Effects of an Oral CRTh2 Antagonist (AZD1981) on Eosinophil Activity and Symptoms in Chronic Spontaneous Urticaria.

BACKGROUND: Approximately 50% of patients with chronic spontaneous urticaria (CSU) experience symptoms that are not fully controlled by antihistamines, indicating an unmet clinical need. OBJECTIVE: To evaluate the effects of the selective CRTh2 antagonist AZD1981 on symptoms and targeted leukocytes in adults with persistent CSU despite treatment with H1-antihistamines. METHODS: We performed a single-center, randomized, placebo-controlled study involving adult CSU subjects with symptoms despite daily antihistamines. The subjects underwent a 2-week placebo run-in and 4 weeks of double-blinded therapy with either AZD1981 40 mg TID or placebo, followed by a 2-week placebo washout. The primary objective was to assess the effect of AZD1981 on CSU signs and symptoms. Secondary objectives included the effects of AZD1981 on prostaglandin D2 (PGD2)-induced eosinophil shape change, circulating leukocyte subsets, CRTh2 expression on blood leukocytes, and total blood leukocyte histamine content. RESULTS: Twenty-eight subjects were randomized to AZD1981 or placebo, with 26 subjects completing the study. The urticaria activity scores declined during the treatment phase in both groups, and they were significantly reduced in the AZD1981 group at the end of washout. AZD1981 treatment increased circulating eosinophils and significantly impaired PGD2-mediated eosinophil shape change. CRTh2 surface expression rose significantly on blood basophils during active treatment. No serious adverse events were observed. CONCLUSIONS: This is the first study to examine the efficacy of a CRTh2 antagonist in antihistamine-refractory CSU. AZD1981 treatment was well tolerated, effectively inhibited PGD2-mediated eosinophil shape change, shifted numbers of circulating eosinophils, and reduced weekly itch scores more than hives during treatment and into washout. Further studies are needed to determine whether inhibition of the PGD2/CRTh2 pathway will be an -effective treatment for CSU.

Cloprostenol administration in the first week postpartum reduces expression of oxytocin receptors in the endometrium in Holstein-Zebu cows / Administração de cloprostenol na primeira semana pós-parto pode reduzir a expressão de receptores de ocitocina no endométrio de vacas mestiças Holandês-Zebu

The present study investigated the hormonal profile and expression of prostaglandin F2α (PGF2α), oxytocin and estrogen receptors in uterine tissues of postpartum cows treated with cloprostenol. Twenty Holstein-Zebu crossbred cows were treated with saline solution (treatment CONT) or cloprostenol (treatment CLO), both administered two and five days postpartum. Blood samples were collected on days two, seven, 14, 21 and 28 postpartum for progesterone, PGF2α metabolite (PGFM) and estradiol determination, and endometrial biopsy was performed in order to quantify the expression of oxytocin receptor (OXTR), prostaglandin F receptor (PTGFR) and estrogen receptor 1 (ERS1) genes. In the CLO treatment, expression of OXTR was reduced (P<0.05) but no difference (P>0.05) between treatments was found for PTGFR and ERS1 expression. Estrogen concentrations increased progressively until day 14 (P<0.05) and the highest OXTR expression and lowest PTGFR expression were observed on day 14 (P<0.05) in both treatments. Serum PGFM concentrations were high throughout the experiment. In conclusion, cloprostenol administration at days two and five of postpartum seems to reduce OXTR expression in the endometrium in crossbred cows.(AU)

O presente estudo avaliou o perfil hormonal e a expressão gênica de receptores de prostaglandina F2α (PGF2α), ocitocina e estrógeno no endométrio de vacas pós-parto tratadas com cloprostenol. Vinte vacas mestiças Holandês-Zebu foram tratadas com solução salina (tratamento CONT, n = 10) ou cloprostenol (tratamento CLO, n = 10), ambos administrados dois e cinco dias após o parto. Amostras de sangue foram coletadas nos dias dois, sete, 14, 21 e 28 pós-parto para mensuração de progesterona, de metabólito de PGF2α (PGFM) e de estradiol, e foram obtidas biópsias endometriais para quantificar a expressão de PTGFR, OXTR e ESR1. No tratamento CLO, a expressão gênica de receptores de ocitocina foi menor (P<0,05). As concentrações de estrógeno aumentaram progressivamente até o dia 14 (P<0,05). A maior expressão de OXTR foi observada no dia 14 (P<0,05). A expressão de ESR1 foi semelhante entre os tratamentos (P>0,05). Os níveis de PGFM foram altos durante todo o estudo. Conclui-se que a administração de cloprostenol nos dias dois e cinco pós-parto parece diminuir a expressão de OXTR no endométrio em vacas mestiças.(AU)

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.

Temporal analysis of prostaglandin F2α receptor, caspase 3, and cyclooxygenase 2 messenger RNA expression and prostaglandin F2α receptor and cyclooxygenase 2 protein expression in endometrial tissue from multiparous Nelore (Bos taurus indicus) cows treated with cloprostenol sodium during puerperium.

The use of cloprostenol sodium in puerperium is questionable, as both favorable and unfavorable responses during the uterine involution process have been reported in the literature. This study is based on the hypothesis that cloprostenol sodium promotes modifications in the prostaglandin F2α receptor (FP), caspase 3 (CASP-3), and cyclooxygenase 2 (COX-2) mRNA expression that may favor the process of postpartum uterine involution in multiparous Nelore (Bos taurus indicus) females. Additionally, we aimed to describe the presence and immunolocalization of the FP and COX-2 protein in endometrial tissue at different postpartum time points in these females. Multiparous Nelore cows (n = 24) were treated with cloprostenol sodium (n = 12) or saline solution (n = 12) on postpartum Days 1 and 4 (Day 0 = birth), and endometrial biopsies were performed with a Yomann biopsy instrument and collected on Days 1, 7, 14, 28, and 42 postpartum. The mRNA expression from samples on the Days 1, 7, 14, 28, and 42 and the protein expression from samples on the Days 1, 14, 28, and 42 were then analyzed. The treated cows had altered FP and CASP-3 mRNA expression, and FP and COX-2 protein were observed in the endometrial surface epithelium, the stroma, and the glandular epithelium, with cytoplasmic immunolocalization. Although we attribute the change in CASP-3 mRNA expression to physiological phenomena, the results obtained for FP mRNA expression opens new doors for the study of hormonal protocols associated with cloprostenol sodium in the puerperium of Zebu females.

The effects of the stromal cell-derived cyclooxygenase-2 metabolite prostaglandin E2 on the proliferation of colon cancer cells.

It is well known that tumor-surrounding stromal tissues support tumor development through secreting soluble factors such as various cytokines, chemokines, and growth factors. It has also been suggested that tumor-associated fibroblast and immune cells have a high expression of cyclooxygenase-2 (COX-2) and produce and secrete several prostaglandins (PGs) to adjacent cancer tissues. From these findings, we assumed that COX-2 inhibition might have an anticancer effect on cancer cells even without COX-2 expression in COX-2-dependent mechanisms through blocking the effect of stroma-derived PGs. Here, because of the complex involvement of various factors in vivo, we investigated this possibility with an in vivo-mimicking model using a Transwell system. To test our hypothesis, we used COX-2-transfected cell lines as stromal cells in our model. When we cocultured cancer cells (LS174T cells without COX-2 expression) with COX-2-high stromal cells in the Transwell membrane system, we observed that the proliferation of cancer cells was promoted and vascular endothelial growth factor synthesis was up-regulated significantly. These effects were blocked completely by COX-2 inhibitors and phosphoinositide-3-kinase inhibitors and partially by the PG E(2) receptor 4 antagonist. Even if some cancer cells did not express COX-2, they were found to have expression of PG receptors and PG-related downstream signaling molecules associated with cell viability. Our observation suggests that these cells can be influenced by PGs derived from stromal tissues. These findings also suggest that COX-2 inhibitors can be used to control the interaction between cancer and surrounding stromal tissues and suppress the proliferation of cancer cells regardless of the expression of COX-2 in cancer cells.

Comprehensive expression analysis of prostanoid enzymes and receptors in the human endometrium across the menstrual cycle.

Prostanoids are well-described primary mediators of inflammatory processes and are essential for the normal physiological function of the female reproductive system. The aim of this study was to determine the temporal expression of the prostanoid biosynthetic enzymes (PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, AKR1C3, CBR1, HPGDS, PTGDS, PTGIS, TBXAS1 and HPGD) and the prostanoid receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGDR, GPR44, PTGIR and TBXA2R) in the human endometrium throughout the menstrual cycle. The analysis identified PTGFR to have a distinct expression profile compared with other components of the prostanoid system, as expression is maximal during the proliferative phase. Immunohistochemical analysis for PTGER1 suggests a dual function for this receptor depending on its temporal (proliferative versus secretory) and spatial (nuclear versus cell membrane) expression. The expression profiles of the PGF(2α) synthases identified AKR1B1 and CBR1 as the likely regulators of PGF(2α) production during the menstrual phase. Immunohistochemical analysis for AKR1B1, CBR1 and AKR1C3 suggest expression to be in the glandular epithelium and vasculature. This study represents the first comprehensive analysis of the components of prostanoid biosynthetic and signalling pathway in the human endometrium. The expression profiles described have the potential to identify specific prostanoid components that may be dysregulated in inflammatory-associated disorders of the endometrium.

Expression and regulation of prostaglandin receptors in the human placenta and fetal membranes at term and preterm.

Prostaglandins (PGs) play an important role in parturition in many species, including humans. The present study examined the distribution of PG receptor subtypes (EP1-4 and FP) in intrauterine tissues at term and preterm birth. Placentas and fetal membranes were collected from patients at term in labour (n = 12) or not in labour (n = 12). Preterm tissue was collected from three different groups of patients: (1) idiopathic preterm labour (PTL) without chorioamnionitis or betamethasone (BM) treatment (n = 9), (2) idiopathic PTL that received BM with no chorioamnionitis (PTL-BM; n = 9) and (3) pregnancies that were complicated with chorioamnionitis and had no BM (PTL-CHA; n = 6). EP1-4 and FP receptors were localised and levels of expression were determined by western blot analysis. All EP receptors and FP were localised to the amnion, placenta and choriodecidua. Moreover, isolated amnion mesenchymal, amnion epithelial, chorion trophoblast and syncytiotrophoblast cells in primary culture also expressed PG receptors. A significant increase was observed in EP1, EP3 and FP expression in placenta, chorion and amnion with labour. Maternal betamethasone treatment increased EP1, EP3 and FP receptor protein expression and chorioamnionitis decreased expression in all the receptor subtypes. These changes in PG receptors in the fetal membranes are consistent with the development of a feed-forwards cascade mediated through PG action that may contribute to the birth process.

CRTH2 expression on T cells in asthma.

Mast cell-derived prostaglandin D2 (PGD2) is the major prostanoid found within the airway of asthmatics immediately following allergen challenge. PGD2 has been shown to have chemokinetic effects on eosinophils and T helper type 2 (Th2) cells in vitro. This occurs through the interaction of PGD2 with the G-protein-coupled chemokine receptor homologous molecule expressed on Th2 lymphocytes (CRTH2). The expression of CRTH2 has been shown to be highly selective for Th2 cells. Using flow cytometry we have studied the expression of CRTH2 on T cells in blood and bronchoalveolar lavage fluid in asthmatics and normal subjects. CRTH2 expression was confined to a small percentage of blood T cells in asthmatics (1.8%+/-0.2) and normal (1.6%+/-0.2) subjects. CRTH2 was enriched significantly on interleukin (IL)-4+/IL-13+ T cells compared to interferon (IFN)-gamma+ T cells (P<0.001). There was a small population of CRTH2+ T cells in the bronchoalveolar lavage (BAL) of asthmatics (2.3%+/-0.6) and normal subjects (0.3%+/-0.1), and there was a significant difference between the two groups (P<0.05). There were similar amounts of PGD2 in the BAL of asthma and normal subjects. Within paired blood-BAL samples from the same subject there was no increase in CRTH2+ T cells in the BAL compared to blood in asthmatics. Enrichment of CRTH2 on IL-4+ and IL-13+ T cells compared to IFN-gamma+ T cells was also seen in BAL from asthmatics (P<0.001). CRTH2 is expressed preferentially by IL-4+/IL-13+ T cells compared to IFN-gamma+ T cells. However, given their small numbers they are unlikely to have a significant involvement in the pathogenesis of asthma. CRTH2 antagonism may not diminish T cell accumulation in the asthmatic lung.

Prostaglandin receptors EP2 and IP are detectable in atherosclerotic arteries and plaques.

AIM: Prostaglandin (PG) receptor agonists are frequently used for the pharmacological treatment of arteriosclerosis obliterans (ASO). In particular, the PG receptors EP2 and IP stimulate vasodilation and inhibit platelet aggregation, biological processes thought to be protective against ASO and important for physiological homeostasis. However it is uncertain whether EP2 and IP exist in diseased arteries, or what their distribution within the artery might be. In this study, we analyzed the distribution of these PG receptors in patients with severe ASO to determine the potential application of stimulation of these receptors as targets for pharmacological treatment. METHODS: We collected segments of atherosclerotic femoral arteries during femoropopliteal bypass surgery and determined the expression levels of EP2 and IP receptors by western blotting. Immunofluorescence was used to observe receptor localization. RESULTS: Findings of western blotting showed an increased Cox-2 expression in patients with ASO. The EP2 as well as IP receptors were each induced approximately 3-fold in comparison to normal samples. The expression of these receptors was increased in the intimal layer as well as the medial layer; their expression was also detectable within the atherosclerotic plaque. CONCLUSION: We observed induction of the PG receptors EP2 and IP in atherosclerotic femoral arteries in the arterial intima, medial layer, as well as the associated atherosclerotic plaque. These results suggest that receptor-selective PG agonists specifically target atherosclerotic arteries and therefore, may find potential application in the pharmacological management of patients with ASO.

The p53 positive Bcl-2 negative phenotype is an independent marker of prognosis in breast cancer.

The purpose of this work was to determine if the immunohistochemical p53 (+) Bcl-2 (-) phenotype predicts survival in breast cancer patients. Tissue from 819 cases of resected primary breast cancer, presented between 1986 and 1998, were assembled in tissue microarray format. Clinicopathological data and prospective disease specific survival data were collected prospectively and immunohistochemical analyses of p53 and Bcl-2 expression were performed using antibodies DO-7 (p53) and 124 (Bcl-2) employing a standard IHC protocol. The expression data were correlated with clinicopathological variables and outcomes in both univariate (chi(2)) and multivariate (Cox's regression) analyses. Abnormal p53 expression and positive Bcl-2 expression were detected in 29% (193/673) and 46% (307/673) of tumours, respectively. On univariate analysis Bcl-2 expression was correlated with the clinicopathological features of less aggressive disease and loss of Bcl-2 expression correlated with a reduction in survival (log rank = 11.91; p < 0.001). p53 expression correlated with the clinicopathological features of aggressive cancers and a reduction in survival (log rank = 17.81; p < 0.001). Nineteen percent (127/673) of tumours displayed a p53 (+) Bcl-2 (-) phenotype. Kaplan-Meier analysis revealed a significant reduction in survival in these cases (log rank 34.01; p < 0.001). Multivariate analysis showed that while neither p53 expression nor Bcl-2 expression alone had independent prognostic significance, the p53 (+) Bcl-2 (-) phenotype remained independently associated with a worse prognosis (HR 1.79 95%CI 1.10-2.89 p = 0.018).

Cyclooxygenase-2 and prostaglandin F2 alpha in Syrian hamster Leydig cells: Inhibitory role on luteinizing hormone/human chorionic gonadotropin-stimulated testosterone production.

We have previously found that cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins (PGs), is present in the testicular interstitial cells of infertile men, whereas it is absent in human testes with no evident morphological changes or abnormalities. To find an animal model for further investigating COX-2 and its role in testicular steroidogenesis, we screened testes from adult species ranging from mice to monkeys. By using immunohistochemical assays, we found COX-2 expression only in Leydig cells of the reproductively active (peripubertal, pubertal, and adult) seasonal breeder Syrian hamster. COX-2 expression in hamster Leydig cells was confirmed by RT-PCR. In contrast, COX-1 expression was not detected in hamster testes. Because COX-2 expression implies PG synthesis, we investigated the effect of various PGs on testosterone production and found that PGF2 alpha stood out because it significantly reduced human chorionic gonadotropin-stimulated testosterone release from isolated hamster Leydig cells in a dose-dependent manner. This mechanism involves a decreased expression of testicular steroidogenic acute regulatory protein and 17beta-hydroxysteroid dehydrogenase. Testicular concentration and content of PGF2 alpha in reproductively active hamsters as well as production of PGF2 alpha from isolated hamster Leydig cells were also determined. Moreover, PGF2 alpha receptors were localized in Leydig cells of hamsters and testicular biopsies from patients with Sertoli cell only and germ arrest syndromes. Thus, in this study, we described a COX-2-initiated pathway that via PGF2 alpha production, PGF2 alpha receptors, steroidogenic acute regulatory protein, and 17beta-hydroxysteroid dehydrogenase represents a physiological local inhibitory system of human chorionic gonadotropin-stimulated testosterone production in the Syrian hamster testes.

Maintenance and polarization of human TH2 central memory T cells by thymic stromal lymphopoietin-activated dendritic cells.

The identity of TH2 memory cells and the mechanism regulating their maintenance during allergic inflammation remain elusive. We report that circulated human CD4+ T cells expressing the prostaglandin D2 receptor (CRTH2) are TH2 central memory T cells, characterized by their phenotype, TH2 cytokine production, gene-expression profile, and the ability to respond to allergens. Only dendritic cells (DCs) activated by thymic stromal lymphopoietin (TSLP) can induce a robust expansion of CRTH2+CD4+ TH2 memory cells, while maintaining their central memory phenotype and TH2 commitments. CRTH2+CD4+ TH2 memory cells activated by TSLP-DCs undergo further TH2 polarization and express cystatin A, Charcot-Leydon crystal protein, and prostaglandin D2 synthase, implying their broader roles in allergic inflammation. Infiltrated CRTH2+CD4+ TH2 effector memory T cells in skin lesion of atopic dermatitis were associated with activated DCs, suggesting that TSLP-DCs play important roles not only in TH2 priming, but also in the maintenance and further polarization of TH2 central memory cells in allergic diseases.

Long-term agonist stimulation of IP prostanoid receptor depletes the cognate G(s)alpha protein in membrane domains but does not change the receptor level.

Iloprost (IP) stimulation (1 microM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous G(s)alpha protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, G(i)alpha and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-G(s)alpha fusion protein. The endogenous G(s)alpha decreased, but the level of Flag-hIPR-G(s)alpha protein did not change. The specific depletion of domain-bound pool of G(s)alpha as consequence of iloprost stimulation was also demonstrated in membrane domains prepared according to alkaline treatment plus sonication protocol (detergent-free procedure of Song et al.). Our data further indicated that in control, quiescent cells only a very small amount of IP prostanoid receptor was present in DIMs together with large amount of its cognate G(s)alpha protein. Expressed in quantitative terms, DIMs contained 30-40% of the total cellular amount of G proteins whereas the content of IP prostanoid receptors was 1-3%. The dominant portion (>95%) of FhIPR as well as FhIPR-G(s)alpha was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-G(s)alpha distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the alpha subunit of Na,K-ATPase, transmembrane form of CD58 and CD44). All these proteins are known to be fully solubilised by detergent and thus unable to float in density gradient. Our data indicate that (i) long-term agonist stimulation of IP prostanoid receptor is associated with preferential decrease of its cognate G protein G(s)alpha from membrane domains; receptor level is unchanged. (ii) Very small fraction (1-3%) of total cellular amount of receptors is recovered in DIMs together with roughly 40% of G proteins. These data suggest a "supra-stoichiometric" arrangement of G proteins and corresponding receptors in DIMs.

Human prostacyclin receptor.

Prostacyclin, a member of the eicosanoid family of lipid mediators, is the major product of arachidonic acid metabolism formed in the marcovascular endothelium. It is a potent vasodilator, antithrombotic, and antiplatelet agent that mediates it effects through a membrane-associated receptor termed the IP. Cloning of the cDNA for IP, from human and other species, indicated its membership of the G protein-coupled receptor superfamily and has allowed detailed examination of the signaling and regulatory pathways utilized by this receptor. This article examines the current state of knowledge of the IP, its signaling and regulation, and its biological role in vivo and examines the possible existence of multiple PGI2 receptor sites.

Prostacyclin is an autocrine regulator in the contraction of oviductal smooth muscle.

BACKGROUND: It was recently discovered that prostacyclin constituted 40-50% of prostaglandins (PG) produced by minced human oviduct. It is well established that prostacyclin relaxes vascular smooth muscle, but whether oviductal smooth muscle synthesizes prostacyclin and whether its contraction is affected by prostacyclin remain unclear. METHODS: Smooth muscle microdissected from human oviducts was used for the study. The expression of prostacyclin synthase (PGIS) and prostacyclin receptor (IP) was confirmed by Western blot analysis. Metabolites of [(3)H]PGH(2) were analysed for prostacyclin. Functional coupling of IP to adenyl cyclase was assessed by the accumulation of intracellular cAMP upon prostacyclin challenge. The presence of saturable, specific binding sites for prostacyclin was confirmed by binding assay. The identity of IP was further confirmed by RT-PCR and nucleotide sequence analysis. Finally, the effects of prostacyclin on muscle contraction were studied. RESULTS: Human oviductal smooth muscle expresses functionally active PGIS and IP. The IP expressed is the same as that cloned from human lung tissue. The ED(50) of prostacyclin to increase intracellular cAMP was 16 nmol/l. Prostacyclin dose-dependently decreased the amplitude of muscle contraction. CONCLUSIONS: Human oviductal smooth muscle produces prostacyclin, which, in turn, decreases its contractility. Prostacyclin may regulate embryo transport.

Secretion of monocyte chemoattractant protein-1 by endothelial cells of the bovine corpus luteum: regulation by cytokines but not prostaglandin F2alpha.

Information regarding the regulation of monocyte chemoattractant protein-1 (MCP-1) in regression of the corpus luteum (CL) is limited. This study tested the hypothesis that endothelial cells derived from bovine CL are a source of MCP-1, and that proinflammatory cytokines, prostaglandin F2alpha (PGF2alpha), and progesterone regulate MCP-1 expression. Endothelial cells were treated without (Control) or with PGF2alpha (1 micro M), TNFalpha (100 ng/ml), interferon-gamma (IFNgamma, 200 IU/ml), and TNFalpha + IFNgamma for 24 and 48 h in the absence or presence of progesterone (P4, 250 ng/ml). Increases in MCP-1 mRNA and protein were observed in response to TNFalpha within 24 and 48 h of culture, respectively (P < 0.05). Interferon-gamma stimulated (P < 0.05) both MCP-1 mRNA and protein after 24 h of culture, and this effect was also sustained through 48 h of culture (P < 0.05). Cotreatment of cultures with TNFalpha + IFNgamma lead to further increases (P < 0.05) in MCP-1 in both 24- and 48-h cultures. Surprisingly, neither PGF2alpha nor P4 affected MCP-1 production. Subsequent experiments revealed that the endothelial cells lacked prostaglandin F2alpha receptor mRNA, and the MAPK pathway, although present and responsive to growth factor stimulation, was unresponsive to PGF2alpha stimulation. In summary, endothelial cells derived from bovine CL respond to TNFalpha and IFNgamma stimulation with an increase in MCP-1 secretion. In contrast, neither PGF2alpha nor P4 directly influenced endothelial expression of MCP-1. These results suggest that cytokines stimulate the synthesis of MCP-1 observed during PGF2alpha-induced luteal regression.

Role of PGF2alpha and oxytocin in parturition in the brushtail possum (Trichosurus vulpecula).

Maturation of the fetal pituitary and adrenal glands allows the secretion of cortisol, which in turn leads to an increase in prostaglandin and mesotocin production. The production of prostaglandin and mesotocin results in an increase in uterine contractions and initiates birth in marsupials. The major metabolite of PGF(2alpha), 13,14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM), has been found in the plasma of the possum at the time of birth and administration of PGF(2alpha) to female possums induced the adoption of the birth position. Evidence that mesotocin is an integral hormone of birth in the tammar wallaby indicates that both PGF(2alpha) and mesotocin or oxytocin are required for marsupial birth. The presence of PGF(2alpha) receptors in the uterus and corpus luteum of the possum, and the in vitro uterine responsiveness to PGF(2alpha) or oxytocin, were examined. PGF(2alpha) receptors were not observed in possum uteri and the inability of PGF(2alpha) to cause contractions indicates that PGF(2alpha) is not involved directly in contraction of the uterus at parturition. The presence of oxytocin and mesotocin receptors in the uterus of possoms and the ability of oxytocin to induce uterine contraction in vitro supports the view that mesotocin is required for expulsion of the young from the uterus. Low numbers of PGF(2alpha) receptors were found in the possum corpus luteum at birth, indicating an involvement of PGF(2alpha) in regression of the corpus luteum.

Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial.

PURPOSE: Expression of ErbB-1 and ErbB-2 (epidermal growth factor receptor and HER2/neu) in breast cancer may cause tamoxifen resistance, but not all studies concur. Additionally, the relationship between ErbB-1 and ErbB-2 expression and response to selective aromatase inhibitors is unknown. A neoadjuvant study for primary breast cancer that randomized treatment between letrozole and tamoxifen provided a context within which these issues could be addressed prospectively. PATIENTS AND METHODS: Postmenopausal patients with estrogen- and/or progesterone receptor-positive (ER+ and/or PgR+) primary breast cancer ineligible for breast-conserving surgery were randomly assigned to 4 months of neoadjuvant letrozole 2.5 mg daily or tamoxifen 20 mg daily in a double-blinded study. Immunohistochemistry (IHC) for ER and PgR was conducted on pretreatment biopsies and assessed by the Allred score. ErbB-1 and ErbB-2 IHC were assessed by intensity and completeness of membranous staining according to published criteria. RESULTS: For study biopsy-confirmed ER+ and/or PgR+ cases that received letrozole, 60% responded and 48% underwent successful breast-conserving surgery. The response to tamoxifen was inferior (41%, P =.004), and fewer patients underwent breast conservation (36%, P =.036). Differences in response rates between letrozole and tamoxifen were most marked for tumors that were positive for ErbB-1 and/or ErbB-2 and ER (88% v 21%, P =.0004). CONCLUSION: ER+, ErbB-1+, and/or ErbB-2+ primary breast cancer responded well to letrozole, but responses to tamoxifen were infrequent. This suggests that ErbB-1 and ErbB-2 signaling through ER is ligand-dependent and that the growth-promoting effects of these receptor tyrosine kinases on ER+ breast cancer can be inhibited by potent estrogen deprivation therapy.

Expression and localization of the contractile prostaglandin F receptor in pregnant rat myometrium in late gestation, labor, and postpartum.

A polyclonal antibody was raised against amino acids 7-18 in the first extracellular loop of rat prostaglandin F (FP) receptor to monitor expression and localization in pregnant rat myometrium at Gestational Days 16, 18, 20, 21, 21.5, 22 (delivery), and 23 (1-day postpartum; n = 5 per group). The antibody recognized a protein of approximately 43 kDa on Western blot analysis in both membrane (soluble and nonsoluble) and cytosolic fractions of myometrium on each day of gestation. Expression of FP protein increased significantly (P < 0.05) during late gestation in both soluble membrane and cytosolic fractions, being significantly greater at Day 21.5 than at Day 20 of gestation in the soluble membrane fraction and in the cytosolic fraction of tissues collected during labor compared with those obtained before labor. The total concentration of FP receptor in the membrane (soluble plus nonsoluble) remained high throughout late gestation and fell significantly (P < 0.05) in the postpartum period. The FP receptor in the soluble membrane fraction (compared to the total membrane FP receptor) was significantly (P < 0.05) higher in late gestation than earlier, whereas the ratio of FP protein in cytosolic to that in the total membrane was significantly (P < 0.05) higher on Day 23 than earlier in gestation, suggesting a dynamic movement of FP with advancing gestational age. Immunoreactive FP receptor localized to circular and longitudinal smooth muscle at all gestational ages, but changes in intracellular localization were observed in late gestation with a staining pattern similar to alpha-actin, suggesting an association with myofibrils. Our study suggests an increase in FP-receptor protein in myometrium with advancing gestation and a marked elevation at term. This supports a role for uterine FP receptors in mediation of uterine contractility at term.