Antibodies against domain I of β2-glycoprotein I: the one and only?

The antiphospholipid syndrome (APS) is diagnosed by the occurrence of thrombosis and/or specific pregnancy morbidity. However, the diagnosis of APS is not easy and is hampered by several problems including high prevalence of clinical symptoms and high variability between different assays resulting in a high false-positive rate. Currently APS can be diagnosed for example by detecting anti-β2-glycoprotein I antibodies by ELISA. It has been reported that β2-glycoprotein I (β2GPI) changes its conformation from a native to an active form and thereby it opens up enabling antibodies to bind a specific epitope. We amongst others have shown that epitope glycine40-arginine43 of domain I of β2GPI is predominantly responsible for binding thrombosis related antibodies. Antibodies with affinity towards other epitopes have not been associated with thrombosis. Despite these results the question remains whether these domain I antibodies are the only antibodies of importance for the detection of APS.     

The significance of autoantibodies against β2-glycoprotein I

The antiphospholipid syndrome (APS) is defined by the persistent presence of antiphospholipid antibodies in patients with a history of thrombosis and/or pregnancy morbidity, including fetal loss. APS is an autoimmune disease with a confusing name because the pathologic auto-antibodies are shown to be directed against the plasma protein β(2)-glycoprotein I and not against phospholipids. In fact, auto-antibodies that recognize phospholipids themselves are not associated with thrombosis but with infectious diseases. One of the intriguing questions is why autoantibodies against β(2)-glycoprotein I are so commonly found in both patients and the healthy. Several potential mechanisms have been suggested to explain the increased thrombotic risk in patients with these autoantibodies. In this overview, we will summarize our knowledge on the etiology of the autoantibodies, and we will discuss the evidence that identify autoantibodies against β(2)-glycoprotein I as the culprit of APS.     

Mutants of β2-glycoprotein I: Their features and potent applications

β2-Glycoprotein I (β2GPI) is a highly-glycosylated plasma protein composed of five homologous domains which regulates coagulation, fibrinolysis, and/or angiogenesis by interacting to negatively charged hydrophobic molecules and/or with plasminogen and its metabolites. The present study focused on structural and functional characterization of β2GPI's domain I (DI) and V (DV). Through N-terminal amino acid sequencing, a novel plasmin-cleaved site at K²⁸⁷C²⁸⁸ was identified in DV. We further modified the intact DV by altering two amino acids at specific proteolytic cleavage sites to generate three stable DV mutants: DV(PP), (PE), and (AA). Results of both SDS-PAGE and MALDI-TOF-MS showed that all three DV mutants were more stable than the intact DV, and DV(PE) was predominantly resistant to proteolysis. Competitive ELISA assessed affinities of intact β2GPI and those mutants to cardiolipin. In culture system, all DV and DI mutants potently inhibited HUVEC's proliferation by 18–30% as compared to control. Only DI and nicked β2GPI showed significant inhibition in HUVEC's tube formation. Moreover, DV(PE)-coated affinity columns demonstrated its binding property towards anionic lipids and could substantially isolate anionic DOPS from zwitterionic DOPC as a purification model. In summary, the proteolytic resistant and unhindered phospholipid (PL) binding properties of DV(PE) have made it an appealing element for subsequent prospective studies. Future in-depth characterization and optimized applications of cleavage-resistant DV(PE) would complement its full capacity as a novel clinical modality in the field of vascular imaging and/or lipidomics studies.     

Single-molecule spectroscopy of the β2 adrenergic receptor: Observation of conformational substates in a membrane protein

Single-molecule studies of the conformations of the intact beta(2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta(2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.     

Altered Helical Structure of a Homotrimer of α1(I)Chains Synthesized by Fibroblasts from a Variant of Osteogenesis Imperfecta

Cultured skin fibroblasts from a variant of osteogenesis imperfecta were previously shown to synthesize a type I procollagen which was a homotrimer of proα1(I) chains. Trimers of α1(I) collagen were isolated by pepsin digestion of culture medium from these fibroblasts. The amino acid composition of the isolated protein indicated that it contained an increased amount of hydroxylysine, apparently because of post-translational over-modification. The thermal stability of the α1(I) trimers was examined by circular dichroism. We found no consistent difference in the melting curve of the α1(I) trimers compared to control type I collagen. We next examined the thermal stability of the α1(I) trimers using digestion with a combination of trypsin and α-chymotrypsin as an alternative probe of helical stability. When enzymatic digestions were carried out at 36° to 40°C, the α1(I) chains in the trimers were cleaved to polypeptides which were shortened by approximately 100 amino acids. Vertebrate collagenase digestion of the shortened molecules indicated that the 100 amino acid segment removed from each α1(I) chain was located at the carboxyl-terminus. The decreased thermal stability of the α1(I) trimers was probably explained by the absence of α2(I) chains in the molecules. The results, however, did not exclude the possibility that the post-translational over-modification of the α1(I) chains contributed to the altered helical structure.     

From the Cover: DIM (3,3′-diindolylmethane) confers protection against ionizing radiation by a unique mechanism

DIM (3,3′-diindolylmethane), a small molecule compound, is a proposed cancer preventive agent that can be safely administered to humans in repeated doses. We report that administration of DIM in a multidose schedule protected rodents against lethal doses of total body irradiation up to 13 Gy, whether DIM dosing was initiated before or up to 24 h after radiation. Physiologic submicromolar concentrations of DIM protected cultured cells against radiation by a unique mechanism: DIM caused rapid activation of ataxia-telangiectasia mutated (ATM), a nuclear kinase that regulates responses to DNA damage (DDR) and oxidative stress. Subsequently, multiple ATM substrates were phosphorylated, suggesting that DIM induces an ATM-dependent DDR-like response, and DIM enhanced radiation-induced ATM signaling and NF-κB activation. DIM also caused activation of ATM in rodent tissues. Activation of ATM by DIM may be due, in part, to inhibition of protein phosphatase 2A, an upstream regulator of ATM. In contrast, DIM did not protect human breast cancer xenograft tumors against radiation under the conditions tested. In tumors, ATM was constitutively phosphorylated and was not further stimulated by radiation and/or DIM. Our findings suggest that DIM is a potent radioprotector and mitigator that functions by stimulating an ATM-driven DDR-like response and NF-κB survival signaling.A diet rich in cruciferous vegetables (e.g., cabbage, broccoli, cauliflower) is linked to a reduced risk of several human cancers (1, 2), and dietary supplementation with indole-3-carbinol (I3C), a phytochemical from cruciferous vegetables, prevents tumors in animals (3–5). I3C is hydrolyzed to various products in the stomach, including DIM (3,3′-diindolylmethane), which is acid stable and is a major bioactive metabolite (6). I3C and DIM are proposed cancer preventive agents and each can be given safely in oral form in repeated doses to rodents and humans (7–12). In humans, oral I3C or DIM at nontoxic doses yielded peak plasma levels of 0.25–2.5 µM (9–12).The mechanism by which DIM prevents cancer is unknown. Most studies have used supraphysiological concentrations of DIM (10–30 μM) and indicate that DIM can inhibit invasion, angiogenesis, and proliferation and induce apoptosis in tumor cells by modulating signaling pathways involving AKT, NF-κB, and FOXO3 (13–17). It can also inhibit estrogen-inducible gene expression and cause an endoplasmic reticulum stress response (17–22). DIM alters estrogen metabolism by shifting metabolism from carcinogenic 16α-hydroxy to inert 2-hydroxy derivatives, and it antagonizes estrogen and androgen receptor activity (17, 20–24).Low concentrations of DIM that can be achieved safely in humans (≤1 μM) protect cells against oxidative stress (25). Protection required the tumor suppressor BRCA1 and, in particular, its ATM (S1387 and S1524). ATM is activated via autophosphorylation in response to DNA double-strand breaks (DSBs), and phospho-ATM then phosphorylates multiple substrates involved in the DNA damage response (DDR), resulting in activation of DNA repair mechanisms, cell cycle checkpoints, antioxidant pathways, and survival pathways (e.g., NF-κB signaling) (26, 27).Here, we describe an activity for DIM as a radioprotector and mitigator; and we establish a unique mechanism, i.e., stimulation of ATM signaling without causing DNA damage.     

RGS2 is a primary terminator of β₂-adrenergic receptor-mediated G(i) signaling

Two major β-adrenergic receptor (βAR) subtypes, β₁AR and β₂AR, are expressed in mammalian heart with β₁AR coupling to G_s and β₂AR dually coupling to G_s and G_i proteins. In many types of chronic heart failure, myocardial contractile response to both β₁AR and β₂AR stimulation is severely impaired. The dysfunction of βAR signaling in failing hearts is largely attributable to an increase in G_i signaling, because disruption of the G_i signaling restores myocardial contractile response to β₁AR as well as β₂AR stimulation. However, the mechanism terminating the β₂AR-G_i signaling remains elusive, while it has been shown activation of the G_i signaling is dependent on agonist stimulation and subsequent PKA-mediated phosphorylation of the receptor. Here we demonstrate that regulator of G protein signaling 2 (RGS2) is a primary terminator of the β₂AR-G_i signaling. Specifically, prolonged absence of agonist stimulation for 24 h impairs the β₂AR-G_i signaling, resulting in enhanced β₂AR- but not β₁AR-mediated contractile response in cultured adult mouse cardiomyocytes. Increased β₂AR contractile response is accompanied by a selective upregulation of RGS2 in the absence of alterations in other major cardiac RGS proteins (RGS3–5) or G_s, G_i or βAR subtypes. Administration of a βAR agonist, isoproterenol (ISO, 1.0 nM), prevents RGS2 upregulation and restores the β₂AR-G_i signaling in cultured cells. Furthermore, RGS2 ablation, similar to βAR agonist stimulation, sustains the β₂AR-G_i signaling in cultured cells, whereas adenoviral overexpression of RGS2 suppresses agonist-activated β₂AR-G_i signaling in cardiomyocytes and HEK293 cells. These findings not only define RGS2 as a novel negative regulator of the β₂AR-G_i signaling but also provide a potential novel target for the treatment of chronic heart failure.     

Separation of Human proα1(I) and proα2(I) Procollagen Chains by Reverse Phase High Performance Liquid Chromatography

Human pro α1(I) and pro α2(I) procollagen chains have been separated on a large por (33 nm) reverse phase high performance liquid chromatography C₁₈ column using an acetonitrile aqueous gradient containing 9 mM trifluoroacetic acid. The separation of the chains was evaluated by gel electrophoresis. The yield of the chromatography, based on radiolabel recovery, is equal to or better than 80%. Absorbance monitoring at 210 nm permits detection of less than 5 µg procollagen.     

Differential Regulation by Glucocorticoids of Mouse Proα2(I) and Proα1(III) DNA-Binding Proteins

Multiple procollagen-specific DNA-binding proteins were identified for the α2(I) and the αl(III) procollagen promotor containing gene fragments. The proteins are genespecific, differing in their relative molecular weights and relative binding specificities. A major finding was the increased DNA-binding with specificity for the procollagen promoter containing DNAs by several nonhistone nuclear proteins in mouse embryo fibroblasts treated with dexamethasone. The previously reported coordinate decrease of type I and type III procollagens by glucocorticoids may involve differential regulation by glucocorticoids of procollagen gene specific DNA-binding proteins.     

Age-related changes of Nrf2 and phosphorylated GSK-3β in a mouse model of accelerated aging (SAMP8)

SAMP8 mice show spontaneously accelerated aging and a short life span with systemic accumulation of oxidative stress. Nrf2 translocates into the nucleus upon oxidative stress and induces the expression of detoxifying and antioxidant enzymes. Recently, several studies reported that Nrf2 is associated with aging and various diseases. In the present study, we investigated the levels of Nrf2 nuclear translocation and phosphorylation of Akt and GSK-3β in livers of SAMP8 and normal aging SAMR1 mice. The protein level of Nrf2 in the nucleus of the liver was significantly decreased in SAMP8 at 10 months old compared with that in age-matched SAMR1. The protein level of Keap1, which anchors Nrf2 in the cytoplasm, did not differ between SAMP8 and SAMR1. In addition, the mRNA expression of Nrf2 in the liver of SAMP8 was significantly lower than that of SAMR1. Moreover, mRNA levels of detoxification and antioxidant enzymes, GSTa1 and NQO1, were significantly decreased in SAMP8 compared with SAMR1. These results indicate that a higher level of oxidative stress in SAMP8 might be caused by a lower level of Nrf2. Furthermore, the phosphorylation of Akt and GSK-3β was significantly decreased in the liver of SAMP8 at 10 months old. Recent studies have suggested that the Akt/GSK-3β signaling pathway is involved in the nuclear translocation of Nrf2. Therefore, it is suggested that the reduction of the translocation of Nrf2 into the nucleus might be induced by a decrease of GSK-3β phosphorylation, resulting in an increase of oxidative stress in SAMP8 mice.     

Preclinical efficacy of THRX-200495, a dual pharmacology muscarinic receptor antagonist and β(2)-adrenoceptor agonist (MABA)

Combinations of a muscarinic receptor antagonist (MA) and a β₂-adrenoceptor agonist (BA) improve bronchodilation in COPD patients to a greater extent than drugs with either mechanism alone. Here, using an in vivo model of bronchoprotection in guinea pigs, we characterize a single agent with dual-acting MA and BA activity, THRX-200495 (MABA). THRX-200495 was compared to a fixed-dose combination of a short-acting muscarinic receptor antagonist (SAMA) and a β₂-adrenoceptor agonist (SABA). The SAMA/SABA combination consisted of a 1:5.7 ratio of ipratropium and albuterol (the components of Combivent®). Conscious guinea pigs received aqueous nebulized solutions of vehicle or test compound by aerosol exposure. Bronchoprotective potency was estimated in anesthetized, tracheotomized and ventilated guinea pigs at predetermined time points after aerosol exposure by measuring changes in ventilation pressure. The individual (MA, BA) and composite (MABA) pharmacologies were assessed by determining protection against bronchoconstrictor responses induced by methacholine in the presence of propranolol (for MA activity), histamine (for BA activity) or methacholine (MABA activity). Bronchoprotection was calculated as percent inhibition of methacholine or histamine response relative to the vehicle group. THRX-200495 exhibited matched MA (ID₅₀ = 11.4 μg/mL) and BA (ID₅₀ = 11.2 μg/mL) potency and potent dual pharmacology (MABA ID₅₀ = 3.5 μg/mL) that persisted for over 24 h. The combination of ipratropium/albuterol exhibited bronchoprotective activity that was 2.6-fold more potent as a BA (ID₅₀ = 5.7 μg/mL) than as an MA (ID₅₀ = 14.6 μg/mL) at 0.5 h post-dose and 37-fold more potent as an MA (ID₅₀ = 4.3 μg/mL) than a BA (ID₅₀ = 159 μg/mL) at 1.5 h post aerosol exposure. Under MABA pharmacological conditions, ipratropium/albuterol produced potent bronchoprotective activity (ID₅₀ = 2.0/11.4 μg/mL) and an apparent additive effect of the two pharmacologies. In conclusion, a dual-acting prototypical MABA, THRX-200495, demonstrated potent, balanced and long-lasting bronchodilation in a guinea pig model of bronchoprotection that was greater than either the MA or BA mechanisms alone.     

24-hour bronchodilation following a single dose of the novel β(2)-agonist olodaterol in COPD

Background

Current guidelines recommend long-acting bronchodilators as maintenance therapy in COPD when symptoms are not adequately controlled with short-acting agents. Olodaterol is a novel long-acting β₂-adrenoceptor agonist with a pre-clinical profile that suggests 24-h bronchodilation may be achieved with once-daily administration.

Objective

To assess dose- and time-response in terms of bronchodilator efficacy, and to evaluate pharmacokinetics, safety and tolerability of single doses of olodaterol administered via Respimat^® Soft Mist™ Inhaler in COPD patients.

Methods

A single-center, double-blind, placebo-controlled, 5-way crossover study including 24-h spirometry (FEV₁, FVC), safety, tolerability and pharmacokinetics (in a subset of patients) following dosing of olodaterol 2 μg, 5 μg, 10 μg and 20 μg; the washout period between test-days was at least 14 days. Primary endpoint of the study was the 24-h post-dosing FEV₁. Patients participating in the pharmacokinetic assessments continued in an open-label extension phase to establish pharmacokinetics of olodaterol 40 μg.

Results

36 patients were assigned to treatment; mean baseline prebronchodilator FEV₁ was 1.01 L (37% predicted normal). All doses of olodaterol provided significantly greater bronchodilation compared to placebo in 24-h FEV₁ post-dose (p < 0.001); a clear dose–response relationship was observed, with values ranging from 0.070 L for olodaterol 2 μg to 0.119 L for olodaterol 20 μg. Similarly, olodaterol was superior to placebo (p < 0.001) in peak FEV₁ (0.121 L to 0.213 L) and average FEV₁ both during the daytime (0–12 h; ranging from 0.099 L to 0.184 L) and night-time (12–24 h; ranging from 0.074 L to 0.141 L). FVC results were consistent with those observed for FEV₁. Pharmacokinetic evaluation of the peak plasma concentrations and renal excretion suggested no obvious deviation from dose-proportionality over the investigated dose range of 2 μg–40 μg; in most patients, no plasma levels could be detected following the 2 μg dose. All treatments were well tolerated with no apparent dose relation in terms of adverse events.

Conclusions

Olodaterol appears to be a promising long-acting β₂-adrenoceptor agonist,with bronchodilation maintained over 24 h that offers an opportunity for once-daily dosing in patients who require maintenance bronchodilator therapy for the management of COPD symptoms.     

Influence of a nucleotide oligomerization domain 1 (NOD1) polymorphism and NOD2 mutant alleles on Crohn's disease phenotype

AIM: To examine genetic variation of nucleotide oligomerization domain 1 (NOD1 ) and NOD2 ,their respective influences on Crohn's disease phenotype and gene-gene interactions. METHODS: (ND1 326561 ) NOD1 polymorphism and SNP8,SNP12 and SNP13 of NOD2 were analyzed in 97 patients and 50 controls. NOD2 variants were determined by reaction restriction fragment length polymorphism analysis. NOD1 genotyping and NOD2 variant confirmation were performed by specific amplification and sequencing. RESULTS: The distribution of NOD1 polymorphism in patients was different from controls (P = 0.045) and not altered by existence of NOD2 mutations. In this cohort,30.92% patients and 6% controls carried at least one NOD2 variant (P < 0.001) with R702W being the most frequent variant. Presence of at least one NOD2 mutation was inversely associated with colon involvement (9.09% with colon vs 36.4% with ileal or ileocolonic involvement,P = 0.04) and indicative of risk of penetrating disease (52.63% with penetrating vs 25.64% with non-penetrating or stricturing behavior,P = 0.02). L1007finsC and double NOD2 mutation conferred the highest risk for severity of disease (26.3% with penetrating disease vs 3.8% with non-penetrating or stricturing behavior presented L1007finsC,P = 0.01 and 21.0% with penetrating disease vs 2.5% with non-penentrating or stricturing behavior carried double NOD2 mutation,P = 0.007). Exclusion of patients with NOD2 mutations from phenotype/NOD1 -genotype analysis revealed higher prevalence of 11 genotype in groups of younger age at onset and colonic location. CONCLUSION: This study suggests population differences in the inheritance of risk NOD1 polymorphism and NOD2 mutations. Although no interaction between NOD1 -NOD2 was noticed,a relationship between disease location and Nod-like receptor molecules was established.