scholarly journals Corticotropin-Releasing Hormone (CRH) and Urocortin Act through Type 1 CRH Receptors to Stimulate Dehydroepiandrosterone Sulfate Production in Human Fetal Adrenal Cells

2005 ◽  
Vol 90 (9) ◽  
pp. 5393-5400 ◽  
Author(s):  
Rosa Sirianni ◽  
Bobbie A. Mayhew ◽  
Bruce R. Carr ◽  
C. Richard Parker ◽  
William E. Rainey
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Outcome Measure ◽  
Dehydroepiandrosterone Sulfate ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Main Outcome Measure ◽  
Sulfate Production ◽  
Estrogen Synthesis

Abstract Context: Near term, the human fetal adrenal increases the production of cortisol and dehydroepiandrosterone sulfate (DHEAS). DHEAS, which acts as substrate for placental estrogen production, induces key changes involved in parturition. Objective: The objective of this study was to determine quantitatively the effect of CRH on mRNA levels of enzymes needed for DHEAS production (steroidogenic acute regulatory protein, CYP11A, CYP17, and SULT2A1), to determine the CRH receptor (CRH-R) subtype(s) responsible for CRH action, and to determine the effect of CRH on CRH-R mRNA expression in human adrenal fetal zone (FZ) cells. Design: Human adrenal FZ cells were treated with CRH, ACTH, urocortin (Unc), and CRH antagonists, and RNA was analyzed by microarray and real-time RT-PCR. Setting: This study was performed at an academic research laboratory. Main Outcome Measure: The main outcome measure was the expression of steroidogenic enzymes and CRH-R. Results: Microarray analysis of human FZ cells treated for 24 h with CRH or ACTH showed increased mRNA expression levels of the genes needed for DHEAS production. Real-time RT-PCR analysis confirmed these data. Induction was lost in the presence of CRH-R1 antagonists, but not CRH-R2 antagonists. Stimulation was reproduced by Unc. The CRH-R1α mRNA splice variant was the only type 1 receptor isoform expressed in the fetal adrenal, and treatment with CRH up-regulates its mRNA levels. Conclusions: CRH, Unc, and ACTH stimulate all elements of the DHEAS synthetic pathway and activate CRH-R1 as well. The resulting increased DHEAS levels can be used for placental estrogen synthesis and contribute to the process leading to parturition in humans.

scholarly journals Dose-dependent immune response in milk cells and mammary tissue after intramammary administration of lipopolysaccharide in dairy cows

2008 ◽  
Vol 52 (No. 6) ◽  
pp. 231-244 ◽  
Author(s):  
C. Werner-Misof ◽  
M.W. Pfaffl ◽  
R.M. Bruckmaier
Keyword(s):  
Immune Response ◽  
Mrna Expression ◽  
Real Time ◽  
Polymorphonuclear Neutrophils ◽  
Mammary Tissue ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Lps Challenge ◽  
Cell Counts ◽  
Cox 2

The immune response in milk cells and the status of mammary tight junctions (TJ) in response to intramammary (IM) infusion of different doses of <i>Escherichia col</i>i lipopolysaccharide (LPS) was investigated. <i>Experiment I</i>: Seven German Braunvieh cows were IM infused into one quarter with 1 &mu;g (LPS-1) and 3 &mu;g (LPS-3) of LPS, respectively, and the contralateral control quarter with saline (9 g/l; C). Milk samples were taken immediately before and 12, 24, 36, 48, 60, 84 and 108 h after infusion and analysed for somatic cell counts (SCC), lactose, sodium (Na) and chloride (Cl) ions, and electrical conductivity (EC). Milk cell mRNA expression of various inflammatory factors was quantified by real-time RT-PCR. Blood samples were taken immediately after milking for the analysis of leukocytes (WBC), polymorphonuclear neutrophils (PMN), Na and Cl. Milk SCC, lactose, Na, Cl and EC did not differ significantly between LPS-1 and C quarters after the challenge. In LPS-3 quarters SCC levels increased within the first 12 h, reached peak levels between 12 and 36 h (<i>P</i> &le; 0.001) and decreased (<i>P</i> &le; 0.05) thereafter to reach baseline at 108 hours. Lactose in LPS-3 quarters decreased (<i>P</i> &le; 0.05) to a minimum at 24 h and increased slightly thereafter while EC, Na, and Cl increased transiently in response to LPS-3. WBC and PMN levels in both groups decreased numerically within 24 h after LPS administration. In LPS-1, WBC at 24, 48 and 108 h were significantly lower whereas in LPS-3 they were significantly higher than at time 0. TNF&alpha;-mRNA expression in both groups did not change in response to IM LPS-challenge. IL-1&beta;-mRNA expression at 12, 24 and 36 h in LPS-1 quarters increased significantly as compared to time 0. In LPS-3 quarters the mRNA expression values of all tested ILs increased significantly as compared to time 0 within 12 h after LPS-challenge. IL-1&beta;-mRNA expression decreased (<i>P</i> &le; 0.05) at 48 and 84 h in LPS quarters. IL-8 mRNA was significantly decreased at 84 h after challenge in LPS-3 quarters. COX-2-mRNA expression in LPS-1 quarters decreased significantly as compared to time 0 at 48, 84 and 108 h, with a minimum at 84 h (<i>P</i> &le; 0.05). In LPS-3 quarters COX-2-mRNA levels increased (<i>P</i> &le; 0.05) within 48 h after the LPS-challenge. <i>Experiment II</i>: Six cows (5 German Braunvieh, 1 Brown Swiss) were injected in one quarter with 100 &mu;g LPS and in the contralateral quarter with saline (9 g/l; C). Mammary biopsy samples of both quarters were taken immediately before and at 3, 6, 9 and 12 h after infusion and mRNA expression of TJ proteins occludin (OCLN) and zonula occludens (ZO-) 1, 2 and 3 were quantified by real-time RT-PCR. OCLN-mRNA expression did not change in response to the IM infusion while that of ZO-1, ZO-2 and ZO-3 decreased significantly within six hours. In conclusion, a dose of 1 &mu;g LPS did not initiate a immune response in the mammary gland. Furthermore the dose of 100 &mu;g of LPS enhanced TJ permeability by reducing TJ plaque proteins density.

scholarly journals mRNA expression profiles for corticotrophin-releasing hormone, urocortin, CRH-binding protein and CRH receptors in human term gestational tissues determined by real-time quantitative RT-PCR

2004 ◽  
Vol 32 (2) ◽  
pp. 339-348 ◽  
Author(s):  
B Sehringer ◽  
HP Zahradnik ◽  
M Simon ◽  
R Ziegler ◽  
C Noethling ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Binding Protein ◽  
Receptor Subtype ◽  
Receptor Subtypes ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Releasing Hormone ◽  
Gestational Tissues ◽  
Crh Receptors

Increasing maternal plasma levels of corticotrophin-releasing hormone (CRH) during the last weeks of pregnancy suggest that this stress hormone plays an important role in the control of human parturition. Little is known about the quantitative contribution of gestational tissues (other than placenta) to intrauterine formation of CRH, urocortin and CRH-binding protein (CRH-BP), or about the distribution of CRH receptors within the uterus. We have investigated the mRNA expression of CRH, urocortin, CRH-BP and CRH receptors 1 and 2 (CRH-R1 and -R2) in gestational tissues by real-time RT-PCR. Placenta, myometrium and choriodecidua were collected after uncomplicated pregnancies at term, before the onset of labour. Distribution of CRH-R1 and CRH-R2 protein was also investigated by immunostaining with receptor subtype-specific antibodies. The placenta was identified as the main site of CRH and CRH-BP mRNA expression, displaying mRNA levels >1000 and >20 times higher than those found in the myometrium and choriodecidua respectively (P<0.05 in each case). mRNA expression of urocortin was low in all tissues investigated. Myometrium and choriodecidua expressed relevant amounts of both receptor subtypes, whereas the CRH receptor population in placenta consisted mainly of CRH-R2. The high expression of CRH in placenta and the substantial expression of CRH receptors in choriodecidua and myometrium suggested that CRH derived from placenta exerts direct or indirect actions on these tissues. Neither CRH produced by myometrium or choriodecidua nor urocortin from other intrauterine sources seem to play a major role in the control of labour.

255 GENE EXPRESSION OF Cox 5a, 5b, AND 6b1 AND THEIR ROLES IN PRE-IMPLANTATION OF MOUSE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 235
Author(s):  
S.-E. Lee ◽  
X.-Y. Li ◽  
X.-S. Cui ◽  
N.-H. Kim
Keyword(s):  
Rna Interference ◽  
Mrna Expression ◽  
Real Time ◽  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Target Mrna ◽  
Protein Levels

Despite clear evidence of regulation of mitochondrial respiration by nuclear encoded genes, cytochrome oxidase (Cox), little information is available on their expression and functional roles during early embryonic development. To examine the role of Cox in oocyte maturation and embryogenesis, we first characterized mRNA and protein levels of nuclear encoded genes, Cox 5a, 5b, and 6b1, in mouse oocytes and during early embryogenesis, using real-time RT-PCR and immunocytochemistry. We then examined the possible role of these genes in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundances of Cox 5a, 5b, and 6b1 transcripts was measured by real time RT-PCR. After normalization by comparison to histone H2a mRNA levels, the mRNA expression of Cox 5a, 5b, and 6b1 were found to be considerable in mature oocytes and zygotes, but reduced slightly in 2-cell embryos. From the 2-cell to the blastocyst stage, mRNA expression is dependent on the number of blastomeres, as expression increases only gradually with development. Immunocytochemical studies revealed that Cox 5a, 5b, and 6b1 proteins were expressed in all blastomeres of the blastocyst. Injection of Cox 5a, 5b, or 6b1 siRNA into GV stage oocytes decreased expression of the target mRNA specifically, while not affecting the expression of mRNAs for the other subunits in mature oocytes. Similarly, each siRNA injection into zygotes specifically reduced target mRNA expression at the 2-cell, morula and blastocyst stages (P < 0.05). Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages. The expression level of mtDNA9, as well as overall levels of mitochondrial mRNAs, was not different following injection of siRNA for Cox 5a, 5b, or 6b1. However, it is evident that the number of mitochondria in siRNA treated blastocysts was greatly reduced, and they appeared to be morphologically abnormal. Significantly higher apoptosis and lower cell numbers were observed in siRNA treated blastocysts. Real time RT PCR revealed that silencing of Cox 5a, 5b, and 6b1 decreased mRNA and protein levels of E-cadherin. These results suggest that the Cox subunits, Cox 5a, 5b, and 6b1, play an important role in mitochondrial function during pre-implantation development. This work was funded by a grant from the National Research Laboratory Program in Korea.

scholarly journals Changes in Placental CRH, Urocortins, and CRH-Receptor mRNA Expression Associated with Preterm Delivery and Chorioamnionitis

2011 ◽  
Vol 96 (2) ◽  
pp. 534-540 ◽  
Author(s):  
Michela Torricelli ◽  
Romina Novembri ◽  
Errico Bloise ◽  
Maria De Bonis ◽  
John R. Challis ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Preterm Delivery ◽  
Outcome Measure ◽  
Perinatal Care ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Premature Rupture Of Membranes ◽  
Premature Rupture ◽  
Dose Dependent

abstract Context: The pathogenesis of preterm delivery (PTD) is not clear, although inflammation/infection play a major role. Corticotropin releasing-hormone (CRH) and Urocortins (Ucns) are involved in the pathophysiology of PTD. Objective: This study evaluates trophoblast mRNA expression of CRH, Ucn, Ucn2, Ucn3, and their receptors [CRH-type 1 receptor (CRH-R1), CRH-R2] in infective conditions. To determine whether infection or glucocorticoids contribute to change their placental mRNA expression, the effects of lipopolysaccharide or dexamethasone was evaluated. Design: Placentas were obtained from spontaneous PTD; premature rupture of membranes (pPROM) and pPROM with chorioamnionitis. Setting: Placental specimens were collected from women receiving perinatal care at our Division of Obstetrics and Gynecology. Patients or Other Participants: Pregnant women delivered preterm were enrolled. Interventions: mRNA expression was evaluated by RT-PCR. Main Outcome Measure: Because CRH and Ucns are involved in immunological functions we evaluated their involvement in PTD with or without infection. Results: CRH, Ucn2, and CRH-R1 mRNA expression were higher, while Ucn and CRHR-2 were lower in pPROM with chorioamnionitis than in PTD and pPROM. Ucn3 mRNA expression was lower in pPROM with and without chorioamnionitis than in PTD. The addition of lipopolysaccharide in trophoblast explants decreased Ucn, Ucn3, and CRH-R2 and increased CRH, Ucn2, and CRH-R1 mRNA expression in a dose-dependent manner. Dexamethasone increased CRH and decreased Ucn2 mRNA expression in a dose dependent manner. Conclusions: Our findings showed a significant impact of pPROM with chorioamnionitis on placental CRH peptides and receptors, suggesting that placental expression of stress-related pathways is activated in infective process.

scholarly journals Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

2002 ◽  
pp. 795-802 ◽  
Author(s):  
F Fallo ◽  
V Pezzi ◽  
L Barzon ◽  
P Mulatero ◽  
F Veglio ◽  
...  
Keyword(s):  
Real Time ◽  
Secretory Activity ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pathophysiological Role ◽  
Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

DETECTION AND DIFFERENTIATION OF THE VACCINE STRAIN AND FIELD ISOLATES OF DUCK HEPATITIS A VIRUS TYPE 1 USING REAL-TIME RT-PCR AND HIGH RESOLUTION MELTING ASSAYS

2015 ◽  
Vol 41 (04) ◽  
pp. 229-235
Author(s):  
Kuang-Po Li ◽  
Shan-Chia Ou ◽  
Jui-Hung Shien ◽  
Poa-Chun Chang
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Virus Type ◽  
Vaccine Strain ◽  
Hepatitis A ◽  
High Resolution Melting ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Duck Hepatitis

Duck hepatitis A virus type 1 (DHAV-1) infection is a highly contagious and fatal disease of young ducklings. A live attenuated vaccine strain designated as 5886 has been used in Taiwan for the control of DHAV-1. Although several molecular biological methods are reported for diagnosis of DHAV-1 infection, none of them is able to discriminate between the vaccine strain and field viruses of DHAV-1. In the present study, a real-time reverse transcriptase polymerase chain reaction (RT-PCR) and high resolution melting (HRM) assay was developed for rapid detection and differentiation between the vaccine strain and field viruses of DHAV-1. This assay is highly specific for DHAV-1 and the detection limit is about 100 copies of the viral RNA. Experiments using fecal samples collected from ducklings experimentally infected with DHAV-1 showed that DHAV-1 could be detected in fecal samples as early as 6 h post-infection. In summary, a real-time RT-PCR and HRM assay is developed in this study and this assay could be valuable for diagnosis and surveillance of DHAV-1 infection in the field.

scholarly journals Activation of Bone Marrow-Derived Cells Angiotensin (Ang) II Type 1 Receptor by Ang II Promotes Atherosclerotic Plaque Vulnerability

2018 ◽  
Vol 19 (9) ◽  
pp. 2621
Author(s):  
Maxime Pellegrin ◽  
Karima Bouzourène ◽  
Jean-François Aubert ◽  
Aimable Nahimana ◽  
Michel Duchosal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mrna Expression ◽  
Atherosclerotic Plaque ◽  
Cholesterol Metabolism ◽  
Mrna Levels ◽  
Ang Ii ◽  
Plaque Vulnerability ◽  
Plaque Development ◽  
Pro Inflammatory Cytokines

Angiotensin (Ang) II triggers vulnerable atherosclerotic plaque development. Bone marrow (BM)-derived cells are key players in atherogenesis but whether Ang II induces plaque vulnerability directly through Ang II type 1 receptor (AT1R) activation on these cells remains to be clarified. In the present study, we investigated whether a lack of AT1R on BM-derived cells might affect Ang II-mediated vulnerable plaque development. The 2-kidney, 1-clip (2K1C) model (Ang II-dependent mouse model of advanced atherosclerosis and vulnerable plaques) was generated in ApoE−/− mice transplanted with AT1aR−/− or AT1aR+/+ BM. Plasma cholesterol as well as hepatic mRNA expression levels of genes involved in cholesterol metabolism were significantly lower in 2K1C mice transplanted with AT1aR−/− BM than in controls. Atherosclerotic lesions were significantly smaller in AT1aR−/− BM 2K1C mice (−79% in the aortic sinus and −71% in whole aorta compared to controls). Plaques from AT1aR−/− BM 2K1C mice exhibited reduced lipid core/fibrous cap and macrophage/smooth muscle cells ratios (−82% and −88%, respectively), and increased collagen content (+70%), indicating a more stable phenotype. Moreover, aortic mRNA levels of pro-inflammatory cytokines IL-12p35, IL-1β, and TNF-α were significantly reduced in AT1aR−/− BM 2K1C mice. No significant differences in either the number of circulating Ly6Chigh inflammatory monocytes and Ly6Clow resident anti-inflammatory monocyte subsets, or in mRNA levels of aortic M1 or M2 macrophage markers were observed between the two groups. No significant differences were observed in splenic mRNA levels of T cell subsets (Th1, Th2, Th17 and Treg) markers between the two groups. In conclusion, direct AT1R activation by Ang II on BM-derived cells promotes hepatic mRNA expression of cholesterol-metabolism-related genes and vascular mRNA expression of pro-inflammatory cytokines that may lead to plaque instability.

scholarly journals Human mononuclear cells express 12-LX: coordinated mRNA regulation with 5-LX and FLAP genes

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 331-340 ◽  
Author(s):  
WE Kaminski ◽  
E Jendraschak ◽  
K Baumann ◽  
R Kiefl ◽  
S Fischer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Time Course ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Constitutive Expression ◽  
Ionophore A23187 ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Human Mononuclear Cells

Abstract Lipoxygenases (LXs) catalyze formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs), proinflammatory, and spasmogenic autacoids that are critical for host defense systems. We studied the expression and regulation of LX genes (12-LX, 5-LX, and 15-LX) and the 5-lipoxygenase activating protein (FLAP) in human mononuclear cells (MNC) and granulocytes using a quantitative reverse transcription polymerase chain reaction (RT-PCR) technique. We show that 12-LX mRNA is constitutively expressed in resting platelet-free MNC. 12-LX gene expression was upregulated by activation with lipopolysaccharide (LPS). The formation of 12-HETE was inducible with ionophore in MNC, as assessed by high-performance liquid chromatography (HPLC) and gas chromatography, and increased after LPS pretreatment. In addition to 12- LX, resting MNC expressed the genes for 5-LX and FLAP constitutively. Quantitative time course analyses of 12-LX, 5-LX, and FLAP gene expression suggested coregulation of 12-LX and FLAP mRNAs, and reciprocal regulation of 5-LX and FLAP mRNAs. During cell stimulation with LPS 5-LX mRNA levels remained unchanged, whereas FLAP gene expression increased. No 15-LX mRNA expression or 15-HETE formation was detectable in unstimulated and activated MNC. In contrast to MNC, quantitative RT-PCR mRNA analysis showed intermittent intraindividual expression of the 5-LX and FLAP genes in resting granulocytes. mRNAs for 12-LX and 15-LX were not expressed. On stimulation of granulocytes ex vivo, mRNA expression of 5-LX and FLAP was upregulated. Stimulation by LPS differed from that by ionophore A23187. Neither LPS nor ionophore induced gene expression of 12-LX or 15-LX in granulocytes. Our data indicate that resting human MNC and granulocytes express LX and FLAP genes in a cell-specific manner. Cell activation induces coordinated upregulation of 12-LX and FLAP genes in MNC, and 5-LX and FLAP genes in granulocytes, respectively. The constitutive expression of 12-LX mRNA, its upregulation on cell activation, and the formation of 12-HETE clearly indicate the presence of a functional 12-LX in human MNC.

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