Acilcarnitinas en plasma y riesgo de insuficiencia cardiaca y fibrilación auricular: el estudio Prevención con dieta mediterránea / Plasma acylcarnitines and risk of incident heart failure and atrial fibrillation: the Prevención con dieta mediterránea study

Introducción y objetivos La desregulación del metabolismo de los ácidos grasos en la mitocondria es un mecanismo involucrado en el desarrollo de insuficiencia cardiaca (IC) y fibrilación auricular (FA). Se evaluó la asociación entre la concentración plasmática de acilcarnitinas y la incidencia de IC o FA y si la dieta mediterránea (DietMed) puede atenuar la asociación entre las acilcarnitinas y el riesgo de IC o FA. Métodos Dos estudios de casos y controles anidados en el ensayo Prevención con dieta mediterránea (PREDIMED). Se incluyó a participantes con elevado riesgo cardiovascular en España: 326 casos incidentes de IC y 509 de FA se emparejaron individualmente con 1 a 3 controles. Las acilcarnitinas en plasma se midieron con espectrometría de masas en tándem con cromatografía líquida de alta resolución. Se ajustaron modelos de regresión logística condicional para estimar las OR multivariables y los IC95%. Se evaluaron interacciones multiplicativas y aditivas por el grupo de intervención, obesidad (índice de masa corporal ≥ 30) y diabetes mellitus tipo 2. Resultados Las altas concentraciones de acilcarnitinas de cadena mediana y larga se asociaron con un mayor riesgo de IC (respectivamente, ORporDE ajustada=1,28; IC95%, 1,09-1,51, y ORporDE ajustada=1,21; IC95%, 1,04-1,42). Se observó una asociación significativa entre las acilcarnitinas de cadena larga y el riesgo de FA: 1,20 (1,06-1,36). Se encontró una interacción aditiva entre las acilcarnitinas de cadena larga y la FA con la DietMed suplementada con aceite de oliva virgen extra (p de interacción=0,036) y con la obesidad (p=0,022) de forma inversa y directa respectivamente. Conclusiones En las personas con alto riesgo cardiovascular, las altas concentraciones de acilcarnitinas de cadena larga se asocian con mayor riesgo de IC y FA incidentes. Una intervención con DietMed+aceite de oliva virgen extra puede reducir el riesgo asociado con las acilcarnitinas de cadena larga (AU)

Introduction and objectives Fatty acid metabolic dysregulation in mitochondria is a common mechanism involved in the development of heart failure (HF) and atrial fibrillation (AF). We evaluated the association between plasma acylcarnitine levels and the incidence of HF or AF, and whether the mediterranean diet (MedDiet) may attenuate the association between acylcarnitines and HF or AF risk. Methods Two case-control studies nested within the Prevención con dieta mediterránea (PREDIMED) trial. High cardiovascular risk participants were recruited in Spain: 326 incident HF and 509 AF cases individually matched to 1 to 3 controls. Plasma acylcarnitines were measured with high-throughput liquid chromatography-tandem mass spectrometry. Conditional logistic regression models were fitted to estimate multivariable OR and 95%CI. Additive and multiplicative interactions were assessed by intervention group, obesity (body mass index ≥ 30 kg/m2), and type 2 diabetes. Results Elevated levels of medium- and long-chain acylcarnitines were associated with increased HF risk (adjusted ORperDE, 1.28; 95%CI, 1.09-1.51 and adjusted ORperDE, 1.21; 95%CI, 1.04-1.42, respectively). A significant association was observed for AF risk with long-chain acylcarnitines: 1.20 (1.06-1.36). Additive interaction of the association between long-chain acylcarnitines and AF by the MediDiet supplemented with extra virgin olive oil (P for additive interaction=.036) and by obesity (P=.022) was observed in an inverse and direct manner, respectively. Conclusions Among individuals at high cardiovascular risk, elevated long-chain acylcarnitines were associated with a higher risk of incident HF and AF. An intervention with MedDiet+extra-virgin olive oil may reduce AF risk associated with long-chain acylcarnitines (AU)

Metabolomics signatures associated with an oral glucose challenge in pregnant women.

AIM: The oral glucose tolerance test (OGTT), widely used as a gold standard for gestational diabetes mellitus (GDM) diagnosis, provides a broad view of glucose pathophysiology in response to a glucose challenge. We conducted the present study to evaluate metabolite changes before and after an oral glucose challenge in pregnancy; and to examine the extent to which metabolites may serve to predict GDM diagnosis in pregnant women. METHODS: Peruvian pregnant women (n=100) attending prenatal clinics (mean gestation 25 weeks) participated in the study with 23% of them having GDM diagnosis. Serum samples were collected immediately prior to and 2-hours after administration of a 75-g OGTT. Targeted metabolic profiling was performed using a LC-MS based metabolomics platform. Changes in metabolite levels were evaluated using paired Student's t-tests and the change patterns were examined at the level of pathways. Multivariate regression procedures were used to examine metabolite pairwise differences associated with subsequent GDM diagnosis. RESULTS: Of the 306 metabolites detected, the relative concentration of 127 metabolites were statistically significantly increased or decreased 2-hours after the oral glucose load (false discovery rate [FDR] corrected P-value<0.001). We identified relative decreases in metabolites in acylcarnitines, fatty acids, and diacylglycerols while relative increases were noted among bile acids. In addition, we found that C58:10 triacylglycerol (ß=-0.08, SE=0.04), C58:9 triacylglycerol (ß=-0.07, SE=0.03), adenosine (ß=0.70, SE=0.32), methionine sulfoxide (ß=0.36, SE=0.13) were significantly associated with GDM diagnosis even after adjusting for age and body mass index. CONCLUSIONS: We identified alterations in maternal serum metabolites, representing distinct cellular and metabolic pathways including fatty acid metabolism, in response to an oral glucose challenge. These findings offer novel perspectives on the pathophysiological mechanisms underlying GDM.

Recurrent Clostridium difficile infection associates with distinct bile acid and microbiome profiles.

BACKGROUND: The healthy microbiome protects against the development of Clostridium difficile infection (CDI), which typically develops following antibiotics. The microbiome metabolises primary to secondary bile acids, a process if disrupted by antibiotics, may be critical for the initiation of CDI. AIM: To assess the levels of primary and secondary bile acids associated with CDI and associated microbial changes. METHODS: Stool and serum were collected from patients with (i) first CDI (fCDI), (ii) recurrent CDI (rCDI) and (iii) healthy controls. 16S rRNA sequencing and bile salt metabolomics were performed. Random forest regression models were constructed to predict disease status. PICRUSt analyses were used to test for associations between predicted bacterial bile salt hydrolase (BSH) gene abundances and bile acid levels. RESULTS: Sixty patients (20 fCDI, 19 rCDI and 21 controls) were enrolled. Secondary bile acids in stool were significantly elevated in controls compared to rCDI and fCDI (P < 0.0001 and P = 0.0007 respectively). Primary bile acids in stool were significantly elevated in rCDI compared to controls (P < 0.0001) and in rCDI compared to fCDI (P = 0.02). Using random forest regression, we distinguished rCDI and fCDI patients 84.2% of the time using bile acid ratios. Stool deoxycholate to glycoursodeoxycholate ratio was the single best predictor. PICRUSt analyses found significant differences in predicted abundances of bacterial BSH genes in stool samples across the groups. CONCLUSIONS: Primary and secondary bile acid composition in stool was different in those with rCDI, fCDI and controls. The ratio of stool deoxycholate to glycoursodeoxycholate was the single best predictor of disease state and may be a potential biomarker for recurrence.

Circulating branched-chain amino acid concentrations are associated with obesity and future insulin resistance in children and adolescents.

UNLABELLED: What is already known about this subject Circulating concentrations of branched-chain amino acids (BCAAs) can affect carbohydrate metabolism in skeletal muscle, and therefore may alter insulin sensitivity. BCAAs are elevated in adults with diet-induced obesity, and are associated with their future risk of type 2 diabetes even after accounting for baseline clinical risk factors. What this study adds Increased concentrations of BCAAs are already present in young obese children and their metabolomic profiles are consistent with increased BCAA catabolism. Elevations in BCAAs in children are positively associated with insulin resistance measured 18 months later, independent of their initial body mass index. BACKGROUND: Branched-chain amino acid (BCAA) concentrations are elevated in response to overnutrition, and can affect both insulin sensitivity and secretion. Alterations in their metabolism may therefore play a role in the early pathogenesis of type 2 diabetes in overweight children. OBJECTIVE: To determine whether paediatric obesity is associated with elevations in fasting circulating concentrations of BCAAs (isoleucine, leucine and valine), and whether these elevations predict future insulin resistance. METHODS: Sixty-nine healthy subjects, ages 8-18 years, were enrolled as a cross-sectional cohort. A subset of subjects who were pre- or early-pubertal, ages 8-13 years, were enrolled in a prospective longitudinal cohort for 18 months (n = 17 with complete data). RESULTS: Elevations in the concentrations of BCAAs were significantly associated with body mass index (BMI) Z-score (Spearman's Rho 0.27, P = 0.03) in the cross-sectional cohort. In the subset of subjects that followed longitudinally, baseline BCAA concentrations were positively associated with homeostasis model assessment for insulin resistance measured 18 months later after controlling for baseline clinical factors including BMI Z-score, sex and pubertal stage (P = 0.046). CONCLUSIONS: Elevations in the concentrations of circulating BCAAs are significantly associated with obesity in children and adolescents, and may independently predict future insulin resistance.

Identification of dual cyclooxygenase-eicosanoid oxidoreductase inhibitors: NSAIDs that inhibit PG-LX reductase/LTB(4) dehydrogenase.

Eicosanoids play key roles in many physiologic and disease processes, and their regulation by nonsteroidal anti-inflammatory drugs (NSAIDs) is critical to many therapeutic approaches. These autacoids are rapidly inactivated by specific enzymes such as 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase/leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH) that act on main series of eicosanoids (i.e., leukotrienes, prostaglandins), and recently found to act in lipoxin inactivation. Here, a panel of NSAIDs was assessed to determine each compound's ability to inhibit eicosanoid-directed activities of either the recombinant 15-PGDH or the PG-LXR/LTB(4)DH. The recombinant 15-PGDH that acts on both prostaglandin E(2) (PGE(2)) and lipoxin A(4) (LXA(4)) was not significantly inhibited by the NSAIDs tested. In contrast, several of the widely used NSAIDs were potent inhibitors of the PG-LXR/LTB(4)DH that metabolizes 15-oxo-PGE(2), and LTB(4) as well as 15-oxo-LXA(4). Diclofenac and indomethacin each inhibited PG-LXR/LTB(4)DH-catalyzed conversion of 15-oxo-PGE(2) to 13,14-dihydro-15-oxo-PGE(2) by 70 and 95%, respectively. Also, a COX-2 inhibitor, niflumic acid, inhibited the PG-LXR/LTB(4)DH eicosanoid oxidoreductase (EOR) by 80% while other COX-2 inhibitors such as nimesulide and NS-398 did not inhibit this enzyme. These results indicate that certain clinically useful NSAIDs such as diclofenac and indomethacin, in addition to inhibiting cyclooxygenases (1 and 2), also interfere with eicosanoid degradation by blocking PG-LXR/LTB(4)DH (EOR) and are members of a new class of dual cyclooxygenase (COX)-EOR inhibitors. Moreover, they suggest that the impact of NSAIDs on PG-LXR/LTB(4)DH activities as targets in the local tissue regulation of eicosanoid-mediated processes should be taken into account.

Lipid mediator class switching during acute inflammation: signals in resolution.

Leukotrienes (LTs) and prostaglandins (PGs) amplify acute inflammation, whereas lipoxins (LXs) have unique anti-inflammatory actions. Temporal analyses of these eicosanoids in clinical and experimental exudates showed early coordinate appearance of LT and PG with polymorphonuclear neutrophil (PMN) recruitment. This was followed by LX biosynthesis, which was concurrent with spontaneous resolution. Human peripheral blood PMNs exposed to PGE2 (as in exudates) switched eicosanoid biosynthesis from predominantly LTB4 and 5-lipoxygenase (5-LO)-initiated pathways to LXA4, a 15-LO product that "stopped" PMN infiltration. These results indicate that first-phase eicosanoids promote a shift to anti-inflammatory lipids: functionally distinct lipid-mediator profiles switch during acute exudate formation to "reprogram" the exudate PMNs to promote resolution.

Novel functional sets of lipid-derived mediators with antiinflammatory actions generated from omega-3 fatty acids via cyclooxygenase 2-nonsteroidal antiinflammatory drugs and transcellular processing.

Aspirin therapy inhibits prostaglandin biosynthesis without directly acting on lipoxygenases, yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins (LXs) epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered LX [ATL]). Here, we report that inflammatory exudates from mice treated with omega-3 polyunsaturated fatty acid and aspirin (ASA) generate a novel array of bioactive lipid signals. Human endothelial cells with upregulated COX-2 treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Each was used by polymorphonuclear leukocytes to generate separate classes of novel trihydroxy-containing mediators, including 5-series 15R-LX(5) and 5,12,18R-triHEPE. These new compounds proved to be potent inhibitors of human polymorphonuclear leukocyte transendothelial migration and infiltration in vivo (ATL analogue > 5,12,18R-triHEPE > 18R-HEPE). Acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on hematologic cells. These findings establish new transcellular routes for producing arrays of bioactive lipid mediators via COX-2-nonsteroidal antiinflammatory drug-dependent oxygenations and cell-cell interactions that impact microinflammation. The generation of these and related compounds provides a novel mechanism(s) for the therapeutic benefits of omega-3 dietary supplementation, which may be important in inflammation, neoplasia, and vascular diseases.

Aspirin-tolerant asthmatics generate more lipoxins than aspirin-intolerant asthmatics.

Asthma is characterized by chronic airway inflammation resulting from overproduction of pro-inflammatory mediators, such as leukotrienes (LT). The authors questioned the biosynthetic capacity of asthmatic patients for lipoxins (LX) and 15-epimer lipoxins (15-epi-LX), endogenous regulators of inflammatory responses that inhibit pro-inflammatory events. Levels of LXA4, 15-epi-LXA4 and LTC4 were determined in 14 clinically characterized aspirin-intolerant asthmatics (AIA), 11 aspirin-tolerant asthmatics (ATA) and eight healthy volunteers using a stimulated whole blood protocol. Both LXA4 and 15-epi-LXA4 were generated in whole blood activated by the divalent cation ionophore, A23187. Higher levels of LXA4 were produced in ATA than either AIA or healthy volunteers. Exposure of AIA whole blood to interleukin-3 prior to A23187 did not elevate their reduced capacity to generate LXA4. Generation of a bronchoconstrictor, LTC4, was similar in both AIA and ATA. Consequently, the ratio of LXA4:LTC4 quantitatively favoured the bronchoconstrictor for AIA and differed from both ATA and healthy subjects. In addition, the capacity for 15-epi-LXA4 generation was also diminished in AIA, since whole blood stimulated in the presence of aspirin gave increased levels only in samples from ATA. The present results indicate that asthmatics possess the capacity to generate both lipoxins and 15-epimer-lipoxins, but aspirin-intolerant asthmatics display a lower biosynthetic capacity than aspirin-tolerant asthmatics for these potentially protective lipid mediators. This previously unappreciated, diminished capacity for lipoxin formation by aspirin-intolerant asthmatic patients may contribute to their more severe clinical phenotype, and represents a novel paradigm for the development of chronic inflammatory disorders.

Lipoxins, aspirin-triggered 15-epi-lipoxin stable analogs and their receptors in anti-inflammation: a window for therapeutic opportunity.

LXs and 15-epimer LXs are generated during cell-cell interactions that occur during multicellular host response to inflammation, tissue injury or host defense. Results indicate that they are present in vivo during human illness and carry predominantly counter-regulatory biological actions opposing the action of well-characterized mediators of inflammation that appear to lead to resolution of the inflammatory response or promotion of repair and wound healing. The first selective receptor of LXA4 was identified by direct ligand binding and was cloned and characterized. Its signaling involves a novel polyisoprenyl-phosphate pathway that directly regulates PLD (Levy et al. 1999a). LX- and 15-epimer-LX-stable analogs that resist metabolic inactivation were designed, synthesized and shown to be potent LX mimetics and novel topically active anti-inflammatory agents in animal models. These new investigational tools enable structure-function studies of LX signal transduction, further elucidation of the role of LX and 15-epimer LX in host responses and exploitation of their potent bioactions in the design of novel pharmacologic agents.

Oxidoreductases in lipoxin A4 metabolic inactivation: a novel role for 15-onoprostaglandin 13-reductase/leukotriene B4 12-hydroxydehydrogenase in inflammation.

The lipoxins (LX) are autacoids that act within a local inflammatory milieu to dampen neutrophil recruitment and promote resolution. 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) and 15-oxoprostaglandin 13-reductase, also termed leukotriene B(4) 12-hydroxydehydrogenase (PGR/LTB(4)DH), are two enzymatic activities appreciated for their roles in the metabolism of prostaglandins and LTB(4). Here, we determined whether these oxidoreductases also catalyze the conversion of lipoxin A(4) (LXA(4)) and assessed the activities of these LXA(4) metabolites. 15-Oxo-LXA(4) was generated by incubating LXA(4) with 15-PGDH and NAD(+) for studies of its further conversion. PGR/LTB(4)DH catalyzed the NADH-dependent reduction of 15-oxo-LXA(4) to yield 13,14-dihydro-15-oxo-LXA(4). With NADH as a cofactor, 15-PGDH acted as a 15-carbonyl reductase and catalyzed the conversion of 13,14-dihydro-15-oxo-LXA(4) to 13, 14-dihydro-LXA(4). Human polymorphonuclear leukocytes (PMN) exposed to native LXA(4), 15-oxo-LXA(4), or 13,14-dihydro-LXA(4) did not produce superoxide anions. At concentrations where LXA(4) and a metabolically stable LXA(4) analog potently inhibited leukotriene B(4)-induced superoxide anion generation, the further metabolites were devoid of activity. Neither 15-oxo-LXA(4) nor 13, 14-dihydro-LXA(4) effectively competed with (3)H-labeled LXA(4) for specific binding to recombinant LXA(4) receptor (ALXR). In addition, introducing recombinant PGR/LTB(4)DH into a murine exudative model of inflammation increased PMN number by approximately 2-fold, suggesting that this enzyme participates in the regulation of PMN trafficking. These results establish the structures of LXA(4) further metabolites and indicate that conversion of LXA(4) to oxo- and dihydro- products represents a mode of LXA(4) inactivation in inflammation. Moreover, they suggest that these eicosanoid oxidoreductases have multifaceted roles controlling the levels of specific eicosanoids involved in the regulation of inflammation.

Lipoxin A(4) analogues inhibit leukocyte recruitment to Porphyromonas gingivalis: a role for cyclooxygenase-2 and lipoxins in periodontal disease.

The potential involvement of the inducible cyclooxygenase isoform (COX-2) and the role of novel lipid mediators were investigated in the pathogenesis of periodontal disease. Crevicular fluids from localized juvenile periodontitis (LJP) patients contained prostaglandin (PG)E(2) and 5-lipoxygenase-derived products, leukotriene B(4), and the biosynthesis interaction product, lipoxin (LX)A(4). Neutrophils from peripheral blood of LJP patients, but not from asymptomatic donors, also generated LXA(4), suggesting a role for this immunomodulatory molecule in periodontal disease. To characterize host responses of interest to periodontal pathogens, Porphyromonas gingivalis was introduced within murine dorsal air pouches. In the air pouch cavity, P. gingivalis elicited leukocyte infiltration, concomitant with elevated PGE(2) levels in the cellular exudates, and upregulated COX-2 expression in infiltrated leukocytes. In addition, human neutrophils exposed to P. gingivalis also upregulated COX-2 expression. Blood borne P. gingivalis gave significant increases in the murine tissue levels of COX-2 mRNA associated with both heart and lungs, supporting a potential role for this oral pathogen in the evolution of systemic events. The administration of metabolically stable analogues of LX and of aspirin-triggered LX potently blocked neutrophil traffic into the dorsal pouch cavity and lowered PGE(2) levels within exudates. Together, these results identify PMN as an additional and potentially important source of PGE(2) in periodontal tissues. Moreover, they provide evidence for a novel protective role for LX in periodontitis, limiting further PMN recruitment and PMN-mediated tissue injury that can lead to loss of inflammatory barriers that prevent systemic tissue invasion of oral microbial pathogens.

A synthetic antagonist for the peroxisome proliferator-activated receptor gamma inhibits adipocyte differentiation.

While searching for natural ligands for the peroxisome proliferator-activated receptor (PPAR) gamma, we identified a synthetic compound that binds to this receptor. Bisphenol A diglycidyl ether (BADGE) is a ligand for PPARgamma with a K(d(app)) of 100 microM. This compound has no apparent ability to activate the transcriptional activity of PPARgamma; however, BADGE can antagonize the ability of agonist ligands such as rosiglitazone to activate the transcriptional and adipogenic action of this receptor. BADGE also specifically blocks the ability of natural adipogenic cell lines such as 3T3-L1 and 3T3-F442A cells to undergo hormone-mediated cell differentiation. These results provide the first pharmacological evidence that PPARgamma activity is required for the hormonally induced differentiation of adipogenic cells.

Anti-microinflammatory lipid signals generated from dietary N-3 fatty acids via cyclooxygenase-2 and transcellular processing: a novel mechanism for NSAID and N-3 PUFA therapeutic actions.

Aspirin therapy inhibits prostaglandin biosynthesis; yet via acetylation of cyclooxygenase 2 (COX-2) it leads to bioactive lipoxins epimeric at carbon 15 (15-epi-LX, also termed aspirin-triggered lipoxin or ATL). Here, we review our findings indicating that inflammatory exudates from mice treated with omega-3 PUFA and aspirin (ASA) generate a novel array of bioactive lipid signals. Also, human endothelial cells, both HUVEC and microvascular, with upregulated COX-2 and treated with ASA converted C20:5 omega-3 to 18R-hydroxyeicosapentaenoic acid (HEPE) and 15R-HEPE. Human PMN activated with serum treated zymosan (STZ) utilized each of these R-HEPEs to generate novel classes of trihydroxy-containing mediators including 5-series 15R-LX and 5,12,18R-triHEPE. The novel products were potent inhibitors of human PMN transendothelial migration and infiltration of PMN in dorsal air pouches in vivo. In addition to ASA, both acetaminophen and indomethacin also permitted 18R-HEPE and 15R-HEPE generation with recombinant human COX-2 as well as omega-5 and omega-9 oxygenations of other fatty acids that act on leukocytes, platelets and endothelial cells. These findings establish new transcellular routes for producing arrays of lipid mediators via COX-2-NSAIDs and cell-cell interactions that impact microinflammation. Moreover, they provide novel mechanism(s) that could underlie the many reported therapeutic benefits of omega-3 dietary supplementation of interest in inflammation, cancer, and vascular disorders.

Deposition of monomeric, not oligomeric, Abeta mediates growth of Alzheimer's disease amyloid plaques in human brain preparations.

Senile plaques composed of the peptide Abeta contribute to the pathogenesis of Alzheimer's disease (AD), and mechanisms underlying their formation and growth may be exploitable as therapeutic targets. To examine the process of amyloid plaque growth in human brain, we have utilized size exclusion chromatography (SEC), translational diffusion measured by NMR, and in vitro models of Abeta amyloid growth to identify the oligomerization state of Abeta that is competent to add onto an existing amyloid deposit. SEC of radiolabeled and unlabeled Abeta over a concentration range of 10(-)(10)-10(-)(4) M demonstrated that the freshly dissolved peptide eluted as a single low molecular weight species, consistent with monomer or dimer. This low molecular weight Abeta species isolated by SEC was competent to deposit onto preexisting amyloid in preparations of AD cortex, with first-order kinetic dependence on soluble Abeta concentration, establishing that solution-phase oligomerization is not rate limiting. Translational diffusion measurements of the low molecular weight Abeta fraction demonstrate that the form of the peptide active in plaque deposition is a monomer. In deliberately aged (>6 weeks) Abeta solutions, a high molecular weight (>100 000 M(r)) species was detectable in the SEC column void. In contrast to the active monomer, assembled Abeta isolated from the column showed little or no focal association with AD tissue. These studies establish that, at least in vitro, Abeta exists as a monomer at physiological concentrations and that deposition of monomers, rather than of oligomeric Abeta assemblies, mediates the growth of existing amyloid in human brain preparations.

Leukotriene B4 receptor transgenic mice reveal novel protective roles for lipoxins and aspirin-triggered lipoxins in reperfusion.

Polymorphonuclear neutrophil (PMN) activation is pivotal in acute inflammation and injury from reperfusion. To elucidate components controlling PMNs in vivo, we prepared novel transgenic mice with the human leukotriene (LT) B4 receptor (BLTR) for functional characterization. Overexpression of BLTR in leukocytes dramatically increased PMN trafficking to skin microabscesses and lungs after ischemia-reperfusion, whereas mice deficient in 5-lipoxygenase (5-LO) showed diminished PMN accumulation in reperfused lungs. Hence, both BLTR expression and LT biosynthesis are critical for PMN infiltration in reperfusion-initiated second-organ injury. Also, in BLTR transgenic mice, 5-LO expression and product formation were selectively increased in exudates, demonstrating that receptor overexpression amplifies proinflammatory circuits. Endogenous lipoxin (LX) A4 was produced in ischemic lungs and elevated by reperfusion. Because LXA4 and aspirin-triggered 15-epimeric LXA4 (ATL) selectively regulate leukocyte responses, they were tested in BLTR transgenic mice. Despite excessive PMN recruitment in BLTR transgenic mice, intravenous injection of ATL sharply diminished reperfusion-initiated PMN trafficking to remote organs, and topical application of LX was protective in acute dermal inflammation. These results demonstrate a direct role for BLTR with positive feedback, involving BLTR and 5-LO signaling in controlling PMNs. Moreover, LXA4 and ATL counter BLTR-amplified networks, revealing a novel protective role for LX and ATL in stress responses that has applications in perioperative medicine.

Local and systemic delivery of a stable aspirin-triggered lipoxin prevents neutrophil recruitment in vivo.

Aspirin (ASA) triggers a switch in the biosynthesis of lipid mediators, inhibiting prostanoid production and initiating 15-epi-lipoxin generation through the acetylation of cyclooxygenase II. These aspirin-triggered lipoxins (ATL) may mediate some of ASA's beneficial actions and therefore are of interest in the search for novel antiinflammatories that could manifest fewer unwanted side effects. Here, we report that design modifications to native ATL structure prolong its biostability in vivo. In mouse whole blood, ATL analogs protected at carbon 15 [15(R/S)-methyl-lipoxin A4 (ATLa1)] and the omega end [15-epi-16-(para-fluoro)-phenoxy-LXA4 (ATLa2)] were recoverable to approximately 90 and 100% at 3 hr, respectively, compared with a approximately 40% loss of native lipoxin A4. ATLa2 retains bioactivity and, at levels as low as approximately 24 nmol/mouse, potently inhibited tumor necrosis factor-alpha-induced leukocyte recruitment into the dorsal air pouch. Inhibition was evident by either local intra-air pouch delivery (approximately 77% inhibition) or systemic delivery by intravenous injection (approximately 85% inhibition) and proved more potent than local delivery of ASA. Rank order for inhibiting polymorphonuclear leukocyte infiltration was: ATLa2 (10 micrograms, i.v.) approximately ATLa2 (10 micrograms, local) approximately dexamethasone (10 micrograms, local) >ASA (1.0 mg, local). Applied topically to mouse ear skin, ATLa2 also inhibited polymorphonuclear leukocyte infiltration induced by leukotriene B4 (approximately 78% inhibition) or phorbol ester (approximately 49% inhibition), which initiates endogenous chemokine production. These results indicate that this fluorinated analog of natural aspirin-triggered lipoxin A4 is bioavailable by either local or systemic delivery routes and is a more potent and precise inhibitor of neutrophil accumulation than is ASA.

Lipoxin and aspirin-triggered 15-epi-lipoxin cellular interactions anti-inflammatory lipid mediators.

Eicosanoids are known to play important roles in inflammation. Recent findings have given rise to several new concepts regulating the generation of eicosanoids, illustrated in Figure 1. Lipoxins (LX) are trihydroxytetraene-containing eicosanoids that are generated within vascular lumen by platelet-leukocyte interactions and at mucosal surfaces by leukocyte-epithelial cell interactions. During these cell-cell interactions, transcellular biosynthetic pathways are used as major routes, and thus, in humans, LX are formed in vivo during multicellular responses such as inflammation, atherosclerosis, and thrombosis. This branch of the eicosanoid cascade generates specific tetraene-containing products that appear to function as stop signals, since they inhibit key steps in leukocyte-mediated inflammation. Of special interest, it appears that aspirin also functions in part via production of novel epimers of lipoxins or 15-epi-lipoxins (Figure 1). Here, we review recent developments on the cellular interactions of these novel anti-inflammatory mediators.