Establishment of fetomaternal tolerance through glycan-mediated B cell suppression.

Discrimination of self from non-self is fundamental to a wide range of immunological processes1. During pregnancy, the mother does not recognize the placenta as immunologically foreign because antigens expressed by trophoblasts, the placental cells that interface with the maternal immune system, do not activate maternal T cells2. Currently, these activation defects are thought to reflect suppression by regulatory T cells3. By contrast, mechanisms of B cell tolerance to trophoblast antigens have not been identified. Here we provide evidence that glycan-mediated B cell suppression has a key role in establishing fetomaternal tolerance in mice. B cells specific for a model trophoblast antigen are strongly suppressed through CD22-LYN inhibitory signalling, which in turn implicates the sialylated glycans of the antigen as key suppressive determinants. Moreover, B cells mediate the MHC-class-II-restricted presentation of antigens to CD4+ T cells, which leads to T cell suppression, and trophoblast-derived sialoglycoproteins are released into the maternal circulation during pregnancy in mice and humans. How protein glycosylation promotes non-immunogenic placental self-recognition may have relevance to immune-mediated pregnancy complications and to tumour immune evasion. We also anticipate that our findings will bolster efforts to harness glycan biology to control antigen-specific immune responses in autoimmune disease.

Exosome poly-ubiquitin inhibits platelet activation, downregulates CD36 and inhibits pro-atherothombotic cellular functions.

INTRODUCTION: Activated platelets shed microparticles from plasma membranes, but also release smaller exosomes from internal compartments. While microparticles participate in athero-thrombosis, little is known of exosomes in this process. MATERIALS & METHODS: Ex vivo biochemical experiments with human platelets and exosomes, and FeCl3 -induced murine carotid artery thrombosis. RESULTS: Both microparticles and exosomes were abundant in human plasma. Platelet-derived exosomes suppressed ex vivo platelet aggregation and reduced adhesion to collagen-coated microfluidic channels at high shear. Injected exosomes inhibited occlusive thrombosis in FeCl3 -damaged murine carotid arteries. Control platelets infused into irradiated, thrombocytopenic mice reconstituted thrombosis in damaged carotid arteries, but failed to do so after prior ex vivo incubation with exosomes.CD36 promotes platelet activation, and exosomes dramatically reduced platelet CD36.CD36 is also expressed by macrophages, where it binds and internalizes oxidized LDL and microparticles, supplying lipid to promote foam cell formation. Platelet exosomes inhibited oxidized-LDL binding and cholesterol loading into macrophages. Exosomes were not competitive CD36 ligands, but instead sharply reduced total macrophage CD36 content. Exosomal proteins, in contrast to microparticle or cellular proteins, were highly adducted by ubiquitin. Exosomes enhanced ubiquitination of cellular proteins, including CD36, and blockade of proteosome proteolysis with MG-132 rescued CD36 expression. Recombinant unanchored K48 poly-ubiquitin behaved similarly to exosomes, inhibiting platelet function, macrophage CD36 expression and macrophage particle uptake. CONCLUSIONS: Platelet-derived exosomes inhibit athero-thrombotic processes by reducing CD36-dependent lipid loading of macrophages and by suppressing platelet thrombosis. Exosomes increase protein ubiquitination and enhance proteasome degradation of CD36.

Pre- and post-surgical factors that predict the provision of rescue analgesia following hysterectomy.

BACKGROUND: To better manage post-surgical pain, standardized analgesic protocols allow for rescue analgesia (RA). This study seeks to determine which pre- and post-surgical clinical and patient-related factors, in addition to post-surgical pain, may influence health care professional decisions on RA administration. METHODS: A consecutive sample of 185 women, submitted to hysterectomy for benign disorders, was assessed 24 h before (time 1; T1) and 48 h after (time 2; T2) surgery. At T1, baseline demographic, clinical and psychological predictors were assessed and at T2, post-surgical pain, anxiety and RA administration were recorded. RESULTS: After controlling for post-surgical acute pain intensity, logistic regression results revealed several pre-surgical (T1) and surgical factors associated with post-surgical RA: having other previous pain states [odds ratio (OR), 4.551; 95% confidence interval (CI), 1.642-12.611, p = 0.004], being anaesthetized with only general or loco-regional anaesthesia (OR, 5.349; 95% CI, 1.976-14.483, p = 0.001) and pre-surgical fear of immediate consequences of surgery (OR, 1.306; 95% CI, 1.031-1.655, p = 0.027). Concerning post-surgical variables, higher pain intensity (OR, 1.591; 95% CI, 1.353-1.871, p < 0.001) and post-surgical anxiety (OR, 1.245; 95% CI, 1.084-1.430, p = 0.002) were significantly associated with RA provision. CONCLUSIONS: Health care decision making to administer RA might be influenced not only by post-surgical pain intensity but also by pre-surgical and surgical clinical factors, such as previous pain and type of anaesthesia. Patient-related psychological characteristics, such as pre-surgical fear and post-surgical anxiety, may also play a role in decision making on RA provision. Implications for practice are discussed.

Acute superior mesenteric artery embolism: reperfusion with AngioJet hydrodynamic suction thrombectomy and pharmacologic thrombolysis with the EKOS catheter.

Acute mesenteric ischemia is commonly treated by surgical exploration and open thrombectomy. Very few reports describe using newer, minimally invasive methods which utilize catheter-based mechanical and pharmacological thrombolysis. Herein, we report a case of acute superior mesenteric embolism successfully treated with AngioJet hydrodynamic mechanical thrombectomy and EKOS catheter pharmacological thrombolysis. A 76-year-old man with new onset atrial fibrillation presented with abdominal pain of 48 hours duration. Subsequent contrast computed tomography scan of the abdomen revealed a filling defect in the superior mesenteric artery (SMA), suggestive of an acute embolus, which was confirmed by SMA angiogram. The AngioJet aspiration device was used for hydrodynamic suction thrombectomy. The repeat angiogram demonstrated only a partial restoration of blood flow, and thus the EKOS tissue plasminogen activator catheter was left in the SMA for continuous thrombolysis. The patient underwent continuous thrombolysis for two days, with two subsequent sessions of angiography. Thereafter, the patient improved symptomatically and serum lactate was normalized. In conclusion, the AngioJet suction thrombectomy and pharmaco-mechanical thrombolysis using the EKOS catheter is associated with minimal morbidity and can be rapidly performed. It may be used as an alternative to open surgical thrombectomy in selected cases of acute SMA embolism.

Prediction of animal clearance using naïve Bayesian classification and extended connectivity fingerprints.

In silico models were developed for predicting high animal clearance using naïve Bayesian classification and extended connectivity fingerprints. Validation and test sets were created from a structurally diverse database of mouse, rat, dog, and monkey clearance (CL) representing approximately 20,000 unique compounds. Model performance was compared with experimental predictors used widely in drug discovery, namely in vitro intrinsic clearance (CL(i)) and CL from a lower preclinical species. The Bayesian model for dog CL was a better predictor than experimental rat or mouse CL. The Bayesian model for rat CL performed at least as well as mouse CL. Bayesian models outperformed mouse, rat, and monkey CL(i) for predicting mouse, rat, and monkey CL, respectively. These models can be used to optimize chemical libraries, direct new chemical synthesis and increase efficiency of screening cascades for lead optimization while reducing overall drug discovery cost, time and animal usage.

Differences in the total body clearance of lead compounds in the rat and mouse: impact on pharmacokinetic screening strategy.

1. The in vivo clearance (CL) for 498 compounds representing more than 40 lead optimization programmes were compared in the rat and mouse. 2. A total of 278 of the compounds had similar CL values in rat and mouse and 41 compounds had a high CL in one rodent species and a low CL in the other (median seven-fold difference). For this latter subset, comparative in vitro plasma protein binding, liver microsomal or hepatocyte intrinsic CL provided plausible explanations for the observed in vivo differences in many cases. 3. A considerable proportion of compounds with substantially different CL in rodents, and those with a high CL in both rat and mouse, had a low-to-moderate CL in dog and/or monkey (43%). A larger proportion (71%) had promising pharmacokinetics in higher species when CL was low in both rat and mouse. 4. Drug-discovery scientists should consider the potential for there to be substantial differences in the disposition of leads in different rodent species and design screening cascades to explore this possibility.

Uteroplacental insufficiency decreases p53 serine-15 phosphorylation in term IUGR rat lungs.

Intrauterine growth restriction (IUGR) increases the incidence of chronic lung disease (CLD). The molecular mechanisms responsible for IUGR-induced acute lung injury that predispose the IUGR infant to CLD are unknown. p53, a transcription factor, plays a pivotal role in determining cellular response to stress by affecting apoptosis, cell cycle regulation, and angiogenesis, processes required for thinning of lung mesenchyme. Because thickened lung mesenchyme is characteristic of CLD, we hypothesized that IUGR-induced changes in lung growth are associated with alterations in p53 expression and/or modification. We induced IUGR through bilateral uterine artery ligation of pregnant rats. Uteroplacental insufficiency significantly decreased serine-15-phosphorylated (serine-15P) p53, an active form of p53, in IUGR rat lung. Moreover, we found that decreased phosphorylation of lung p53 serine-15 localized to thickened distal air space mesenchyme. We also found that IUGR significantly decreased mRNA for targets downstream of p53, specifically, proapoptotic Bax and Apaf, as well as Gadd45, involved in growth arrest, and Tsp-1, involved in angiogenesis. Furthermore, we found that IUGR significantly increased mRNA for Bcl-2, an antiapoptotic gene downregulated by p53. We conclude that in IUGR rats, uteroplacental insufficiency induces decreased lung mesenchymal p53 serine-15P in association with distal lung mesenchymal thickening. We speculate that decreased p53 serine-15P in IUGR rat lungs alters lung phenotype, making the IUGR lung more susceptible to subsequent injury.

Nurses' attitudes to a medical emergency team service in a teaching hospital.

BACKGROUND: Cultural barriers including allegiance to traditional models of ward care and fear of criticism may restrict use of a medical emergency team (MET) service, particularly by nursing staff. A 1-year preparation and education programme was undertaken before implementing the MET at the Austin Hospital, Melbourne, Australia. During the 4 years after introduction of the MET, the programme has continued to inform staff of the benefits of the MET and to overcome barriers restricting its use. OBJECTIVE: To assess whether nurses value the MET service and to determine whether barriers to calling the MET exist in a 400-bed teaching hospital. METHODS: Immediately before hand-over of ward nursing, we conducted a modified personal interview, using a 17-item Likert agreement scale questionnaire. RESULTS: We created a sample of 351 ward nurses and obtained a 100% response rate. This represents 50.9% of the 689 ward nurses employed at the hospital. Most nurses felt that the MET prevented cardiac arrests (91%) and helped manage unwell patients (97%). Few nurses suggested that they restricted MET calls because they feared criticism of their patient care (2%) or criticism that the patient was not sufficiently unwell to need a MET call (10%). 19% of the respondents indicated that MET calls are required because medical management by the doctors has been inadequate; many ascribed this to junior doctors and a lack of knowledge and experience. Despite hospital MET protocol, 72% of nurses suggested that they would call the covering doctor before the MET for a sick ward patient. However, 81% indicated that they would activate the MET if they were unable to contact the covering doctor. In line with hospital MET protocol, 56% suggested that they would make a MET call for a patient they were worried about even if the patient's vital signs were normal. Further, 62% indicated that they would call the MET for a patient who fulfilled MET physiological criteria but did not look unwell. CONCLUSIONS: Nurses in the Austin Hospital value the MET service and appreciate its potential benefits. The major barrier to calling the MET appears to be allegiance to the traditional approach of initially calling parent medical unit doctors, rather than fear of criticism for calling the MET service. A further barrier seems to be underestimation of the clinical significance of the physiological perturbations associated with the presence of MET call criteria.

Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells.

Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element-containing (ARE-containing) 3' untranslated region (3'UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3'UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.

Oxidized LDL contains inflammatory PAF-like phospholipids.

Atherosclerosis has an underlying inflammatory component. Oxidation of low-density lipoprotein (LDL) particles to modified forms promotes atherogenesis by supplying cholesterol and through the oxidative generation of agents that activate macrophages, smooth muscle and endothelial cells. A primary target of oxidizing compounds, derived from cigarette smoke, dietary sources, exuberant inflammatory cell responses and normal cellular metabolism among other sources, are the esterified polyunsaturated fatty acids in the phospholipid shell that surrounds the insoluble lipids of the lipoprotein core. One type of phospholipid oxidation product mimics the structure of the potent inflammatory mediator platelet-activating factor (PAF), and these oxidation products activate the PAF receptor found on platelets, monocytes and leukocytes. Production of such PAF mimetics is, in contrast to the physiologic generation of PAF, uncontrolled. PAF mimetics and other phospholipid oxidation products are found in atherosclerotic lesions or even in blood after exposure to cigarette smoke. Here we summarize our data describing the structure, activity and metabolism of the PAF-like lipids found in atherogenic LDL particles.

Escherichia coli Braun lipoprotein induces a lipopolysaccharide-like endotoxic response from primary human endothelial cells.

All bacteria contain proteins in which their amino-terminal cysteine residue is modified with N-acyl S-diacylglycerol functions, and peptides and proteins bearing this modification are immunomodulatory. The major outer membrane lipoprotein of Escherichia coli, the Braun lipoprotein (BLP), is the prototypical triacylated cysteinyl-modified protein. We find it is as active as LPS in stimulating human endothelial cells to an inflammatory phenotype, and a BLP-negative mutant of E. coli was less inflammatory than its parental strain. While the lipid modification was essential, the lipidated protein was more potent than a lipid-modified peptide. BLP associates with CD14, but this interaction, unlike that with LPS, was not required to elicit endothelial cell activation. BLP stimulated endothelial cell E-selectin surface expression, IL-6 secretion, and up-regulation of the same battery of cytokine mRNAs induced by LPS. Quantitative microarray analysis of 4400 genes showed the same 30 genes were induced by BLP and LPS, and that there was near complete concordance in the level of gene induction. We conclude that the lipid modification of at least one abundant Gram-negative protein is essential for endotoxic activity, but that the protein component also influences activity. The equivalent potency of BLP and LPS, and their complete concordance in the nature and extent of endothelial cell activation show that E. coli endotoxic activity is not due to just LPS. The major outer membrane protein of E. coli is a fully active endotoxic agonist for endothelial cells.

Characterization of human UMP-CMP kinase enzymatic activity and 5' untranslated region.

We have cloned a cDNA for human UMP-CMP kinase from a macrophage cDNA library. Sequence analysis showed that this cDNA is derived from the same gene as a previously reported EST-derived cDNA. Here we show that a conspicuous difference between these two clones, 73 additional 5' nucleotides in the EST clone, including a putative translational start site, is not functionally significant. This work shows that the additional 5'sequence in the EST clone was unnecessary for enzymatic activity and nonfunctional in the initiation of translation. Specifically, we found that protein expressed by both the macrophage-derived cDNA and the extended cDNA had the same relative molecular mass, consistent with use of an ATG internal to the macrophage-derived clone as the functional start site. In addition, this work more precisely defines the catalytic activity of UMP-CMP kinase. Here, we show a 3-fold greater substrate preference for CMP relative to UMP, identify ATP and UTP as the preferred phosphate donors for the reaction, and demonstrate that the reaction is Mg2+-dependent. In addition, investigation of UMP-CMP-kinase expression revealed two mRNA products in immune tissues and cancer cell lines. The smaller RNA product was previously undescribed.

Differential regulation of matrix metalloproteinase-9 by monocytes adherent to collagen and platelets.

Circulating monocytes adhere to platelets and matrix proteins at sites of vascular injury, where engagement of specific surface tethering molecules mediates outside-in signaling and synthesis of gene products by the leukocytes. Here we demonstrate that interaction of isolated human monocytes with collagen induces matrix metalloproteinase-9 (MMP-9; gelatinase B) synthesis by monocytes, a process that is greatly enhanced in the presence of platelets. MMP-9 is a potent matrix degrading enzyme implicated in atherosclerotic plaque rupture, aneurysm formation, and other vascular syndromes. Synthesis of MMP-9 by monocytes is tightly regulated and synergistically increased following adhesion to collagen and platelets. Adhesion to control matrix proteins alone did not result in MMP-9 protein production and, similarly, adhesion of monocytes to platelets activated with thrombin in suspension was not sufficient to induce MMP-9 synthesis in the absence of monocyte adhesion to collagen. Interruption of intercellular contact between platelets and monocytes dramatically inhibited MMP-9 synthesis. These observations demonstrate that discrete adhesion-dependent signaling pathways govern MMP-9 synthesis by monocytes. The synthesis of MMP-9 by monocytes may be critical in vascular syndromes and other pathological processes that are dependent on dysregulated cell-cell and cell-matrix interactions.

Activated platelets mediate inflammatory signaling by regulated interleukin 1beta synthesis.

Platelets release preformed mediators and generate eicosanoids that regulate acute hemostasis and inflammation, but these anucleate cytoplasts are not thought to synthesize proteins or cytokines, or to influence inflammatory responses over time. Interrogation of an arrayed cDNA library demonstrated that quiescent platelets contain many messenger RNAs, one of which codes for interleukin 1beta precursor (pro-IL-1beta). Unexpectedly, the mRNA for IL-1beta and many other transcripts are constitutively present in polysomes, providing a mechanism for rapid synthesis. Platelet activation induces rapid and sustained synthesis of pro-IL-1beta protein, a response that is abolished by translational inhibitors. A portion of the IL-1beta is shed in its mature form in membrane microvesicles, and induces adhesiveness of human endothelial cells for neutrophils. Signal-dependent synthesis of an active cytokine over several hours indicates that platelets may have previously unrecognized roles in inflammation and vascular injury. Inhibition of beta3 integrin engagement markedly attenuated the synthesis of IL-1beta, identifying a new link between the coagulation and inflammatory cascades, and suggesting that antithrombotic therapies may also have novel antiinflammatory effects.

Cell adhesion regulates gene expression at translational checkpoints in human myeloid leukocytes.

Engagement of adhesion molecules on monocytes and other myeloid leukocytes, which are effector cells of the innate immune system, not only tethers the leukocytes in place but also transmits outside-in signals that induce functional changes and alter gene expression. We found that a subset of mRNAs that are induced or amplified by adhesion of human monocytes to P-selectin via its surface ligand, P-selectin glycoprotein 1, have characteristics that suggest specialized translational control. One of these codes for urokinase plasminogen activator receptor (UPAR), a critical surface protease receptor and regulator of cell adhesion and migration. Although UPAR transcripts are induced by adhesion, rapid synthesis of the protein uses constitutive mRNA without a requirement for new transcription and is regulated by mammalian target of rapamycin, demonstrating new biologic roles for the signal-dependent translation pathway controlled by this intracellular kinase. The synthesis of UPAR in monocytic cells is also regulated by eukaryotic translation initiation factor 4E, a second key translational checkpoint, and phosphorylation of eukaryotic translation initiation factor 4E is induced by adhesion of monocytes to P-selectin. Translationally controlled display of UPAR by monocytes confers recognition of the matrix protein, vitronectin. Adhesion-dependent signaling from the plasma membrane to translational checkpoints represents a previously unrecognized mechanism for regulating surface phenotype that may be particularly important for myeloid leukocytes and other cells that are specialized for rapid inflammatory and vascular responses.

Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids.

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist. We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Identification of platelet-activating factor as the inflammatory lipid mediator in CCl4-metabolizing rat liver.

Unmitigated oxidative stress is deleterious, as epitomized by CCl4 intoxication. In this well-characterized model of free radical-initiated damage, liver metabolism of CCl4 to CCl3. causes lipid peroxidation, F-ring isoprostane formation, and pathologic leukocyte activation. The nature of the mediator that couples oxidation to the hepatotoxic inflammatory response is uncharacterized. We found that oxidatively modified phosphatidylcholines were present in the livers of CCl4-exposed rats and not in livers from control animals, that CCl4 metabolism generated lipids that activated 293 cells stably transfected with the human platelet-activating factor (PAF) receptor, and that this PAF-like activity was formed as rapidly as isoprostane-containing phosphatidylcholine (iPC) during oxidation. iPC and the PAF-like activity also had similar chromatographic properties. The potential for iPC activation of the PAF receptor has been unexplored, but we conclude that iPC themselves did not activate the PAF receptor, as phospholipase A1 hydrolysis completely destroyed iPC, but none of the PAF-like bioactivity. Oxidatively fragmented phospholipids are potent agonists of the PAF receptor, but mass spectrometry characterized PAF as the major inflammatory component coeluting with iPC. Oxidatively fragmented phospholipids and iPC are markers of free radical generation in CCl4-intoxicated liver, but PAF generation by activated hepatic cells generated the inflammatory agent.

Oxidized alkyl phospholipids are specific, high affinity peroxisome proliferator-activated receptor gamma ligands and agonists.

Synthetic high affinity peroxisome proliferator-activated receptor (PPAR) agonists are known, but biologic ligands are of low affinity. Oxidized low density lipoprotein (oxLDL) is inflammatory and signals through PPARs. We showed, by phospholipase A(1) digestion, that PPARgamma agonists in oxLDL arise from the small pool of alkyl phosphatidylcholines in LDL. We identified an abundant oxidatively fragmented alkyl phospholipid in oxLDL, hexadecyl azelaoyl phosphatidylcholine (azPC), as a high affinity ligand and agonist for PPARgamma. [(3)H]azPC bound recombinant PPARgamma with an affinity (K(d)((app)) approximately 40 nm) that was equivalent to rosiglitazone (BRL49653), and competition with rosiglitazone showed that binding occurred in the ligand-binding pocket. azPC induced PPRE reporter gene expression, as did rosiglitazone, with a half-maximal effect at 100 nm. Overexpression of PPARalpha or PPARgamma revealed that azPC was a specific PPARgamma agonist. The scavenger receptor CD36 is encoded by a PPRE-responsive gene, and azPC enhanced expression of CD36 in primary human monocytes. We found that anti-CD36 inhibited azPC uptake, and it inhibited PPRE reporter induction. Results with a small molecule phospholipid flippase mimetic suggest azPC acts intracellularly and that cellular azPC accumulation was efficient. Thus, certain alkyl phospholipid oxidation products in oxLDL are specific, high affinity extracellular ligands and agonists for PPARgamma that induce PPAR-responsive genes.

A novel syndrome of variant leukocyte adhesion deficiency involving defects in adhesion mediated by beta1 and beta2 integrins.

Leukocyte adhesion deficiency type I (LAD-1) is a disorder associated with severe and recurrent bacterial infections, impaired extravascular targeting and accumulation of myeloid leukocytes, altered wound healing, and significant morbidity that is caused by absent or greatly diminished surface expression of integrins of the beta2 class. We report clinical features and analysis of functions of cells from a patient with a myelodysplastic syndrome and infectious complications similar to those in the severe form of LAD-1, but whose circulating neutrophils displayed normal levels of beta2 integrins. Analysis of adhesion of these cells to immobilized ligands and to endothelial cells and assays of cell-cell aggregation and chemotaxis demonstrated a profound defect in adhesion mediated by beta2 integrins indicative of a variant form of LAD-1. A novel cell line established from Epstein-Barr virus-transformed lymphoblasts from the subject demonstrated deficient beta2 integrin-dependent adhesive function similar to that of the primary leukocytes. In addition, these cells had markedly impaired beta1 integrin-dependent adhesion. Sequence analysis and electrophoretic mobility of beta1 and beta2 proteins from the cell line demonstrated that the defects were not a result of structural abnormalities in the integrin subunit chains themselves and suggest that the adhesive phenotype of these cells is due to one or more abnormalities of inside-out signaling mechanisms that regulate the activity of integrins of these classes. These features define a unique LAD-1 variant syndrome that may reveal important insights that are generally relevant to inside-out signaling of integrins, a molecular process that is as yet incompletely understood.