination botanical product (ClearGuard) for allergy: a pilot randomized double-blind placebo-controlled trial.

BACKGROUND: Botanical products are frequently used for treatment of nasal allergy. Three of these substances, Cinnamomum zeylanicum, Malpighia glabra, and Bidens pilosa, have been shown to have a number of anti-allergic properties in-vitro. The current study was conducted to determine the effects of these combined ingredients upon the nasal response to allergen challenge in patients with seasonal allergic rhinitis. METHODS: Twenty subjects were randomized to receive the combination botanical product, (CBP) 2 tablets three times a day, loratadine, 10 mg once a day in the morning, or placebo, using a randomized, double-blinded crossover design. Following 2 days of each treatment and during the third day of treatment, subjects underwent a nasal allergen challenge (NAC), in which nasal symptoms were assessed after each challenge dose and every 2 hours for 8 hours. Nasal lavage fluid was assessed for tryptase, prostaglandin D2, and leukotriene E4 concentrations and inflammatory cells. RESULTS: Loratadine significantly reduced the total nasal symptom score during the NAC compared with placebo (P = 0.04) while the CBP did not. During the 8 hour period following NAC, loratadine and the CBP both reduced NSS compared with placebo (P = 0.034 and P = 0.029, respectively). Analysis of nasal lavage fluid demonstrated that the CBP prevented the increase in prostaglandin D2 release following NAC, while neither loratadine nor placebo had this effect. None of the treatments significantly affected tryptase or leukotriene E4 release or inflammatory cell infiltration. CONCLUSION: The CBP significantly reduced NSS during the 8 hours following NAC and marginally inhibited the release of prostaglandin D2 into nasal lavage fluid, suggesting potential clinical utility in patients with allergic rhinitis.

Regulation of inflammatory pathways in schizophrenia: A comparative study with bipolar disorder and healthy controls.

BACKGROUND: Immune-inflammatory processes have been implicated in schizophrenia (SCH), but their specificity is not clear. MAIN AIM: To identify potential differential intra-/intercellular biochemical pathways controlling immune-inflammatory response and their oxidative-nitrosative impact on SCH patients, compared with bipolar disorder (BD) patients and healthy controls (HC). METHODS: Cross-sectional, naturalistic study of a cohort of SCH patients (n=123) and their controls [BD (n=102) and HC (n=80)]. STATISTICAL ANALYSIS: ANCOVA (or Quade test) controlling for age and gender when comparing the three groups, and controlling for age, gender, length of illness, cigarettes per day, and body mass index (BMI) when comparing SCH and BD. RESULTS: Pro-inflammatory biomarkers: Expression of COX-1 was statistically higher in SCH and BD than HC (P<0.0001; P<0.0001); NFκB and PGE2 were statistically higher in SCH compared with BD (P=0.001; P<0.0001) and HC (P=0.003; P<0.0001); NLRP3 was higher in BD than HC (P=0.005); and CPR showed a gradient among the three groups. Anti-inflammatory biomarkers: BD patients had lower PPARγ and higher 15d-PGJ2 levels than SCH (P=0.005; P=0.008) and HC (P=0.001; P=0.001). Differences between SCH and BD: previous markers of SCH (NFκB and PGE2) and BD (PPARγ and 15d-PGJ2) remained statistically significant and, interestingly, iNOS and COX-2 (pro-inflammatory biomarkers) levels were statistically higher in SCH than BD (P=0.019; P=0.040). CONCLUSIONS: This study suggests a specific immune-inflammatory biomarker pattern for established SCH (NFκB, PGE2, iNOS, and COX-2) that differentiates it from BD and HC. In future, their pharmacological modulation may constitute a promising therapeutic target.

PGD2 metabolism in plasma: kinetics and relationship with bioactivity on DP1 and CRTH2 receptors.

Prostaglandin (PG)D(2), an important mediator in allergic diseases, is rapidly transformed in plasma to active metabolites that bind and activate two distinct receptors, DP1 and CRTH2. Since the rate of PGD(2) degradation and the bioactivity of the resulting metabolites are still unclear, the aim of our study was to analyze the kinetics and biological effects of PGD(2) metabolites formed in plasma. Eosinophil shape change was taken as a parameter of chemotactic activation mediated by CRTH2 whereas inhibition of platelet aggregation served as a measure of DP1 activity. PGD(2) was degraded in plasma with an apparent half-life of approximately 30 min, accompanied by a loss of potency in inhibiting platelet aggregation as well as inducing eosinophil stimulation. Incubation of PGD(2) in plasma for 120 min caused an increase in the IC(50) for platelet aggregation by a factor of 6.5 and an increase of the EC(50) for eosinophil shape change by a factor of 7.2. However, tandem mass spectrometry analysis showed that incubation of PGD(2) in plasma for 120 min resulted in clearance of PGD(2) of more than 92%, which was mirrored by a continuous formation of Delta(12)-PGD(2) and Delta(12)-PGJ(2), whereas only small amounts of 15d-PGD(2) and 15d-PGJ(2) were detected. Interestingly, a rapid degradation of PGD(2) was also observed in serum, which was not prevented by pepsin digestion of serum preceding the addition of PGD(2). Therefore, despite extensive non-enzymatic metabolization of PGD(2) in plasma, its biological activity with respect to DP1 and CRTH2 is maintained through the formation of bioactive metabolites.

Development of enzyme-linked immunosorbent assay for prostaglandin D2 using the stable isosteric analogue as a hapten mimic and its application.

For the determination of prostaglandin (PG) D(2) produced by cultured cells in response to external stimuli, immunological methods would be convenient and useful. However, PGD(2) is unstable under the physiological conditions, so that it has been difficult to get a specific antibody for the parent PGD(2). In an attempt to get a specific antibody for PGD(2), we tried to prepare monoclonal antibodies for 11-deoxy-11-methylene-PGD(2), a novel, chemically stable, isosteric analogue of PGD(2). We successfully cloned a hybridoma cell line secreting a monoclonal antibody reacting specifically with the parent PGD(2). To develop the enzyme-linked immunosorbent assay (ELISA) for PGD(2), the immobilized antigen using the stable PGD(2) derivative was immunoreacted in a competitive manner with the monoclonal antibody in presence of free PGD(2). The optimization of the assay provided a sensitive calibration curve for PGD(2) from 0.32 pg to 0.18 ng with a value of 7.6 pg at 50% displacement. PGD(2) was almost stable during the ELISA condition. The developed assay method was useful for applying to the direct determination of PGD(2) in the culture medium of mouse 3T3-L1 adipocytes. The incubation of PGD(2) in the maturation medium of adipocytes at 37 degrees C caused the chemical conversion into PGJ(2) derivatives. The conversion became more evident after 6 h of the incubation. These findings indicate the importance of considering the optimal time for collecting the samples to be determined for PGD(2) before the conversion starts to occur.

Niacin ameliorates ulcerative colitis via prostaglandin D2-mediated D prostanoid receptor 1 activation.

Niacin, as an antidyslipidemic drug, elicits a strong flushing response by release of prostaglandin (PG) D2 However, whether niacin is beneficial for inflammatory bowel disease (IBD) remains unclear. Here, we observed niacin administration-enhanced PGD2 production in colon tissues in dextran sulfate sodium (DSS)-challenged mice, and protected mice against DSS or 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in D prostanoid receptor 1 (DP1)-dependent manner. Specific ablation of DP1 receptor in vascular endothelial cells, colonic epithelium, and myeloid cells augmented DSS/TNBS-induced colitis in mice through increasing vascular permeability, promoting apoptosis of epithelial cells, and stimulating pro-inflammatory cytokine secretion of macrophages, respectively. Niacin treatment improved vascular permeability, reduced apoptotic epithelial cells, promoted epithelial cell update, and suppressed pro-inflammatory gene expression of macrophages. Moreover, treatment with niacin-containing retention enema effectively promoted UC clinical remission and mucosal healing in patients with moderately active disease. Therefore, niacin displayed multiple beneficial effects on DSS/TNBS-induced colitis in mice by activation of PGD2/DP1 axis. The potential efficacy of niacin in management of IBD warrants further investigation.

The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation.

The importance of inflammation in initiating the sequence of events that lead to liver fibrosis is increasingly recognized. In this study, we tested the effects of SC-236, a selective cyclooxygenase (COX)-2 inhibitor, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Livers from CCl4-treated rats showed increased COX-2 expression and 15-deoxy-prostaglandin (PG)J2 (15d-PGJ2) formation, as well as decreased peroxisome proliferator-activated receptor (PPAR)gamma expression. In these animals, SC-236 reduced liver fibrosis as revealed by histological analysis and by a reduction in hepatic hydroxyproline levels, metalloproteinase-2 activity, and alpha-smooth muscle actin expression. Interestingly, SC-236 normalized 15d-PGJ2 levels and restored PPARgamma expression in the liver of CCl4-treated rats. In isolated hepatic stellate cells (HSCs)--the major player in liver fibrogenesis--and Kupffer cells--the cell type primarily responsible for increased hepatic COX-2-SC-236 exhibited remarkable pro-apoptotic and growth inhibitory properties. Of interest, SC-236 decreased HSC viability to a similar extent than the PPARgamma ligand rosiglitazone. Moreover, SC-236 significantly induced PPARgamma expression in HSCs and acted as a potent PPARgamma agonist in a luciferase-reporter trans-activation assay. These data indicate that, by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation, the selective COX-2 inhibitor SC-236 might have therapeutic potential for prevention of liver fibrosis.