Complete Genome Sequence of a Bovine Ephemeral Fever Virus Isolate from Israel.

Here, we report the first complete genome of a bovine ephemeral fever virus (BEFV) isolate from an infected bovine in Israel. The genome shares 95.3% identity with a Turkish genomic sequence but contains α3 and γ open reading frames that are truncated compared to those of existing BEFV genome sequences.

Molecular characterization of equine infectious anaemia virus strains detected in England in 2010 and 2012.

Equine infectious anaemia virus (EIAV) is a retrovirus with worldwide distribution which is notifiable to the OIE. Despite its importance to the equine industry, most information regarding its biology have been obtained using only two strains (EIAVWYO and EIAVLIA ) from the USA and China, respectively. Recently full genome sequences from Ireland, Italy and Japan have been published; however, this is still not representative of the number of EIAV outbreaks experienced globally each year. The limited availability of published sequences makes design of a universal EIAV PCR difficult, hence diagnosis is solely reliant on serology. Accordingly, it is important to further investigate the re-emerging cases in other areas of the world. Here, we provide information regarding the outbreaks of EIA in England in 2010 and 2012 including the molecular characterization of strains. Full genome was obtained for two symptomatic cases but could not be resolved for the asymptomatic cases. The two British genomes from 2010 (EIAVDEV ) and 2012 (EIAVCOR ) each represent a new phylogenetic group, each differing genetically from the other available full genome sequences by 21.1%-25.5%. That the majority of new EIAV full genome sequences to be published adds another phylogenetic group indicates that the surface of EIAV global diversity is just being scratched. These data highlight that further work is needed to fully understand EIAV genetic diversity, namely the full genome sequencing of EIAV cases from a variety of locations and time points. This would aid both the use of phylogenetics in parallel with horse tracing as the epidemiological tool of disease tracking and the design of a universally applicable molecular diagnostic method.

Quasispecies evolution of the prototypical genotype 1 porcine reproductive and respiratory syndrome virus early during in vivo infection is rapid and tissue specific.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious threat to the pig industry worldwide. Increasing evidence suggests that microevolution within a quasispecies population can give rise to high sequence heterogeneity in PRRSV; potentially impacting the pathogenicity of the virus. Here, we report on micro-evolutionary events taking place within the viral quasispecies population in lung and lymph node 3 days post infection (dpi) following experimental in vivo infection with the prototypical Lelystad PRRSV (LV). Sequence analysis revealed 16 high frequency single nucleotide variants (SNV) or differences from the reference LV genome which are assumed to be representative of the consensus inoculum genome. Additionally, 49 other low frequency SNVs were also found in the inoculum population. At 3 dpi, a total of 9 and 10 SNVs of varying frequencies could already be detected in the LV population infecting the lung and lymph nodes, respectively. Interestingly, of these, three and four novel SNVs emerged independently in the two respective tissues when compared to the inoculum. The remaining variants, though already present at lower frequencies in the inoculum, were positively selected and their frequency increased within the quasispecies population. Hence, we were able to determine directly from tissues infected with PRRSV the repertoire of genetic variants within the viral quasispecies population. Our data also suggest that microevolution of these variants is rapid and some may be tissue-specific.

Resting interhemispheric functional magnetic resonance imaging connectivity predicts performance after stroke.

OBJECTIVE: Focal brain lesions can have important remote effects on the function of distant brain regions. The resulting network dysfunction may contribute significantly to behavioral deficits observed after stroke. This study investigates the behavioral significance of changes in the coherence of spontaneous activity in distributed networks after stroke by measuring resting state functional connectivity (FC) using functional magnetic resonance imaging. METHODS: In acute stroke patients, we measured FC in a dorsal attention network and an arm somatomotor network, and determined the correlation of FC with performance obtained in a separate session on tests of attention and motor function. In particular, we compared the behavioral correlation with intrahemispheric FC to the behavioral correlation with interhemispheric FC. RESULTS: In the attention network, disruption of interhemispheric FC was significantly correlated with abnormal detection of visual stimuli (Pearson r with field effect = -0.624, p = 0.002). In the somatomotor network, disruption of interhemispheric FC was significantly correlated with upper extremity impairment (Pearson r with contralesional Action Research Arm Test = 0.527, p = 0.036). In contrast, intrahemispheric FC within the normal or damaged hemispheres was not correlated with performance in either network. Quantitative lesion analysis demonstrated that our results could not be explained by structural damage alone. INTERPRETATION: These results suggest that lesions cause state changes in the spontaneous functional architecture of the brain, and constrain behavioral output. Clinically, these results validate using FC for assessing the health of brain networks, with implications for prognosis and recovery from stroke, and underscore the importance of interhemispheric interactions.

Right hemisphere dominance during spatial selective attention and target detection occurs outside the dorsal frontoparietal network.

Spatial selective attention is widely considered to be right hemisphere dominant. Previous functional magnetic resonance imaging studies, however, have reported bilateral blood-oxygenation-level-dependent responses in dorsal frontoparietal regions during anticipatory shifts of attention to a location (Kastner et al., 1999; Corbetta et al., 2000; Hopfinger et al., 2000). Right-lateralized activity has mainly been reported in ventral frontoparietal regions for shifts of attention to an unattended target stimulus (Arrington et al., 2000; Corbetta et al., 2000). However, clear conclusions cannot be drawn from these studies because hemispheric asymmetries were not assessed using direct voxelwise comparisons of activity in left and right hemispheres. Here, we used this technique to measure hemispheric asymmetries during shifts of spatial attention evoked by a peripheral cue stimulus and during target detection at the cued location. Stimulus-driven shifts of spatial attention in both visual fields evoked right-hemisphere dominant activity in temporoparietal junction (TPJ). Target detection at the attended location produced a more widespread right hemisphere dominance in frontal, parietal, and temporal cortex, including the TPJ region asymmetrically activated during shifts of spatial attention. However, hemispheric asymmetries were not observed during either shifts of attention or target detection in the dorsal frontoparietal regions (anterior precuneus, medial intraparietal sulcus, frontal eye fields) that showed the most robust activations for shifts of attention. Therefore, right hemisphere dominance during stimulus-driven shifts of spatial attention and target detection reflects asymmetries in cortical regions that are largely distinct from the dorsal frontoparietal network involved in the control of selective attention.

Interaction of stimulus-driven reorienting and expectation in ventral and dorsal frontoparietal and basal ganglia-cortical networks.

Shifts of attention to unattended stimuli (stimulus-driven reorienting) are often studied by measuring responses to unexpected stimuli, confounding reorienting and expectation. We separately measured the blood-oxygenation-level-dependent signal for both factors by manipulating the probability of salient visual cues that either shifted attention away from or maintained attention on a stream of visual stimuli. The results distinguished three networks recruited by reorienting. Right temporoparietal junction (TPJ), the posterior core of a ventral frontoparietal network, was activated more by cues for shifting than maintaining attention independently of cue location and probability, acting as a switch. TPJ was separately modulated by low probability cues, which signaled a breach of spatial expectation, independently of whether they shifted attention. Under resting conditions, TPJ activity was correlated [resting-state functional connectivity magnetic resonance imaging, (rs-fcMRI)] with right inferior frontal gyrus (IFG), an anterior component of the ventral network. Nevertheless, IFG was activated only by unexpected shifts of attention, dissociating its function from TPJ. Basal ganglia and frontal/insula regions also were activated only when reorienting was unexpected but showed strong rs-fcMRI among themselves, not with TPJ/IFG, defining a distinct network that may retrieve/activate commands for shifting attention. Within dorsal frontoparietal regions, shifting attention produced sustained spatially selective modulations in intraparietal sulcus (IPS) and frontal-eye field (FEF), and transient less selective modulations in precuneus and FEF. Modulations were observed even when reorienting was likely, but increased when reorienting was unexpected. The latter result may partly reflect interactions with lateral prefrontal components of the basal-ganglia/frontal/insula network that showed significant rs-fcMRI with the dorsal network.

Social appraisal of adult ADHD: stigma and influences of the beholder's Big Five personality traits.

OBJECTIVE: This study investigates social stigma associated with a diagnosis of ADHD in adulthood and whether Big Five personality traits predict appraisals of affected individuals. METHOD: A sample of 257 undergraduates rate the desirability of targets with ADHD, minor medical problems, and with no appreciable weakness, across several social engagement contexts. RESULTS: Participants exhibit significantly less desire to engage with those with ADHD (as compared to both controls). Agreeableness, Extraversion, and Conscientiousness are found to predict appraisals of targets with and without ADHD, moderated by sex of the target and the beholder. CONCLUSION: It is suggested that fairly subtle, negative bias toward ADHD contributes to rejection of individuals with the disorder, particularly in academic and work settings. Findings also suggest peers' personalities do have some effect on appraisals of targets with ADHD

The use of miniscrews for orthodontic anchorage.

The incorporation of miniscrews into orthodontic treatment planning has allowed for predictable control of anchorage and has increased the ability to correct severe skeletal discrepancy with nonsurgical orthodontics. It is the goal of this article to review the terminology, design, placement, and activation of miniscrews, as well as the advantages and complications associated with their use so that the multidisciplinary team will become familiar and comfortable with this novel treatment modality.

Ex utero intrapartum treatment (EXIT) for congenital giant ranula.

UNLABELLED: Congenital giant ranula causing life-threatening upper airway obstruction is a rare condition. A well-planned ex utero intrapartum treatment (EXIT) procedure for aspiration and decompression of the cystic lesion is simple, efficacious and effective in securing a patent airway before the fetus is completely delivered, and may prevent lifelong neurodevelopmental complications associated with perinatal asphyxia in such cases. CONCLUSION: EXIT could be a life-saving procedure for congenital giant ranula.

Salivary IgA in response to periodontal treatment.

There is evidence that the quantity of antigen load is crucial for the activation of IgA immune responses. In order to investigate the relevance of these findings in aggressive periodontitis, salivary antibody responses were measured during non-surgical and antibiotic treatment. Twenty-one patients with generalized aggressive periodontitis were monitored for total salivary IgA and IgA reactive to Porphyromonas gingivalis in resting and stimulated whole saliva. Non-surgical treatment included full-mouth professional tooth cleaning and subgingival scaling and root planing (SRP) under local anesthesia. Patients were recalled at 3 months and 6 months following systemic antibiotic treatment. Non-parametric statistics showed significant improvements in the clinical parameters in all patients. Between baseline and 4 wk following SRP, median concentrations of total IgA decreased both in resting (-46%) and in stimulated (-33%) saliva. The P. gingivalis-specific IgA activity showed a twofold increase at 4 wk after SRP. In addition to these changes, periodontal treatment of aggressive periodontitis did not appear to affect salivary IgA, and there were no significant correlations of IgA to the clinical parameters. In conclusion, salivary IgA responses during periodontal treatment were not found to have a diagnostic or prognostic significance.

A prospective randomized trial comparing patient-controlled sedation using propofol and alfentanil and physician-administered sedation using diazepam and pethidine during transvaginal ultrasound-guided oocyte retrieval.

BACKGROUND: This open label study compared the effectiveness of patient-controlled sedation (PCS), with physician-administered sedation (PAS) during transvaginal ultrasound-guided oocyte retrieval (TUGOR). METHODS: A total of 106 patients was randomized using a computer model to receive either PCS (n = 51) or PAS (n = 55). Intra-operative sedation and co-operation were measured on a 5-point scale while severity of pain, and doctor and patient satisfaction were assessed using a 100 mm visual analogue scale. Number of oocytes retrieved and fertilization, cleavage and clinical pregnancy rates were also recorded. RESULTS: Levels of sedation and co-operation were similar between groups. The pain score was higher in the PCS group during (mean +/- SD, 53 +/- 23 versus 35 +/- 24; P < 0.01) and 2 h after the procedure (29 +/- 27 versus 17 +/- 22; P < 0.05). Doctors were less satisfied with PCS than PAS (62 +/- 25 versus 71 +/- 26; P < 0.05) while patients were highly satisfied with both methods (76 +/- 23 versus 74 +/- 21; not significant). There were no oversedation or peri-operative complications. Fertility outcomes were similar. Patients tended to prefer PCS when given the choice of sedation method. CONCLUSION: Although PCS provides less analgesia then PAS during TUGOR, it is safe, satisfactory and accepted by patients.

Phospholipase D is required in the signaling pathway leading to p38 MAPK activation in neutrophil-like HL-60 cells, stimulated by N-formyl-methionyl-leucyl-phenylalanine.

Human acute myelogenous leukemia cells (HL-60 cells) can be induced to differentiate to neutrophils by exposure to dibutyryl-cyclic AMP. The differentiation of HL-60 cells allowed the mitogen-activated protein kinases p38 and p44/p42 to be rapidly and transiently activated upon stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Western blot analysis using phosphospecific p38 and p44/p42 mitogen-activated protein kinase antibodies showed that increasing concentrations of ethanol or 1-butanol but not 2-butanol (0.05-0.5%) inhibited fMLP-induced p38 activation but did not inhibit p44/p42 activation. These data indicated that activation of phospholipase D (PLD) was required for activation of p38 but not p44/p42. We compared the effect of fMLP with those of tumor necrosis factor alpha (TNF alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We found that ethanol did not inhibit p38 phosphorylation upon stimulation with either GM-CSF or TNF alpha. These results suggested that in cells stimulated with fMLP, PLD was upstream of p38. To further test the involvement of PLD, we used antisense inhibition of human PLD1 expression. Treatment with antisense oligonucleotides inhibited p38 but not p44/p42 phosphorylation. These data supported a role for human PLD1 in fMLP-induced p38 activation in neutrophil-like HL-60 cells. In addition, the results obtained with TNF alpha and GM-CSF demonstrated that p38 activation occurred independently of PLD activation.

Protein kinases C translocation responses to low concentrations of arachidonic acid.

Arachidonic acid (AA) directly activates protein kinases C (PKC) and may thereby serve as a regulatory signal during cell stimulation. The effect, however, requires a > or =20 microm concentration of the fatty acid. We find that human polymorphonuclear neutrophils (PMN) equilibrated with a ligand for the diacylglycerol receptor on PKC, [(3)H]phorbol dibutyrate (PDB), increased binding of [(3)H]PDB within 15 s of exposure to > or =10-30 nm AA. Other unsaturated fatty acids, but not a saturated fatty acid, likewise stimulated PDB binding. These responses, similar to those caused by chemotactic factors, resulted from a rise in the number of diacylglycerol receptors that were plasma membrane-associated and therefore accessible to PDB. Unlike chemotactic factors, however, AA was fully active on cells overloaded with Ca(2+) chelators. The major metabolites of AA made by PMN, leukotriene B(4) and 5-hydroxyicosatetraenoate, did not mimic AA, and an AA antimetabolite did not block responses to AA. AA also induced PMN to translocate cytosolic PKCalpha, beta(II), and delta to membranes. This response paralleled PDB binding with respect to dose requirements, time, Ca(2+)-independence, resistance to an AA antimetabolite, and induction by another unsaturated fatty acid but not by a saturated fatty acid. Finally, HEK 293 cells transfected with vectors encoding PKCbeta(I) or PKCdelta fused to the reporter enhanced green fluorescent protein (EGFP) were studied. AA caused EGFP-PKCbeta translocation from cytosol to plasma membrane at > or =0.5 microm, and EGFP-PKCdelta translocation from cytosol to nuclear and, to a lesser extent, plasma membrane at as little as 30 nm. We conclude that AA induces PKC translocations to specific membrane targets at concentrations 2-4 orders of magnitude below those activating the enzymes. These responses, at least as they occur in PMN, do not require changes in cell Ca(2+) or oxygenation of the fatty acid. AA seems more suited for signaling the movement than activation of PKC.

Phospholipase D, tumor promoters, proliferation and prostaglandins.

Phosphatidylcholine hydrolysis by phospholipase D is a widespread response to cellular stimulation. However, the downstream signaling events subsequent to phosphatidylcholine hydrolysis are just beginning to be determined. Initially it was proposed that diglyceride formation by phospholipase D and phosphatidate phosphohydrolase resulted in long-term stimulation of protein kinase C. However, recent studies indicate that phosphatidic acid is the relevant signaling molecule in some signaling pathways. The present review will summarize studies of phospholipase D in the response of cells to the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, which causes cells to mimic the phenotype of oncogenic transformation. The role of phospholipase D in stimulation of Raf-1 and prostaglandin H synthase type-2 is emphasized.

Quaternary ammonium analogs of ether lipids inhibit the activation of protein kinase C and the growth of human leukemia cell lines.

PURPOSE: ET-18-OCH3 (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is a representative of the first generation of antitumor ether lipids and is a model in the development of new compounds including a series of quaternary ammonium analogs (QAA). In the present study, we evaluated the QAA as inhibitors of cell growth and studied their mechanism of action. METHODS: We compared the effects of the QAA on the proliferation of human leukemia cell lines which are sensitive (HL-60) or resistant to ET-18-OCH3 (HL-60R and K562). In addition we used cell fractionation and enzymatic assays to determine the effects of QAA on protein kinase C (PKC) translocation in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA). RESULTS: The QAA and ET-18-OCH3 were approximately equally effective inhibitors of HL-60 cell growth. However, the QAA were more effective inhibitors of K562 and HL-60R cell proliferation. The HL-60R cells, which were selected for resistance to ET-18-OCH3, were also resistant to BM 41.440 which is structurally similar. In serum-free medium, the phosphorus-containing compounds (ET-18-OCH3, BM 41.440 and He-PC) were much more effective inhibitors (8-20-fold) of the K562 cell line while the activities of the QAA were only moderately increased (1.2-2.3-fold). When serum albumin was added to the serum-free medium, the K562 cells became resistant to ET-18-OCH3, suggesting that albumin is responsible for the differential sensitivity. The QAA compounds, which inhibit PKC activity in vitro, inhibited cell proliferation. However, a QAA which did not inhibit PKC did not inhibit cell proliferation. The phorbol ester TPA stimulates PKC translocation and causes HL-60 cell differentiation to adherent macrophage-type cells. The QAA inhibited TPA-induced cell differentiation and PKC translocation indicating that they also inhibit PKC in intact cells. CONCLUSIONS: The cellular effects of the QAA appear to be due to inhibition of PKC. In addition, these data indicate that albumin, which is important as a mediator of the uptake of ET-18-OCH3, has only a small effect on the uptake of QAA. Together these data indicate that the QAA are potential anticancer agents, showing a significant ability to inhibit growth of leukemia cell lines that are resistant to ET-18-OCH3.

Protein kinase CbetaII activation by 1-beta-D-arabinofuranosylcytosine is antagonistic to stimulation of apoptosis and Bcl-2alpha down-regulation.

1-beta-D-Arabinofuranosylcytosine (ara-C) stimulates the formation of both diglyceride and ceramide in the acute myelogenous leukemia cell line HL-60 (Strum, J. C., Small, G. W., Pauig, S. B., and Daniel, L. W. (1994) J. Biol. Chem 269, 15493-15497). ara-C also causes apoptosis in HL-60 cells which can be mimicked by exogenous ceramide. However, the signaling role for ara-C-induced diacylglycerol (DAG) is not defined. We found that Bcl-2 levels were increased by treatment of HL-60 cells with exogenous DAG or 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, exogenous ceramide treatment caused a decrease in cellular Bcl-2 levels. Thus, ara-C stimulates the synthesis of two second messengers with opposing effects on Bcl-2. Since the effects of ara-C-induced DAG could be due to protein kinase C (PKC) activation, we determined the effects of ara-C on PKC isozymes. ara-C caused an increase in membrane-bound PKCbetaII (but not PKCalpha or PKCdelta). ara-C or TPA-induced translocation of PKCbetaII was inhibited by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3), and ara-C-induced apoptosis was stimulated by pretreatment of the cells with ET-18-OCH3. ET-18-OCH3 also inhibited stimulation of Bcl-2 by TPA and enhanced the decrease in Bcl-2 observed in ara-C-treated cells. These data indicate that ara-C-induced apoptosis is limited by ara-C-stimulated PKCbetaII through effects on Bcl-2. To further determine the role of PKC, we used antisense oligonucleotides directed toward PKCbetaII. The antisense, but not the sense, oligonucleotide inhibited PKCbetaII activation and enhanced ara-C-induced apoptosis. These data demonstrate that the stimulation of apoptosis by ara-C is self-limiting and can be enhanced by inhibition of PKC.

Comparison of alkylacylglycerol vs. diacylglycerol as activators of mitogen-activated protein kinase and cytosolic phospholipase A2 in human neutrophil priming.

In human neutrophils, the choline-containing phosphoglycerides contain almost equal amounts of alkylacyl- and diacyl-linked subclasses. In contrast to phosphatidylinositol hydrolysis which yields diacylglycerol, hydrolysis of choline-containing phosphoglycerides by phospholipase D coupled with phosphohydrolase yields both alkylacyl- and diacylglycerol. While diacylglycerol activates protein kinase C, alkylacylglycerol does not, and its role is unclear. Yet previous studies have shown that exogenous alkylacyl- and diacylglycerols can prime for the release of radiolabeled arachidonic acid (AA) in intact neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine. We have now examined the effects of both diacylglycerol (1-oleoyl-2-acetylglycerol; OAG) and alkylacylglycerol (1-O-hexadecyl-2-acetylglycerol; EAG) on the activation of mitogen-activated protein (MAP) kinase and the 85-kDa cytosolic phospholipase A2 (cPLA2) in human neutrophils. We observed that while OAG could effectively activate p42 and p44 MAP kinases along with cPLA2 in a time- and concentration-dependent manner, EAG could not. A novel p40 MAP kinase isoform is also present and activated in response to OAG treatment; the behavior of this MAP kinase isoform is discussed. The activation of cPLA2 and MAP kinase by 20 microM OAG could be inhibited by pretreatment with 1 microM GF-109203X, a selective inhibitor of protein kinase C. Although only OAG activated cPLA2, both OAG and EAG primed for the release of AA mass as determined by gas chromatography/mass spectrometry. The priming of AA release by OAG may be explained by the phosphorylation of cPLA2 through the activation of protein kinase C linked to MAP kinase. However, priming by EAG appears to involve a separate mechanism that is dependent on a different PLA2. Our results support a role for phospholipase D-derived products modulating the activation of cPLA2, further supporting the idea of cross-talk among various phospholipases.

Characterization of an HL-60 cell variant resistant to the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine.

A resistant cell line (HL-60R) was selected by incubating HL-60 cells with increasing concentrations of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) and used to examine the mechanism of resistance to the antineoplastic ether-linked lipid. The HL-60R cells exhibited a > 10-fold increase in resistance when measured by [3H]-thymidine incorporation in comparison to the HL-60 cell line. ET-18-OCH3 binding occurred at 4 degrees C and was not saturable at the concentrations tested (1-100 microM), indicating that the binding was receptor-independent. At 4 degrees C, association of ET-18-OCH3 was low for each cell line. AT 37 degrees C, uptake in the HL-60 cells was approximately 5-fold greater in comparison to HL-60R cells at each concentration tested. However, when the cellular content of ET-18-OCH3 was equal, both cell lines experienced similar declines in cell growth. Cellular incorporation of ether lipid was determined using serum-free media and in the presence of serum albumin or lipoproteins. Reduced uptake by the resistant cell line was observed only in the presence of albumin. A greater proportion of ether lipid could be removed from prelabeled HL-60R cells than from HL-60 cells, by an albumin wash procedure, indicating an increased rate of internalization and retention by the sensitive cell line. ET-18-OCH3 uptake in the HL-60 cell line was also more sensitive to treatment with endocytic (chloroquine, monensin) or metabolic (NaF, KCN) inhibitors. These results suggest that uptake is the principal determinant influencing sensitivity of the resistant cell line and consists of receptor-independent binding followed by internalization. Differential uptake requires the presence of serum albumin and is dependent on the energy-dependent endocytosis of the ether lipid.

5-Oxo-eicosatetraenoate is a broadly active, eosinophil-selective stimulus for human granulocytes.

5-Oxo-eicosatetraenoate (5-oxoETE) is gaining recognition as a chemotactic factor for eosinophilic (Eo) as well as neutrophilic (Neu) polymorphonuclear leukocytes. We found that the eicosanoid was far stronger than C5a, platelet-activating factor (PAF), leukotriene B4 (LTB4), or FMLP in stimulating Eo chemotaxis. Moreover, it had weak intrinsic degranulating effects on otherwise unstimulated Eo, produced prominent degranulation responses in Eo primed by granulocyte-macrophage CSF, and enhanced the Eo-degranulating potencies of PAF, C5a, LTB4, and FMLP by up to 10,000-fold. Low picomolar levels of 5-oxoETE also induced Eo to activate mitogen-activated protein kinases (MAPKs), as defined by shifts in the electrophoretic mobility and tyrosine phosphorylation of two immunodetectable proteins, p44 and p42. 5-OxoETE was > or = 100-fold weaker or unable to stimulate any of these responses in Neu. Finally, 5-oxo-15-hydroxy-ETE and 5-hydroxy-ETE activated both cell types, but were weaker than 5-oxoETE and had Eo/Neu potency ratios approaching unity. 5-OxoETE, thus, is uniquely potent and selective in promoting Eo not only to migrate, but also to release granule enzymes and activate MAPKs. By triggering MAPK activation, the eicosanoid may also influence the production of anaphylactoid lipids (e.g., PAF), arachidonic acid metabolites, and cytokines. 5-OxoETE therefore possesses a biologic profile well suited for mediating Eo-dominated allergic reactions in vivo.

5-Oxo-eicosanoids and hematopoietic cytokines cooperate in stimulating neutrophil function and the mitogen-activated protein kinase pathway.

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.