Chronic obstructive pulmonary disease (COPD) is characterized by the degradation of elastin, the major insoluble protein of lung tissues. The degradation of elastin gives rise to desmosine (DES) and ...isodesmosine (IDES), two major urinary products typified by a hydrophilic pyridinium-based cross-linker structure. A high sensitivity method based on nanoflow liquid chromatography tandem mass spectrometry with multiple reaction monitoring was developed for the analysis of urinary DES and IDES. The analytes were derivatized with propionic anhydride and deuterated DES (D4-DES) was used as an internal standard. This method enables the quantification of DES and IDES in as little as 50 μL of urine and provides a detection limit of 0.10 ng/mL (0.95 fmol on-column). We report the analysis of DES and IDES in a cohort of 40 urine specimens from four groups of individuals: (a) COPD rapid decliners (11.8 ± 3.7 ng/mg creatine (crea)), (b) COPD slow decliners (16.0 ± 3.1 ng/mg crea), (c) healthy smokers (13.2 ± 1.9 ng/mg crea), and (d) healthy nonsmokers (14.9 ± 2.9 ng/mg crea). Our analysis reveals a statistically significant decrease in the level of urinary DES and IDES in COPD rapid decliner patients compared to healthy nonsmoker controls and COPD slow decliner patients. This methodology may be useful for monitoring DES and IDES levels in well controlled animal models for COPD or for longitudinal studies in COPD patients.
In Huntington disease, polyglutamine expansion of the protein huntingtin (Htt) leads to selective neurodegenerative loss of medium spiny neurons throughout the striatum by an unknown apoptotic ...mechanism. Binding of Hip-1, a protein normally associated with Htt, is reduced by polyglutamine expansion. Free Hip-1 binds to a hitherto unknown polypeptide, Hippi (Hip-1 protein interactor), which has partial sequence homology to Hip-1 and similar tissue and subcellular distribution. The availability of free Hip-1 is modulated by polyglutamine length within Htt, with disease-associated polyglutamine expansion favouring the formation of pro-apoptotic Hippi-Hip-1 heterodimers. This heterodimer can recruit procaspase-8 into a complex of Hippi, Hip-1 and procaspase-8, and launch apoptosis through components of the 'extrinsic' cell-death pathway. We propose that Htt polyglutamine expansion liberates Hip-1 so that it can form a caspase-8 recruitment complex with Hippi. This novel non-receptor-mediated pathway for activating caspase-8 might contribute to neuronal death in Huntington disease.
Background: PGD2 is the major prostanoid released by mast cells during an allergic response. Its role in the allergic response, however, remains unclear. Objective: Because the accumulation of ...eosinophils is a feature of allergic reactions, we investigated the role of PGD2 in the modulation of eosinophil function. Methods: Circulating human eosinophils were isolated and challenged with PGD2. The effects of PGD2 on various eosinophil functions were then analyzed. Results: PGD2 binds with high affinity preferentially to 2 receptors, DP and chemoattractant receptor-homologous molecule expressed on TH2 cells (CRTH2). We show that both DP and CRTH2 are detectable on circulating eosinophils. We demonstrate that PGD2 (1-10 nmol/L) induces a rapid change in human eosinophil morphology and an increase in chemokinesis and promotes eosinophil degranulation. These effects are induced by the CRTH2-selective agonist 13-14-dihydro-15-keto-PGD2 (DK-PGD2) but not by the DP-selective agonist BW245C. These results suggest a role for CRTH2 in the modulation of eosinophil movement and in triggering the release of cytotoxic proteins. Finally, we demonstrate that BW245C, but not DK-PGD2, can delay the onset of apoptosis in cultured eosinophils, presumably through interaction with DP. Conclusion: These data support the hypothesis that PGD2 controls eosinophil functions through 2 pharmacologically distinct receptors with independent functions. Blockade of PGD2-mediated effects on human eosinophils may reduce the damage caused by these cells during an allergic response, but inhibition of both receptors may be required. (J Allergy Clin Immunol 2001;108:982-8.)
In this manuscript we wish to report the discovery of MK-7246 (4), a potent and selective CRTH2 (DP2) antagonist. SAR studies leading to MK-7246 along with two synthetic sequences enabling the ...preparation of this novel class of CRTH2 antagonist are reported. Finally, the pharmacokinetic and metabolic profile of MK-7246 is disclosed.
The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor that has been reported to modulate inflammatory responses in various rodent ...models of asthma, allergic rhinitis and atopic dermatitis. In this study, we describe the biological and pharmacological properties of {(7R)-7-(4-fluorophenyl)sulfonyl(methyl)amino-6,7,8,9-tetrahydropyrido1,2-aindol-10-yl}acetic acid (MK-7246), a novel synthetic CRTH2 antagonist. We show that MK-7246 1) has high affinity for the human, monkey, dog, rat, and mouse CRTH2, 2) interacts with CRTH2 in a reversible manner, 3) exhibits high selectivity over all prostanoid receptors as well as 157 other receptors and enzymes, 4) acts as a full antagonist on recombinant and endogenously expressed CRTH2, 5) demonstrates good oral bioavailability and metabolic stability in various animal species, 6) yields ex vivo blockade of CRTH2 on eosinophils in monkeys and sheep, and 7) significantly blocks antigen-induced late-phase bronchoconstriction and airway hyper-responsiveness in sheep. MK-7246 represents a potent and selective tool to further investigate the in vivo function of CRTH2.
The recombinant human prostaglandin D
2
(PGD
2
) receptor, hCRTH2, has been expressed in HEK293(EBNA) and characterized with respect to radioligand binding and signal transduction properties. High ...and low affinity binding sites for PGD
2
were identified in the CRTH2 receptor population by saturation analysis with respective equilibrium dissociation constants (
K
D
) of 2.5 and 109 n
M
. This revealed that the affinity of PGD
2
for CRTH2 is eight times less than its affinity for the DP receptor.
Equilibrium competition binding assays revealed that of the compounds tested, only PGD
2
and several related metabolites bound with high affinity to CRTH2 (
K
i
values ranging from 2.4 to 34.0 n
M
) with the following rank order of potency: PGD
2
>13,14‐dihydro‐15‐keto PGD
2
>15‐deoxy‐Δ
12,14
‐PGJ
2
>PGJ
2
>Δ
12
‐PGJ
2
>15(S)‐15 methyl‐PGD
2
. This is in sharp contrast with the rank order of potency obtained at DP : PGD
2
>PGJ
2
>Δ
12
‐PGJ
2
>15‐deoxy‐Δ
12,14
‐PGJ
2
>>>13,14‐dihydro‐15‐keto‐PGD
2
.
Functional studies demonstrated that PGD
2
activation of recombinant CRTH2 results in decrease of intracellular cAMP in a pertussis toxin‐sensitive manner. Therefore, we showed that CRTH2 can functionally couple to the G‐protein G
αi/o
. PGD
2
and related metabolites were tested and their rank order of potency followed the results of the membrane binding assay.
By Northern blot analysis, we showed that, besides haemopoietic cells, CRTH2 is expressed in many other tissues such as brain, heart, thymus, spleen and various tissues of the digestive system. In addition,
in situ
hybridization studies revealed that CRTH2 mRNA is expressed in human eosinophils. Finally, radioligand binding studies demonstrated that two eosinophilic cell lines, butyric acid‐differentiated HL‐60 and AML 14.3D10, also endogenously express CRTH2.
British Journal of Pharmacology
(2002)
137
, 1163–1172. doi:
10.1038/sj.bjp.0704973
Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the ...allergic activity of this antigen after direct application to the lungs of Brown Norway rats.
Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats.
We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production.
Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.
The anaphylatoxin C3a is an important immune regulator with a number of distinct functions in both innate and adaptive immunity. Many of these roles have been ascribed to C3a based on studies in mice ...genetically modified to lack its precursor, C3, or its receptor, C3aR. However, other presumed functions of C3a are based on results obtained with a recently described small molecule ligand of C3aR, SB 290157. Although this compound was originally described as an antagonist and appears to act as such in some systems, it has recently been shown to have effects that cannot be explained by simple antagonism of C3aR. In the current study, SB 290157 is shown to have full agonist activity on C3aR in a variety of cell systems, including a calcium mobilization assay in transfected RBL cells, a β-lactamase assay in CHO-NFAT-
bla-Gα
16 cells and an enzyme-release assay in differentiated U-937 cells. On the other hand, the compound lacks agonist activity in guinea pig platelets, cells known to express C3aR at very low levels. SB 290157 agonism of C3aR is consistent with recent discrepant data obtained using this molecule. These results caution against attributing novel roles to C3a based on data obtained with SB 290157 and highlight a continuing need for the identification of true small molecule C3aR antagonists.
Huntington disease is a devastating neurodegenerative disease caused by the expansion of a polymorphic glutamine tract in huntingtin. The huntingtin interacting protein (HIP-1) was identified by its ...altered interaction with mutant huntingtin. However, the function of HIP-1 was not known. In this study, we identify HIP-1 as a proapoptotic protein. Overexpression of HIP-1 resulted in rapid caspase 3-dependent cell death. Bioinformatics analyses identified a novel domain in HIP-1 with homology to death effector domains (DEDs) present in proteins involved in apoptosis. Expression of the HIP-1 DED alone resulted in cell death indistinguishable from HIP-1, indicating that the DED is responsible for HIP-1 toxicity. Furthermore, substitution of a conserved hydrophobic phenylalanine residue within the HIP-1 DED at position 398 eliminated HIP-1 toxicity entirely. HIP-1 activity was found to be independent of the DED-containing caspase 8 but was significantly inhibited by the antiapoptotic protein Bcl-xL, implicating the intrinsic pathway of apoptosis in HIP-1-induced cell death. Co-expression of a normal huntingtin fragment capable of binding HIP-1 significantly reduced cell death. Our data identify HIP-1 as a novel proapoptotic mediator and suggest that HIP-1 may be a molecular accomplice in the pathogenesis of Huntington disease.
ABSTRACT
Chronic obstructive pulmonary disease (COPD) is a prevalent pulmonary disease characterized by a progressive decline in lung function. The identification of biomarkers capable of predicting ...the rate of lung function decline or capable of giving an early read on drug efficacy in clinical trials would be very useful. The aim of this study was to identify plasma biomarkers capable of accurately distinguishing patients with COPD from healthy controls. Eighty-nine plasma markers in 40 COPD patients and 20 healthy smoker controls were analyzed. The COPD patients were divided into two subgroups, rapid and slow decliners based on their rate of lung function decline measured over 15 years. Univariate analysis revealed that 25 plasma markers were statistically different between rapid decliners and controls, 4 markers were different between slow decliners and controls, and 10 markers were different between rapid and slow decliners (p < 0.05). Multivariate analysis led to the identification of groups of plasma markers capable of distinguishing rapid decliners from controls (signature 1), slow decliners from controls (signature 2) and rapid from slow decliners (signature 3) with over 90%% classification accuracy. Importantly, signature 1 was shown to be longitudinally stable using plasma samples taken a year later from a subset of patients. This study describes a novel set of plasma markers differentiating slow from rapid decline of lung function in COPD. If validated in distinct and larger cohorts, the signatures identified will have important implications in both disease diagnosis, as well as the clinical evaluation of new therapies.
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