Vanadium promotes hydroxyl radical formation by activated human neutrophils

This study was undertaken to investigate the effects of vanadium in the +2, +3, +4, and +5 valence states on superoxide generation, myeloperoxidase (MPO) activity, and hydroxyl radical formation by activated human neutrophils in vitro, using lucigenin-enhanced chemiluminescence (LECL), autoiodination, and electron spin resonance with 5,5-dimethyl-l-pyrroline N-oxide as the spin trap, respectively. At concentrations of up to 25 microM, vanadium, in the four different valence states used, did not affect the LECL responses of neutrophils activated with either the chemoattractant, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), or the phorbol ester, phorbol 12-myristate 12-acetate (25 ng/ml). However, exposure to vanadium in the +2, +3, and +4, but not the +5, valence states was accompanied by significant augmentation of hydroxyl radical formation by activated neutrophils and attenuation of MPO-mediated iodination. With respect to hydroxyl radical formation, similar effects were observed using cell-free systems containing either hydrogen peroxide (100 microM) or xanthine/xanthine oxidase together with vanadium (+2, +3, +4), while the activity of purified MPO was inhibited by the metal in these valence states. These results demonstrate that vanadium in the +2, +3, and +4 valence states interacts prooxidatively with human neutrophils, competing effectively with MPO for hydrogen peroxide to promote formation of the highly toxic hydroxyl radical.     

Photo-ionization spectroscopy and mass spectrometry of some molecular and supramolecular asymmetric systems in the isolated state

Asymmetric molecular and supramolecular systems are characterized by: i. the circular dicroism in the angular distribution of valence photoelectrons emitted from randomly oriented chiral molecules by their interaction with circularly polarized VUV light; ii. the different stability and reactivity of diastereomeric aggregates. Both these aspects may have some relationship with the "chiral enrichment mechanism" of chirogenesis, based on the preferential destruction of one enantiomer of a racemate by interaction with a chiral agent, whether a massive species or a circularly polarized photon. The most recent spectroscopic and mass spectrometric studies on this topic are reported in the present mini-review.     

Novel imidazole-based combretastatin A-4 analogues: evaluation of their in vitro antitumor activity and molecular modeling study of their binding to the colchicine site of tubulin

The in vitro antitumor activity of novel combretastatin-like 1,5- and 1,2-diaryl-1H-imidazoles was evaluated against the NCI 60 human tumor cell lines panel. Compounds 2d and 2g proved to be more cytotoxic than CA-4 in tests involving their evaluation over a 10(-4)-10(-8) range. Docking experiments showed a good correlation between the MG_MID Log GI(50) values of all these compounds and their calculated interaction energies with the colchicine binding site of alphabeta-tubulin.     

Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains

Proteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration.     

The complementarity determining region 2 of BV8S2 (V beta 8.2) contributes to antigen recognition by rat invariant NKT cell TCR

Invariant NKT cells (iNKT cells) are characterized by a semi-invariant TCR comprising an invariant alpha-chain paired with beta-chains with limited BV gene usage which are specific for complexes of CD1d and glycolipid Ags like alpha-galactosylceramide (alpha-GalCer). iNKT cells can be visualized with alpha-GalCer-loaded CD1d tetramers, and the binding of mouse CD1d tetramers to mouse as well as to human iNKT cells suggests a high degree of conservation in recognition of glycolipid Ags between species. Surprisingly, mouse CD1d tetramers failed to stain a discrete cell population among F344/Crl rat liver lymphocytes, although comprised iNKT cells are indicated by IL-4 and IFN-gamma secretion after alpha-GalCer stimulation. The arising hypothesis that rat iNKT TCR recognizes alpha-GalCer only if presented by syngeneic CD1d was then tested with the help of newly generated rat and mouse iNKT TCR-transduced cell lines. Cells expressing mouse iNKT TCR reacted to alpha-GalCer presented by rat or mouse CD1d and efficiently bound alpha-GalCer-loaded mouse CD1d tetramers. In contrast, cells expressing rat iNKT TCR responded only to alpha-GalCer presented by syngeneic CD1d and bound mouse CD1d tetramers only poorly or not at all. Finally, CD1d-dependent alpha-GalCer reactivity and binding of mouse CD1d tetramers was tested for cells expressing iNKT TCR comprising either rat or mouse AV14 (Valpha14) alpha-chains and wild-type or mutated BV8S2 (Vbeta8.2) beta-chains. The results confirmed the need of syngeneic CD1d as restriction element for rat iNKT TCR and identified the CDR2 of BV8S2 as an essential site for ligand recognition by iNKT TCR.     

Low copy number of the FCGR3B gene and rheumatoid arthritis: a case-control study and meta-analysis

Introduction  

Low copy number (CN) of the Fc gamma receptor 3B (FCGR3B) gene has been associated with systemic autoimmune disease. This receptor for IgG is present almost exclusively on neutrophils and plays a role in their interaction with immune complexes. At present the relationship between FCGR3B and rheumatoid arthritis (RA) is unclear. The aim of the present study was to investigate whether low CN of the FCGR3B gene is associated with susceptibility to RA.     

Polymorphisms in cytochrome P450 2C19 enzyme and cessation of leflunomide in patients with rheumatoid arthritis

Introduction

Rational selection of disease modifying anti-rheumatic drugs in the treatment of rheumatoid arthritis (RA) has many potential advantages, including rapid disease control, reduced long-term disability and reduced overall cost to the healthcare system. Inter-individual genetic differences are particularly attractive as markers to predict efficacy and toxicity, as they can be determined rapidly prior to drug selection. The aims of this study, therefore, were to investigate the association between differences in genes associated with the metabolism, clearance and efficacy of leflunomide with its cessation in a group of rheumatoid arthritis patients who were treated with an intensive contemporary, treat-to-target approach.

Methods

This retrospective cohort study identified all individuals who received leflunomide and were enrolled in the Early Arthritis inception cohort at the Royal Adelaide Hospital between 2001 and July 2011. Inclusion criteria were age (>18) and a diagnosis of rheumatoid arthritis. Patients were excluded if a DNA sample was not available, if they withdrew from the cohort or if clinical data were insufficient. Subjects were followed for 12 months or until either another disease modifying antirheumatic drug was added or leflunomide was ceased. The following single nucleotide polymorphisms (SNPs) were determined: CYP2C19*2 (rs4244285), CYP2C19*17 (rs12248560), ABCG2 421C>A (rs2231142), CYP1A2*1F (rs762551) and DHODH 19C>A (rs3213422). The effects of variables on cessation were assessed with Cox Proportional Hazard models.

Results

Thirty-three of 78 (42.3%) patients ceased leflunomide due to side effects. A linear trend between cytochrome P450 2C19 (CYP2C19) phenotype and leflunomide cessation was observed, with poor and intermediate metabolizers ceasing more frequently (adjusted Hazard Ratio = 0.432 for each incremental change in phenotype, 95% CI 0.237 to 0.790, P = 0.006). Previously observed associations between cytochrome P450 1A2 (CYP1A2) and dihydro-orotate dehydrogenase (DHODH) genotype and toxicity were not apparent, but there was a trend for ATP-binding cassette sub-family G member 2 (ABCG2) genotype to be associated with cessation due to diarrhea.

Conclusions

CYP2C19 phenotype was associated with cessation due to toxicity, and since CYP2C19 intermediate and poor metabolizers have lower teriflunomide concentrations, it is likely that they have a particularly poor risk:benefit ratio when using this drug.     

N-terminal pro-brain natriuretic peptide in a novel screening algorithm for pulmonary arterial hypertension in systemic sclerosis: a case-control study

Introduction

Pulmonary arterial hypertension is a major cause of mortality in systemic sclerosis. N-terminal pro-brain natriuretic peptide (NT-proBNP) has emerged as a candidate biomarker that may enable the early detection of systemic sclerosis-related pulmonary arterial hypertension (SSc-PAH). The objective of our study was to incorporate NT-proBNP into a screening algorithm for SSc-PAH that could potentially replace transthoracic echocardiography (TTE) as a more convenient and less costly "first tier" test.

Methods

NT-proBNP levels were measured in patients from four clinical groups: a group with right heart catheter (RHC)-diagnosed SSc-PAH before commencement of therapy for PAH; a group at high risk of SSc-PAH based on TTE; a group with interstitial lung disease; and systemic sclerosis (SSc) controls with no cardiopulmonary complications. NT-proBNP levels were compared by using ANOVA and correlated with other clinical variables by using simple and multiple linear regression. ROC curve analyses were performed to determine the optimal cut point for NT-proBNP and other clinical variables in prediction of PAH.

Results

NT-proBNP was highest in the PAH group compared with other groups (P < 0.0001), and higher in the risk group compared with controls (P < 0.0001). NT-proBNP was positively correlated with systolic pulmonary artery pressure (PAP) on TTE (P < 0.0001), and mean PAP (P = 0.013), pulmonary vascular resistance (P = 0.005), and mean right atrial pressure (P = 0.006) on RHC. A composite model wherein patients screened positive if NT-proBNP was ≥ 209.8 pg/ml, and/or DLCO_corr was < 70.3% with FVC/DLCO_corr ≥ 1.82, had a sensitivity of 100% and specificity of 77.8% for SSc-PAH.

Conclusion

We have proposed a screening algorithm for SSc-PAH, incorporating NT-proBNP level and PFTs. This model has high sensitivity and specificity for SSc-PAH and, if positive, should lead to TTE and confirmatory testing for PAH. This screening algorithm must be validated prospectively.