The role of the FcGRIIIa polymorphism in modifying the association between treatment and outcome in patients with rheumatoid arthritis treated with rituximab versus TNF-α antagonist therapies.

OBJECTIVES: There is an association between the FcGRIIIa polymorphism and the development of rheumatoid arthritis (RA). Studies in non-Hodgkin lymphoma demonstrated a relationship between the FcGRIIIa polymorphism and response to anti-CD20 therapies. However, there are currently no published studies evaluating the relationship between this polymorphism and therapeutic response to treatment with anti-CD20 agents such as rituximab in RA. We conducted a study to identify if the FcGRIIIa polymorphism is associated with rituximab efficacy in patients with RA. METHODS: Subjects with RA treated with rituximab (cases, n=158) or TNF-α antagonist (controls, n=390) were recruited from the Consortium of Rheumatology Researchers of North America. The FcGRIIIa variant was genotyped for all subjects and longitudinal patient outcomes were assessed using the clinical disease activity index (CDAI). We used a linear regression random effects model to estimate CDAI scores over time with multiple time points nested within patient. RESULTS: Similar changes in CDAI were observed across the three FcGRIIIa genotypes for the rituximab treated group (VV [4.56, SD 14.5]), VF (7.44, SD 14.9) and FF (4.75, SD 10.8) (p >0.05)] and the TNF-α antagonist therapy treated group [VV (5.12, SD 14.6), VF (6.77, SD 15.9), and FF (4.36, SD 18.2) (p >0.05). Overall, there were similar changes in CDAI at 6 months for rituximab (-5.91, SD 14.1) and anti-TNFs (-5.77, SD 15.5) (p >0.05). The FcGRIIIa genotype was not significantly associated (p=0.86) with treatment response in rituximab treated RA patients compared with TNF-α antagonist therapy treated patients. Baseline CDAI and number of prior biologics were significant predictors of clinical response over time. CONCLUSIONS: Our finding emphasises the idea that determinants of response to treatment are complex and may be dependent upon genetic and phenotypic interactions. Future studies should analyse the interaction between the FcGRIIIa gene, other neighbouring polymorphisms and other phenotypic and environmental factors.

Circulating endothelial and endothelial progenitor cells in patients with severe sepsis.

Elevated circulating endothelial cell (CEC) and circulating endothelial progenitor cell (CEPC) counts may indicate vascular damage and disease status, but data on these cell populations in patients with severe sepsis are limited. This study compared CEC and CEPC counts in patients with and without severe sepsis following intensive care unit (ICU) admission. Venous blood samples were collected within 24 h, 48-72 h, and 120-144 h. Baseline demographics, 28-day mortality, ICU and hospital days, and Sequential Organ Failure Assessment (SOFA) scores were recorded. Patients with (n=18) and without (n=28) severe sepsis were balanced for mean age (63.7 and 61.3 years, respectively) and gender. There were no differences in 28-day mortality, ICU days, or hospital days. Baseline SOFA scores were higher in the sepsis group. At 48-72 h, patients with severe sepsis had significantly higher median CEC counts (51.5 vs. 28.0 cells/4 ml of blood, P=0.02). CEC values for all ICU patients were significantly (P<0.05) higher than in healthy volunteers. CEPC counts in both cohorts ranged from 0 to >21 colonies/4 ml blood (mean=1.13±2.25; median=0) without significant differences at any time point. This study demonstrates the ability to quantify CECs and CEPCs using consensus methodology. Understanding the relationship between CEC/CEPC counts and outcomes may provide insight into the mechanisms of endothelial cell changes in severe sepsis.

ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor.

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605-M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13-mediated cleavage of VWF. Recombinant human full-length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1.5 mol/l urea, and analyzed via multimer migration distance. The glutathione-S-transferase (GST) and histidine-tagged construct, E1554-R1668 of VWF (VWF115) was assayed via enzyme-linked immunosorbent assay: VWF115 was bound to anti-GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase-labelled anti-histidine tag antibody. All alterations examined in the Y1605-M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A-domain changes in VWF alter ADAMTS13-mediated proteolysis.

Treatment of sepsis-induced acquired protein C deficiency reverses Angiotensin-converting enzyme-2 inhibition and decreases pulmonary inflammatory response.

The protein C (PC) pathway plays an important role in vascular and immune function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. However, the association of acquired PC deficiency with the pathophysiology of lung injury is unclear. We hypothesized that low PC induced by sepsis would associate with increased pulmonary injury and that replacement with activated protein C (APC) would reverse the activation of pathways associated with injury. Using a cecal ligation and puncture (CLP) model of polymicrobial sepsis, we examined the role of acquired PC deficiency on acute lung injury assessed by analyzing changes in pulmonary pathology, chemokine response, inducible nitric-oxide synthase (iNOS), and the angiotensin pathway. Acquired PC deficiency was strongly associated with an increase in lung inflammation and drivers of pulmonary injury, including angiotensin (Ang) II, thymus and activation-regulated chemokine, plasminogen activator inhibitor (PAI)-1, and iNOS. In contrast, the protective factor angiotensin-converting enzyme (ACE)-2 was significantly suppressed in animals with acquired PC deficiency. The endothelial protein C receptor, required for the cytoprotective signaling of APC, was significantly increased post-CLP, suggesting a compensatory up-regulation of the signaling receptor. Treatment of septic animals with APC reduced pulmonary pathology, suppressed the macrophage inflammatory protein family chemokine response, iNOS expression, and PAI-1 activity and up-regulated ACE-2 expression with concomitant reduction in AngII peptide. These data demonstrate a clear link between acquired PC deficiency and pulmonary inflammatory response in the rat sepsis model and provide support for the concept of APC as a replacement therapy in acute lung injury associated with acquired PC deficiency.

Activated protein C decreases tumor necrosis factor related apoptosis-inducing ligand by an EPCR- independent mechanism involving Egr-1/Erk-1/2 activation.

UNLABELLED: Background- APC is an antithrombotic and antiinflammatory serine protease that plays an important role in vascular function. We report that APC can suppress the proapoptotic mediator TRAIL in human umbilical vein endothelial cells, and we have investigated the signaling mechanism. METHODS AND RESULTS: APC inhibited endothelial TRAIL expression and secretion and its induction by cell activation. To explore the mechanism, we examined factors associated with TRAIL regulation and demonstrated that APC increased the level of EGR-1, a transcriptional factor known to suppress the TRAIL promoter. APC also induced a significant increase in phosphorylation of ERK-1/2, required to activate EGR-1 expression. Activation of ERK-1/2 was dependent on the protease activated receptor-1 (PAR-1), but independent of the endothelial protein C receptor (EPCR). Using siRNA, we found that the effect of APC on the EGR-1/ERK signaling required for TRAIL inhibition was dependent on the S1P1 receptor and S1P1 kinase. CONCLUSIONS: Our data suggest that APC may provide cytoprotective activity by activating the ERK pathway, which upregulates EGR-1 thereby suppressing the expression of TRAIL. Moreover, we provide evidence that APC can induce a cell signaling response through a PAR-1/S1P1-dependent but EPCR-independent mechanism.

Negative regulation of inducible nitric-oxide synthase expression mediated through transforming growth factor-beta-dependent modulation of transcription factor TCF11.

Inducible nitric-oxide synthase (iNOS) plays a central role in the regulation of vascular function and response to injury. A central mediator controlling iNOS expression is transforming growth factor-beta (TGF-beta), which represses its expression through a mechanism that is poorly understood. We have identified a binding site in the iNOS promoter that interacts with the nuclear heterodimer TCF11/MafG using chromatin immunoprecipitation and mutation analyses. We demonstrate that binding at this site acts to repress the induction of iNOS gene expression by cytokines. We show that this repressor is induced by TGF-beta1 and by Smad6-short, which enhances TGF-beta signaling. In contrast, the up-regulation of TCF11/MafG binding could be suppressed by overexpression of the TGF-beta inhibitor Smad7, and a small interfering RNA to TCF11 blocked the suppression of iNOS by TGF-beta. The binding of TCF11/MafG to the iNOS promoter could be enhanced by phorbol 12-myristate 13-acetate and suppressed by the protein kinase C inhibitor staurosporine. Moreover, the induction of TCF11/MafG binding by TGF-beta and Smad6-short could be blocked by staurosporine, and the effect of TGF-beta was blocked by the selective protein kinase C inhibitor calphostin C. Consistent with the in vitro data, we found suppression of TCF11 coincident with iNOS up-regulation in a rat model of endotoxemia, and we observed a highly significant negative correlation between TCF11 and nitric oxide production. Furthermore, treatment with activated protein C, a serine protease effective in septic shock, blocked the down-regulation of TCF11 and suppressed endotoxin-induced iNOS. Overall, our results demonstrate a novel mechanism by which iNOS expression is regulated in the context of inflammatory activation.

Activated protein C and sepsis.

Protein C is a plasma protease that when activated plays a central role in modulating the function of the vascular endothelium and its interface with the innate immune system. A recombinant form of human activated protein C (APC), drotrecogin alfa (activated), has shown efficacy in a number of preclinical models of thrombosis and ischemia and reduces mortality in patients that have a high risk of dying from severe sepsis. Studies have begun to elucidate the mechanism for the multifunctional role of APC in modulating not only coagulation, but also inflammation and apoptotic processes. From gene profiling to pharmacology studies, drotrecogin alfa (activated) appears to directly modulate endothelial dysfunction by blocking cytokine signaling, functional cell adhesion expression, vascular permeability and preventing the induction of apoptosis. Moreover, APC, via endothelial protein C receptor/protease activated receptor-1 mediated mechanisms, also appears to directly modulate leukocyte migration and adhesion. The ability of APC to suppress pro-inflammatory pathways and enhance cellular survival suggests that APC has a role in the adaptive response at the vessel wall, in which it protects the wall from vascular insult and prolongs endothelial, cellular, and organ survival. The emerging data further suggest that APC effectively modulates the complex changes that occur during multi-system activation and dysfunction in sepsis.

A comparison of methods for modeling quantitative structure-activity relationships.

A large number of methods are available for modeling quantitative structure-activity relationships (QSAR). We examine the predictive accuracy of several methods applied to data sets of inhibitors for angiotensin converting enzyme, acetylcholinesterase, benzodiazepine receptor, cyclooxygenase-2, dihydrofolate reductase, glycogen phosphorylase b, thermolysin, and thrombin. Descriptors calculated with CoMFA, CoMSIA, EVA, HQSAR, and traditional 2D and 2.5D descriptors were used for developing models with partial least squares (PLS). In addition, the genetic function approximation algorithm, genetic PLS, and back-propagation neural networks were used for deriving models from 2.5D descriptors (i.e., 2D descriptors and 3D descriptors calculated from CORINA structures and Gasteiger-Marsili charges). Predictive accuracy was assessed using designed test sets. It was found that HQSAR generally performs as well as CoMFA and CoMSIA; other descriptor sets performed less well. When 2.5D descriptors were used, only neural network ensembles were found to be similarly or more predictive than PLS models. In addition, we show that many cross-validation procedures yield similar estimates of the interpolative accuracy of methods. However, the lack of correspondence between cross-validated and test set predictive accuracy for four sets underscores the benefit of using designed test sets.

Molecular modeling of the von Willebrand factor A2 Domain and the effects of associated type 2A von Willebrand disease mutations.

A homology model for the A2 domain of von Willebrand factor (VWF) is presented. A large number of target-template alignments were combined into a consensus alignment and used for constructing the model from the structures of six template proteins. Molecular dynamics simulation was used to study the structural and dynamic effects of eight mutations introduced into the model, all associated with type 2A von Willebrand disease. It was found that the group I mutations G1505R, L1540P and S1506L cause significant deviations over multiple regions of the protein, coupled to significant thermal fluctuations for G1505R and L1540P. This suggests that protein instability may be responsible for their intracellular retention. The group II mutations R1597W, E1638K and G1505E caused single loop displacements near the physiologic VWF proteolysis site between Y1605-M1606. These modest structural changes may affect interactions between VWF and the ADAMTS13 protease. The group II mutations I1628T and L1503Q caused no significant structural change in the protein, suggesting that inclusion of the protease in this model is necessary for understanding their effect. [Figure: see text]. Homology model of the von Willebrand factor A2 domain

Pruned receptor surface models and pharmacophores for three-dimensional database searching.

A pharmacophore represents the 3D arrangement of chemical features that are shared by molecules exhibiting activity at a protein receptor. Pharmacophores are routinely used in 3D database searching for identifying potential lead compounds. The lack of shape constraints causes the query to identify compounds that could not fit into the active site. In the absence of structural information, a receptor surface model (RSM) can be used to represent the active site. The RSM consists of a surface that envelops a set of known actives after these have been aligned using their common features. When used for database searching, a RSM is overconstraining as it restricts access to regions that could be occupied by ligands, such as the solvent-protein interface or unexplored pockets. We describe a protocol for developing pruned RSMs using information gleaned from 3D quantitative structure-activity relationship (QSAR) models. We examined the performance of queries that consist of pharmacophores used alone or with pruned or unpruned RSMs by performing searches on six databases containing known actives distributed among inactives. The pruned RSMs yield an average selectivity 1.8 times greater than that for pharmacophore queries, compared to 1.6 times for unpruned RSMs. However, the pruned RSMs retrieve on average 73% of the actives identified using the pharmacophores, compared to 40% for the unpruned RSMs. As such, pruned RSMs represent a useful compromise between the high sensitivity of pharmacophores and the high selectivity of unpruned RSMs.

A novel type 2A von Willebrand factor mutation located at the last nucleotide of exon 26 (3538G>A) causes skipping of 2 nonadjacent exons.

In this manuscript, we describe a case of type 2A von Willebrand disease (VWD) caused by the novel heterozygous G>A transition at nucleotide 3538, which should result in the putative, nonconservative substitution of G1180R. This mutation was reproduced by site-directed mutagenesis; however, the recombinant mutant protein was efficiently secreted from cells and assembled correctly into multimers. Because the substitution is located at the last nucleotide of exon 26, the patient's platelet von Willebrand factor (VWF) mRNA was analyzed and 3 transcripts were observed: the normal transcript without the 3538G>A transition, a transcript with the in-frame deletion of exon 26, and a transcript with the in-frame deletions of exons 23 and 26. These deletion VWF cDNA constructs were created and the resulting recombinant proteins were analyzed following transfection into COS-7 cells. Cotransfection results demonstrate that the exon-skipped transcripts led to intracellular retention, and the levels of VWF antigen (VWF:Ag) produced by these constructs were as follows: del23/26A transition at nucleotide (nt) 3538 does not result in the expression of the G1180R missense mutation, but rather leads to exon skipping, which is the pathogenic basis of the patient's phenotype. This is the first report of a coding region mutation resulting in the skipping of 2 nonadjacent exons.

Spline-fitting with a genetic algorithm: a method for developing classification structure-activity relationships.

Classification methods allow for the development of structure-activity relationship models when the target property is categorical rather than continuous. We describe a classification method which fits descriptor splines to activities, with descriptors selected using a genetic algorithm. This method, which we identify as SFGA, is compared to the well-established techniques of recursive partitioning (RP) and soft independent modeling by class analogy (SIMCA) using five series of compounds: cyclooxygenase-2 (COX-2) inhibitors, benzodiazepine receptor (BZR) ligands, estrogen receptor (ER) ligands, dihydrofolate reductase (DHFR) inhibitors, and monoamine oxidase (MAO) inhibitors. Only 1-D and 2-D descriptors were used. Approximately 40% of compounds in each series were assigned to a test set, "cherry-picked" from the complete set such that they lie outside the training set as much as possible. SFGA produced models that were more predictive for all but the DHFR set, for which SIMCA was most predictive. RP gave the least predictive models for all but the MAO set. A similar trend was observed when using training and test sets to which compounds were randomly assigned and when gradually eliminating compounds from the (designed) training set. The stability of models was examined for the random and reduced sets, where stability means that classification statistics and the selected descriptors are similar for models derived from different sets. Here, SIMCA produced the most stable models, followed by SFGA and RP. We show that a consensus approach that combines all three methods outperforms the single best model for all data sets.

Glycosaminoglycans bind factor Xa in a Ca2+-dependent fashion and modulate its catalytic activity.

Recent studies have demonstrated the existence of a Ca(2+)-dependent heparin-binding site on factor Xa. To characterize this heparin-binding site, the extrinsic fluorescence of fluorescein-labeled, active site-blocked factor Xa was monitored as it was titrated with glycosaminoglycans of various sulfate content and chain length. The binding of glycosaminoglycans to factor Xa appears to be charge-dependent because affinity is correlated with degree of glycosaminoglycan sulfation. All glycosaminoglycans bind factor Xa with higher affinity in the presence of Ca(2+) than in its absence. In contrast, when Gla-domainless factor Xa was substituted for factor Xa, glycosaminoglycans bound with similar affinities in the absence and presence of Ca(2+). These results support the hypothesis that the anionic Gla domain impairs glycosaminoglycan binding in the absence of Ca(2+). The changes in fluorescence intensity of factor Xa when titrated with glycosaminoglycans suggest that glycosaminoglycans induce conformational changes in the active site environment of factor Xa. To explore the consequences of these conformational changes, the effect of glycosaminoglycans on the catalytic activity of factor Xa was examined. Glycosaminoglycans influenced the ability of factor Xa to cleave chromogenic substrates and attenuated the capacity of factor Xa to activate factor VII. The potency of glycosaminoglycans in these assays reflected their affinity for factor Xa. These studies suggest that glycosaminoglycan binding perturbs exosites on the surface of factor Xa, potentially modifying interactions with cofactors or substrates.

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease.

To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312-19A>C) and exon 28 at Tyr1584Cys (4751A>G). The Tyr1584Cys variation was identified in 14.3% (10 of 70) of the families and was in phase with the 5312-19A>C variation in 7 (10.0%) families. Both variants were observed in 2 of 10 UK families with type 1 VWD, but neither variant was found in 200 and 100 healthy, unrelated persons, respectively. Mean von Willebrand factor antigen (VWF:Ag), VWF ristocetin cofactor (VWF:RCo), and factor VIII coagulant activity (FVIII:C) for the index cases in these families are 0.4 U/mL, 0.36 U/mL, and 0.54 U/mL, respectively, and VWF multimer patterns show no qualitative abnormalities. Aberrant VWF splicing was not observed in these patients, and both alleles of the VWF gene are expressed as RNA. Molecular dynamic simulation was performed on a homology model of the VWF-A2 domain containing the Tyr1584Cys mutation. This showed that no significant structural changes occur as a result of the substitution but that a new solvent-exposed reactive thiol group is apparent. Expression studies revealed that the Tyr1584Cys mutation results in increased intracellular retention of the VWF protein. We demonstrate that all the families with the Tyr1584Cys mutation share a common, evolved VWF haplotype, suggesting that this mutation is ancient. This is the first report of a mutation that segregates in a significant proportion of patients with type 1 VWD.