Role of FLAP and PDE4D in myocardial infarction and stroke: Target discovery and future treatment options

Opinion statement Biomarkers such as C-reactive protein (CRP) and myeloperoxidase (MPO) are elevated in patients with coronary artery disease and confer risk of acute cardiovascular events, such as myocardial infarction (MI) and stroke. More recently, variants in the 5-lipoxygenase-activating protein (FLAP) gene were shown to confer risk to both MI and stroke, effects that appear to be mediated through elevated LTB₄, a chemoattractant mediator shown to be upregulated in patients with MI. Another gene in the leukotriene (LT) pathway, LTA ₄ hydrolase, was subsequently found to confer increased risk to MI, effects that were ethnicity-specific with an approximately threefold higher risk in African Americans than in whites. In another study, markers in the phosphodiesterase (PDE) 4D gene were found to confer risk to large-vessel occlusive and cardiogenic stroke. Interestingly, there is a cross-link between the 5-LO and the PDE4D pathways with converging biology. To address the role of an inhibitor of FLAP on biomarkers of MI risk, a randomized placebocontrolled phase II trial was conducted in patients with MI. This trial showed that LTB₄ and MPO production was reduced in whole blood leukocytes that were stimulated with ionomycin and the effects of the inhibitor were dose dependent. Serum CRP and plasma MPO were also reduced at the highest dose, which was well tolerated. These data suggest that LTB₄ is a risk factor of MI and that inhibition of FLAP and the LT pathway produces suppression of biomarkers that are associated with MI risk, including but not limited to LTB₄, MPO, and CRP, supporting the notion that the LTB₄ arm of the LT pathway may play a fundamental role in heart attacks and stroke.     

Transcellular biosynthesis of cysteinyl leukotrienes in vivo during mouse peritoneal inflammation

Leukotrienes (LTs) are lipid mediators of inflammation formed by enzymatic oxidation of arachidonic acid. One intriguing aspect of LT production is transcellular biosynthesis: cells expressing 5-lipoxygenase (5LO) form LTA₄ and transfer it to cells expressing LTA₄ hydrolase (LTA₄H) or LTC₄ synthase (LTC₄S) to produce LTB₄ or LTC₄. This process has been demonstrated in vivo for LTB₄, but not for cysteinyl LTs (cysLTs). We examined transcellular cysLT synthesis during zymosan-induced peritonitis, using bone marrow transplants with transgenic mice deficient in key enzymes of LT synthesis and analyzing all eicosanoids by liquid chromatography/tandem mass spectrometry. WT mice time-dependently produced LTB₄ and cysLTs (LTC₄, LTD₄, and LTE₄). 5LO^−/− mice were incapable of producing LTs. WT bone marrow cells restored this biosynthetic ability, but 5LO^−/− bone marrow did not rescue LT synthesis in irradiated WT mice, demonstrating that bone marrow-derived cells are the ultimate source of all LTs in this model. Total levels of 5LO-derived products were comparable in LTA₄H^−/− and WT mice, but were reduced in LTC₄S^−/− animals. No differences in prostaglandin production were observed between these transgenic or chimeric mice. Bone marrow cells from LTA₄H^−/− or LTC₄S^−/− mice injected into 5LO^−/− mice restored the ability to synthesize LTB₄ and cysLTs, providing unequivocal evidence of efficient transcellular biosynthesis of cysLTs. These results highlight the potential relevance of transcellular exchange of LTA₄ for the synthesis of LTs mediating biological activities during inflammatory events in vivo.     

Regulation of the expression of 5‐lipoxygenase–activating protein/5‐lipoxygenase and the synthesis of leukotriene B4 in osteoarthritic chondrocytes: Role of transforming growth factor β and eicosanoids

Objective

To explore the modulation of 5‐lipoxygenase–activating protein (FLAP) and 5‐lipoxygenase (5‐LOX) expression in human osteoarthritic (OA) chondrocytes, their relative implications in leukotriene B₄ (LTB₄) production, the effect of different factors on this system, and the influence of increased LTB₄ production on the synthesis of catabolic factors of cartilage.

Methods

FLAP and 5‐LOX expression and LTB₄ production were monitored following treatment with transforming growth factor β1 (TGFβ1; 5 ng/ml) and 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃; 50 nM) alone or in combination with selective or nonselective cyclooxygenase (COX) inhibitors, naproxen (90 μg/ml), NS‐398 (10 μM), or FR122047 (5 μM), or a dual inhibitor of COX/5‐LOX activity, licofelone (2.6 μM). LTB₄, prostaglandin E₂ (PGE₂), and matrix metalloprotease 1 (MMP‐1) production were measured by specific enzyme‐linked immunosorbent assays, nitric oxide by the Griess reaction, and FLAP and 5‐LOX expression by quantitative polymerase chain reaction.

Results

Human OA chondrocytes expressed both FLAP and 5‐LOX. TGFβ1 and/or 1,25(OH)₂D₃ induced a rapid and marked enhancement (∼4–13‐fold) in FLAP messenger RNA (mRNA) levels, which was associated with a subsequent and late increase in LTB₄ production and PGE₂ synthesis. Treatment with COX inhibitors in the absence or presence of TGFβ1 and 1,25(OH)₂D₃ induced a rapid increase in LTB₄ production; this response was mediated by the sustained and significant (P < 0.01) up‐regulation (∼1.5‐fold) of 5‐LOX mRNA levels. Conversely, treatment with licofelone showed no effect on 5‐LOX but significantly reduced FLAP expression levels. Coincubation of licofelone with TGFβ1 plus 1,25(OH)₂D₃ did not affect FLAP or 5‐LOX levels. In the presence of TGFβ1 plus 1,25(OH)₂D₃, naproxen, but not licofelone, induced MMP‐1 production and both drugs decreased nitric oxide levels.

Conclusion

Both the eicosanoids PGE₂ and LTB₄ are important cofactors in regulating FLAP/5‐LOX expression; the inhibition of PGE₂ up‐regulates 5‐LOX while down‐regulating FLAP gene expression, and LTB₄ appears to be an up‐regulating factor on the 5‐LOX gene. Importantly, nonsteroidal antiinflammatory drugs up‐regulate the synthesis of LTB₄, supporting the shunt hypothesis from COX to 5‐LOX. We also demonstrated that LTB₄ likely contributes to the up‐regulation of important catabolic factors involved in the pathophysiology of OA, such as MMP.

     

Participation of 5-lipoxygenase-derived LTB4 in 4-hydroxynonenal-enhanced MMP-2 production in vascular smooth muscle cells

5-Lipoxygenase (5-LO) has been suggested as a modulator of atherosclerotic plaque instability, however, its role in MMP production in vascular smooth muscle cells (VSMC) is still unclear. Thus, this study investigated the role of 5-LO in HNE-enhanced MMP-2 production in VSMC, and the mechanisms by which this enzyme could be activated by HNE. VSMC stimulated with HNE (1 μM) produced MMP-2, which was markedly attenuated in 5-LO-deficient VSMC as well as in cells pretreated with a FLAP inhibitor, MK886, confirming a role for 5-LO metabolites in HNE-enhanced MMP-2 production. Related to these results, HNE increased nuclear translocation of 5-LO promoting 5-LO activity, which was attenuated not only by SB203580, a p38 MAPK inhibitor, but also by PD98059, an ERK inhibitor. In parallel, phosphorylation of p38 MAPK and ERK occurred as early as 15 min after exposure to HNE, suggesting a potential role for p38 MAPK and ERK pathways in HNE-induced activation of 5-LO. Among leukotriene (LT) receptor antagonists, U-75302, a BLT receptor antagonist, but not MK-571 and Rev-5901, cysLT receptor antagonists, showed an inhibitory effect on HNE-enhanced MMP-2 production. Moreover, MMP-2 production in VSMC was also significantly increased by LTB₄, but not by LTC₄ and LTD₄. Collectively, these data suggest that 5-LO mediates HNE-enhanced MMP-2 production via LTB₄-BLT receptor pathways, consequently leading to atherosclerotic plaque instability.     

Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

Leukotriene (LT) A₄ hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB₄. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA₄ hydrolase. Here, we determined the crystal structure of LTA₄ hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA₄ hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB₄ synthesis in human neutrophils (IC₅₀ of ∼0.5 μM) and conversion of LTA₄ into LTB₄ by purified LTA4H with a K_i of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA₄ hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA₄, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA₄ into LTB₄, although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA₄ hydrolase inhibitor that holds promise for improved anti-inflammatory properties.Leukotriene (LT) A₄ hydrolase/aminopeptidase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the formation of the potent chemotactic agent LTB₄, a key lipid mediator in the innate immune response (1, 2). Previous work has shown that LTA₄ hydrolase (LTA4H) is an aminopeptidase with high affinity for N-terminal arginines of various synthetic tripeptides (3, 4). The two enzyme activities of LTA4H are exerted via distinct but overlapping active sites and depend on the catalytic zinc, bound within the signature HEXXH-(X)₁₈-E, typical of M1 metallopeptidases (5–7). In LTA4H, His295, His299, and Glu318 are the zinc-binding ligands, whereas Glu296 is the general base catalyst for peptide hydrolysis (8, 9).LTA4H’s crystal structure has been determined (10). The enzyme folds into an N-terminal domain, a catalytic domain, and a C-terminal domain, each with ∼200 amino acids. The interface of the domains forms a cavity, where the active site is located (Fig. 1). The cavity narrows at the zinc-binding site, forming a tunnel into the catalytic domain. The opening and wider parts of the cavity are highly polar; the tunnel is more hydrophobic. The cavity is mostly defined by the catalytic and C-terminal domains; part of the tunnel is defined by the N-terminal domain. Bound substrate is in contact with all three domains.Open in a separate windowFig. 1.Position and extension of the active center in LTA4H. Cartoon representation of the structure of LTA4H with a tunnel for LTA₄ (red mesh) and peptide substrates (blue mesh). The catalytic zinc (yellow sphere) is located in a wide section of the active site from which a narrow, L-shaped, hydrophobic tunnel protrudes ∼15 Å deeper into the protein. LTA₄ is believed to bind with its ω-end at the end of the hydrophobic tunnel. The volume of the active center was calculated in CAVER (31).Recently, it was discovered that LTA4H cleaves and inactivates the chemotactic tripeptide Pro-Gly-Pro, thus identifying a previously unrecognized endogenous, physiologically significant aminopeptidase substrate (11). Inasmuch as Pro-Gly-Pro attracts neutrophils and promotes inflammation, these data also suggest that LTA4H plays dual and opposite roles during an inflammatory response (i.e., production of chemotactic LTB₄, as well as inactivation of chemotactic Pro-Gly-Pro). Previous efforts to develop inhibitors of LTA4H have used the aminopeptidase activity for screening purposes, and these molecules therefore block both catalytic activities of LTA4H (12).Here, we used crystallography, MS, and a stable peptide analog to determine the binding mode of Pro-Gly-Pro at the active site of LTA4H, as well as the mechanism of peptide cleavage. Based on the structure, we also designed a lead compound that selectively blocks the conversion of LTA₄ into LTB₄, although sparing the hydrolysis of Pro-Gly-Pro.     

Effects of a FLAP inhibitor,GSK2190915, in asthmatics with high sputum neutrophils

Patients with refractory asthma frequently have neutrophilic airway inflammation and respond poorly to inhaled corticosteroids. This study evaluated the effects of an oral 5-lipoxygenase-activating protein (FLAP) inhibitor, GSK2190915, in patients with asthma and elevated sputum neutrophils.Patients received 14 (range 13–16) days treatment with GSK2190915 100 mg and placebo with a minimum 14 day washout in a double-blind, cross-over, randomised design (N = 14). Sputum induction was performed twice pre-dose in each treatment period to confirm sputum neutrophilia, and twice at the end of each treatment period. The primary endpoint was the percentage and absolute sputum neutrophil count, averaged for end-of-treatment visits.GSK2190915 did not significantly reduce mean percentage sputum neutrophils (GSK2190915-placebo difference [95% CI]: −0.9 [−12.0, 10.3]), or mean sputum neutrophil counts (GSK2190915/placebo ratio [95% CI]: 1.06 [0.43, 2.61]). GSK2190915 resulted in a marked suppression (>90%) of sputum LTB₄ and urine LTE₄, but did not alter clinical endpoints. There were no safety issues.Despite suppressing the target mediator LTB₄, FLAP inhibitor GSK2190915 had no short-term effect on sputum cell counts or clinical endpoints in patients with asthma and sputum neutrophilia.     

Elevated levels of leukotriene B4 and thromboxane B2 distinguish chest pain of cardiac and non cardiac origin

ObjectiveMyocardial infarction (MI) is often preceded by severe chest pain. The use of inflammatory markers to distinguish between chest pain of cardiac and non cardiac origin are not well reported. The aim of the study was to distinguish the chest pain of non cardiac and cardiac origin by using reliable inflammatory markers.MethodsThe present study enrolled 80 subjects including chest pain which lead to myocardial infarction (n=40), non-cardiac chest pain (CP) patients (n=20) and healthy volunteers (N) (n=20). Leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) levels were analyzed along with hs-CRP.ResultsReceiver operating characteristic (ROC) curve analysis showed LTB₄ and TXB₂ to be a good discriminator between patients with chest pain of cardiac and non cardiac in origin. The area under the curve was found to be 0.988 and 0.925 for LTB₄ and TXB₂, respectively when compared with hs-CRP. The sensitivity and specificity of LTB₄ and TXB₂ were found to be 90, 85% and 95, 90%, respectively.ConclusionThe measurement of LTB₄ and TXB₂ levels may therefore be useful to distinguish the chest pain leading to MI from that of non cardiac in origin and for the management of the disease.     

A distinctive role of the leukotriene B4 receptor BLT1 in osteoclastic activity during bone loss

Although leukotriene B₄ (LTB₄) is produced in various inflammatory diseases, its functions in bone metabolism remain unknown. Using mice deficient in the high-affinity LTB₄ receptor BLT1, we evaluated the roles of BLT1 in the development of two bone resorption models, namely bone loss induced by ovariectomy and lipopolysaccharide. Through observations of bone mineral contents and bone morphometric parameters, we found that bone resorption in both models was significantly attenuated in BLT1-deficient mice. Furthermore, osteoclasts from BLT1-deficient mice showed reduced calcium resorption activities compared with wild-type osteoclasts. Osteoclasts expressed BLT1, but not the low-affinity LTB₄ receptor BLT2, and produced LTB₄. LTB₄ changed the cell morphology of osteoclasts through the BLT1-Gi protein-Rac1 signaling pathway. Given the causal relationship between osteoclast morphology and osteoclastic activity, these findings suggest that autocrine/paracrine LTB₄ increases the osteoclastic activity through the BLT1-Gi protein-Rac1 signaling pathway. Inhibition of BLT1 functions may represent a strategy for preventing bone resorption diseases.     

Study of the role of leukotriene B4 in abnormal function of human subchondral osteoarthritis osteoblasts: Effects of cyclooxygenase and/or 5‐lipoxygenase inhibition

Objective

To compare the effect of licofelone, NS‐398 (an inhibitor of cyclooxygenase 2 [COX‐2]), and BayX‐1005 (an inhibitor of 5‐lipoxygenase activating protein) on the production of leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), and on cell biomarkers by human osteoarthritis (OA) subchondral osteoblasts.

Methods

Primary in vitro osteoblasts were prepared from subchondral bone specimens obtained from OA patients and autopsy subjects. LTB₄ and PGE₂ levels were measured by enzyme‐linked immunosorbent assay in conditioned media of osteoblasts incubated in the presence or absence of licofelone, NS‐398, or BayX‐1005. The effect of these drugs or of the addition of LTB₄ on alkaline phosphatase (AP) activity and osteocalcin release by OA and normal osteoblasts was determined. The presence of LTB₄ receptors in normal and OA osteoblasts was evaluated by Western blot analysis.

Results

OA osteoblasts produced variable levels of PGE₂ and LTB₄ compared with normal osteoblasts. Licofelone, at the maximal dose used, inhibited production of PGE₂ and LTB₄ by OA osteoblasts by a mean ± SEM of 61.2 ± 6.4% and 67.0 ± 7.6%, respectively. NS‐398 reduced PGE₂ production by 75.8 ± 5.3%. BayX‐1005 inhibited LTB₄ production in OA osteoblasts by 38.7 ± 14.5% and marginally affected PGE₂ levels (reduction of 14.8 ± 5.3%). Licofelone dose‐dependently stimulated 1,25‐dihydroxyvitamin D‐induced AP activity while inhibiting osteocalcin release. BayX‐1005 partly reproduced these effects, but NS‐398 failed to affect them. LTB₄ dose‐dependently inhibited AP activity in OA osteoblasts, while its effect on osteocalcin depended on endogenous LTB₄ levels in these cells. In normal osteoblasts, LTB₄ dose‐dependently stimulated osteocalcin, whereas it failed to influence AP. LTB₄ receptors BLT1 and BLT2 were present in normal and OA osteoblasts.

Conclusion

Licofelone inhibits the production of PGE₂ and LTB₄. Selective effects of licofelone on AP and osteocalcin occur via its role on LTB₄ production. Because LTB₄ can modify cell biomarkers in OA and normal osteoblasts, our results suggest licofelone could modify abnormal bone remodeling in OA.

     

Polymorphisms of LTA, LGALS2, and PSMA6 genes and coronary atherosclerosis: a pathological study of 1503 consecutive autopsy cases

ObjectiveRecent genome-wide association studies have identified polymorphisms of lymphotoxin-α (LTA), galectin-2 (LGALS2), and proteasome subunit a type 6 (PSMA6) genes as genetic risk factors for myocardial infarction (MI). However, their effects on coronary atherosclerosis, an intermediate phenotype of MI, remain largely unknown.MethodsWe investigated the correlation between polymorphisms of the LTA, LGALS2, and PSMA6 genes and the severity of pathological coronary stenosis index (CSI) and MI in 1503 consecutive autopsy cases of Japanese elderly patients.ResultsThe polymorphisms LTA rs1041981 and LGALS2 rs7291467 were associated with CSI with odds ratios of 1.54 (95% CI, 1.17–2.01; AA + CA over CC) and 1.62 (95% CI, 1.11–2.37; TT over CC + CT), respectively. PSMA6 rs1048990 was not associated with CSI. None of the SNPs was associated with MI in our sample.ConclusionOur findings indicate that the LTA and LGALS2 polymorphisms affect the subclinical phenotype of the coronary artery, which predisposes to the incidence of MI.     

Bile salts determine leukotriene B4 synthesis in a human intestinal cell line (CaCo-2)

The ability of a human colonic epithelial cell line (CaCo-2) to synthesize leukotriene B₄ (LTB₄) in response to bile salt stimulation was examined, as was the dependency of such stimulation on the hydrophobic-hydrophilic balance of the bile salts. We demonstrate for the first time in this human intestinal epithelial cell line the ability of bile salts to stimulate synthesis of LTB₄. CaCo-2 cell monolayers were incubated with a series of bile salts ranging in concentration from 0.5 µM to 1 mM. This resulted in a dose- and hydrophobicity-dependent increase in LTB₄ synthesis. Hydrophobic bile salts (glycine and taurine conjugates of lithocholate and deoxycholate) caused LTB₄ synthesis to be stimulated 27% and 35%, respectively, above control levels. In contrast, hydrophilic bile salts (glycine and taurine conjugates of ursodeoxycholate) increased LTB₄ synthesis only 11.2% and 16.1%. Under basal conditions pretreatment with dexamethasone significantly inhibited bile salt-induced LTB₄ synthesis by 38% compared to control. With more hydrophobic bile salts, chenodeoxycholate and deoxycholate, dexamethasone inhibited LTB₄ synthesis to levels significantly below those observed with dexamethasone under basal conditions. Unlike A23187 calcium ionophore-induced LTB₄ synthesis, bile salt-induced stimulation of LTB₄ synthesis was not found to be dependent on the presence of extracellular calcium. Variations in bile salt stimulation of LTB₄ by intestinal epithelial cells could be important in modulating cellular responses. The synthesis of chemotactic factors, such as LTB₄, by the human colonic adenocarcinoma epithelial cell line now needs to be extended to normal human intestinal epithelium, as it may play a role in many of the functional disturbances which characterize intestinal inflammatory conditions.This work was funded by the Canadian Foundation for Ileitis and Colitis (CFIC).     

A review on leukotrienes and their receptors with reference to asthma

Abstract

Objective and methods: Leukotrienes (LTs) including cysteinyl leukotrienes (CysLTs) and LTB₄ are the most potent inflammatory lipid mediators and play a central role in the pathophysiology of asthma and other inflammatory diseases. These biological molecules mediate a plethora of contractile and inflammatory responses through specific interaction with distinct G protein-coupled receptors (GPCRs). The main objective of this review is to present an overview of the biological effects of CysLTs and their receptors, along with the current knowledge of mechanisms and role of LTs in the pathogenesis of asthma. Results: CysLTs including LTC₄, LTD₄ and LTE₄ are ligands for CysLT₁ and CysLT₂ receptors, and LTB₄ is the agonist for BLT₁ and BLT₂ receptors. The role of CysLT₁ receptor is well established, and most of the pathophysiological effects of CysLTs in asthma are mediated by CysLT₁ receptor. Several CysLT₁ antagonists have been developed to date and are currently in clinical practice. Most common among them are classical CysLT₁ receptor antagonists such as montelukast, zafirlukast, pranlukast, pobilukast, iralukast, cinalukast and MK571. The pharmacological role of CysLT₂ receptor, however, is less defined and there is no specific antagonist available so far. The recent demonstration that mice lacking both known CysLT receptors exhibit full/augmented response to CysLT points to the existence of additional subtypes of CysLT receptors. LTB₄, on the other hand, is another potent inflammatory leukotriene, which acts as a strong chemoattractant for neutrophils, but weaker for eosinophils. LTB₄ is known to play an important role in the development of airway hyper-responsiveness in severe asthma. However there is no LTB₄ antagonist available in clinic to date. Conclusion: This review gives a recent update on the LTs including their biosynthesis, biological effects and the role of anti-LTs in the treatment of asthma. It also discusses about the possible existence of additional subtypes of CysLT receptors.     

Factors Affecting the Genetic Diagnostic Rate in Congenital Heart Disease

Background: Over 400 genes contribute to the development of congenital heart disease (CHD). Additionally, multisystemic manifestations accompanying syndromic CHD pose a higher risk of genetic diseases. This study investigated the diagnostic yield of whole-exome sequencing (WES) in patients with sporadic syndromic CHD and the phenotypic factors affecting the genetic diagnostic rate. Methods: Sixty-four patients with sporadic syndromic CHD aged <18 years underwent WES between May 2018 and December 2020 in a single tertiary center, and the association between genetic testing data and extracardiac phenotypes was analyzed. Results: Extracardiac phenotypes were measured as 3.66 ± 3.05 (standard deviation, interquartile range: 2–5) items per patient. WES detected diagnostic variants in 19 (29.7%) patients: seven (36.8%), seven (36.8%), and five (26.3%) with pathogenic variants, likely pathogenic variants, and variants of unknown significance, respectively. Post-diagnosis surveillance identified the extracardiac phenotype in 54.5% (6/11) of patients. De novo variants accounted for 76.2% (15/19) of variants and autosomal dominant inheritance for 94.7% (18/19). Most diseases were ultra-rare. No significant differences were noted in cardiac and extracardiac phenotypes, single or combined (all P > 0.05), between the groups with and without a diagnostic variant. However, patients with ≥3 extracardiac phenotypes had a significantly higher likelihood of having a diagnostic variant than those with ≤2 (38.3% vs. 5.9%, odds ratio = 9.93, 95% confidence interval = 1.21–81.44, P = 0.013). Conclusions: The number of extracardiac phenotypes is important in predicting the possibility of genetic diagnosis. Physicians will be able to select patients with a high probability of genetic diagnosis and provide appropriate genetic counseling based on the results of this study.     

Genetic variation in the first-pass metabolism of ethinylestradiol,sex hormone binding globulin levels and venous thrombosis risk

BackgroundUse of ethinylestradiol, one of the active ingredients in combined oral contraceptives, affects the incidence of venous thrombosis. To explain why some women develop thrombosis when using oral contraceptives and others do not, we hypothesized a role for the first-pass metabolism of ethinylestradiol in the liver. We set out to determine the association between genetic variation in the first-pass metabolism of ethinylestradiol, venous thrombosis risk and the effect on Sex-hormone-binding-globulin (SHBG) levels.MethodsPremenopausal women were included from two case-control studies: LETS (103 cases; 159 controls) and MEGA (397 cases; 796 controls). Haplotype-tagging SNPs were selected in 11 candidate genes; COMT, CYP1A2, CYP2C9, CYP3A4, CYP3A5, SULT1A1, SULT1E1, UGT1A1, UGT1A3, UGT1A9, UGT2B7. Venous thrombosis risk was expressed as odds ratios (OR) with 95% confidence intervals (CI). For SHBG levels, mean differences with 95%CI were estimated in combined oral contraceptive-using control subjects from the MEGA study.ResultsTwo copies of haplotype D in the UGT2B7 gene increased venous thrombosis risk (ORLETS: 3.78; OR_MEGA: 2.61) as well as SHBG levels (mean difference 27.6 nmol/L, 95%CI: − 61.7 to 116.9 compared with no copies) in oral contraceptive users and not in non-users. In oral contraceptive users, haplotype A and B in the CYP3A4 gene were associated with venous thrombosis risk, but not in non-users; however, the effect on SHBG levels was not directional with the risk. None of the other haplotypes were associated with venous thrombosis.ConclusionGenetic variation in the UGT2B7 gene may, in part, explain venous thrombosis risk in combined oral contraceptive users.     

Variability of exhaled breath condensate leukotriene B4 and 8-isoprostane in COPD patients

The reproducibility of exhaled breath condensate (EBC) mediators is not well documented in chronic obstructive pulmonary disease (COPD). This study assessed within assay (WA), within (WD) and between day (BD) reproducibility of EBC leukotriene B₄ (LTB₄) and 8-isoprostane. Three EBC samples were collected from 24 COPD patients separated by 1 h and 1 wk, to assess WD and BD reproducibility. WA reproducibility was assessed by sample analysis by enzyme immunoassay in triplicate. WA coefficient of variation for LTB₄ and 8-isoprostane (18.2% and 29.2%, respectively) was lower than corresponding values for WD (47.7% and 65.3%, respectively) and BD (75.7% and 79.1%, respectively). Repeatability coefficient for 8-isoprostane and LTB₄ assays were 18.6 pg/ml and 13.2 pg/ml, respectively. Group mean differences for WD and BD were small and statistically nonsignificant. Using the Bland Altman method, there were wide limits of agreement for WD (−51.6 to 47.2 for 8-isoprostane and −31.8 to 31.4 for LTB₄) and BD reproducibility (−61.4 to 75.7 for 8-isoprostane and −29.3 to 38.6 for LTB₄). This is the first study to fully report the variability of EBC 8-isoprostane and LTB₄ in COPD. WA variability and group mean changes were small. However, we observed considerable WD and BD variability for these biomarkers.     

Inhibitory effects of theophylline,terbutaline, and hydrocortisone on leukotriene B4 and C4 generation by human leukocytes in vitro

Leukotriene B₄ (LTB₄) and leukotriene C₄ (LTC₄) are considered to be important mediators in the pathophysiology of asthma. Theophylline, terbutaline, and hydrocortisone are drugs commonly used in the treatment of asthma. In the present study we have investigated the in vitro inhibitory effects of theophylline, terbutaline, and hydrocortisone on LTB₄ and LTC₄ generation from human leukocytes. After preincubation in the presence of these drugs, the cells were stimulated with the calcium ionophore A 23187 and the supernatants were analyzed for their LTB, and LTC, content using reverse-phase high-performance liquid chromatography (HPLC). Total leukotriene (LT) production (the combined amounts of LTB₄ and LTC₄) was dose-dependently inhibited by pretreatment with theophylline, terbutaline or hydrocortisone. Therapeutic levels of hydrocortisone (5 × 10^?6 M) plus theophylline (5 × 10^?5 M) inhibited LTB₄ and LTC₄ production in an additive way, as did the combination of hydrocortisone plus terbutaline (5 × 10^?8 M). A statistically significant effect of diminished LTB, generation was obtained after preincubation with therapeutic levels of theophylline plus terbutaline, but no such effect was seen for LTC₄ levels. The in vitro inhibitory effects on LTB₄ and LTC₄ generation from human leukocytes by theophylline, terbutaline, and hydrocortisone, as well as the additive effect of hydrocortisone plus theophylline or terbutaline, add to our understanding of the therapeutic effects of these drugs in the treatment of bronchopulmonary obstruction. Pediatr Pulmonol. 1994;18:129–134. © 1994 Wiley-Liss, Inc.     

Higher genetic susceptibility to inflammation in mild disease activity of systemic lupus erythematosus

In order to test the hypothesis that stratification of Mexican Modification of the Systemic Lupus Erythematosus Disease Activity Index (MEX-SLEDAI) simplifies the genetic study of SLE, we evaluated the genetic susceptibility to inflammation and defects in clearance of immune complexes among SLE patients in Taiwan. SLE phenotypes were stratified according to the MEX-SLEDAI scores into two subgroups (≤10 and >10), and then according to renal disorder and neurological disorder, aiming to minimize any loss of power associated with disease heterogeneity. Upon stratification, IL1-β polymorphism and LTA were significantly associated with SLE within the MEX-SLEDAI ≤10 subgroup. When SLE patients were classified into two subgroups with or without renal disorder to stratify the genetic study, we could find that the stratification with renal disorder could partially confirm the hypothesis that stratification of MEX-SLEDAI score simplifies the genetic study of complex diseases such as SLE. So we concluded that in the mild disease state of SLE, stratification of disease phenotypes, especially IL1-β and LTA, according to MEX-SLEDAI scores could reveal new associations between candidate genes and disease activity index of SLE. Li-Jen Tsai and Sheng-Hsiung Hsiao have contributed equally to this study.     

Radioimmunoassay for leukotriene B4

A rabbit immunized with leukotriene B₄ [LTB₄; (5S,12R)-6, 14-cis-8, 10-trans-icosatetraenoic acid] coupled to bovine serum albumin via the 12-oxy function of the lipid produced antibodies having an average association constant (K_a) for [14,15-³H]LTB₄ of 3.2 × 10⁹ M^-1 at 37°C and in a concentration of 0.37 μg/ml of the immune plasma. When 10 μl of anti-LTB₄ and 3.9 nCi of [14,15-³H]LTB₄ (28 Ci/mmol; 1 Ci = 3.7 × 10¹⁰ becquerels) were incubated in a volume of 250 μl, 50% inhibition of radioligand binding was achieved with 0.31 ng of LTB₄ and with 1.95 ng of (5S,12S)-6-trans-8-cis-LTB₄. The sulfidopeptide leukotrienes, LTC₄ and LTD₄, displaced the radioligand from this antibody with less than 1/100th the activity of LTB₄, and the diastereoisomers of 6-trans-LTB₄, 5-L-hydroxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HETE), and three prostaglandins were minimally effective. The specificity of this radioimmunoassay was further shown by assessment of the immunoreactive products generated from calcium ionophore (A23187)-activated rat serosal mast cells and human neutrophils after reversed-phase HPLC. Resolution of the supernatants from each cell type yielded a single immunoreactive peak that coeluted with synthetic LTB₄ and quantitatively correlated with the physical measurement by integrated A₂₆₉ in that peak; UV-absorbing peaks eluting at other retention times were not immunoreactive. The immunoreactive LTB₄ generated averaged 4.6 ng per 10⁶ rat mast cells and resolution of the supernatants by reversed-phase HPLC without a prior extraction step gave a recovery of 54%, validating the direct applicability of this sensitive and specific assay for LTB₄, a highly potent chemotactic factor, to unfractionated biologic fluids.