The extracellular loop of the membrane permease VraG interacts with GraS to sense cationic antimicrobial peptides in Staphylococcus aureus.

Host defense proteins (HDPs), aka defensins, are a key part of the innate immune system that functions by inserting into the bacterial membranes to form pores to kill invading and colonizing microorganisms. To ensure survival, microorganism such as S. aureus has developed survival strategies to sense and respond to HDPs. One key strategy in S. aureus is a two-component system (TCS) called GraRS coupled to an efflux pump that consists of a membrane permease VraG and an ATPase VraF, analogous to the BceRS-BceAB system of Bacillus subtilis but with distinct differences. While the 9 negatively charged amino acid extracellular loop of the membrane sensor GraS has been shown to be involved in sensing, the major question is how such a small loop can sense diverse HDPs. Mutation analysis in this study divulged that the vraG mutant phenocopied the graS mutant with respect to reduced activation of downstream effector mprF, reduction in surface positive charge and enhanced 2 hr. killing with LL-37 as compared with the parental MRSA strain JE2. In silico analysis revealed VraG contains a single 200-residue extracellular loop (EL) situated between the 7th and 8th transmembrane segments (out of 10). Remarkably, deletion of EL in VraG enhanced mprF expression, augmented surface positive charge and improved survival in LL-37 vs. parent JE2. As the EL of VraG is rich in lysine residues (16%), in contrast to a preponderance of negatively charged aspartic acid residues (3 out of 9) in the EL of GraS, we divulged the role of charge interaction by showing that K380 in the EL of VraG is an important residue that likely interacts with GraS to interfere with GraS-mediated signaling. Bacterial two-hybrid analysis also supported the interaction of EL of VraG with the EL of GraS. Collectively, we demonstrated an interesting facet of efflux pumps whereby the membrane permease disrupts HDP signaling by inhibiting GraS sensing that involves charged residues in the EL of VraG.

Differential Enhancement of Neutrophil Phagocytosis by Anti-Bactericidal/Permeability-Increasing Protein Antibodies.

Bactericidal/permeability-increasing protein (BPI) plays a major role in innate immunity through the ability of the N-terminal domain (NTD) to bind LPS, mediate cytotoxicity, and block LPS-induced inflammation. The C-terminal domain mediates phagocytosis of bacteria bound to the NTD. These two domains are linked by a surface-exposed loop at amino acids 231-249 for human BPI, known as the "hinge region." Autoantibodies to human BPI are prevalent in many chronic lung diseases; their presence is strongly correlated with Pseudomonas aeruginosa and with worse lung function in patients with cystic fibrosis and bronchiectasis. Although prior literature has reported BPI neutralization effect with autoantibodies targeting either NTD or C-terminal domain, the functionality of BPI Ab to the hinge region has never been investigated. Here, we report that Ab responses to the BPI hinge region mediate a remarkably selective potentiation of BPI-dependent phagocytosis of P. aeruginosa with both human and murine neutrophils in vitro and in vivo. These findings indicate that autoantibodies to the BPI hinge region might enhance bacterial clearance.

Filgotinib in combination with methotrexate or as monotherapy versus methotrexate monotherapy in patients with active rheumatoid arthritis and limited or no prior exposure to methotrexate: the phase 3, randomised controlled FINCH 3 trial.

OBJECTIVES: To investigate efficacy and safety of the Janus kinase-1 inhibitor filgotinib in patients with active rheumatoid arthritis (RA) with limited or no prior methotrexate (MTX) exposure. METHODS: This 52-week, phase 3, multicentre, double-blind clinical trial (NCT02886728) evaluated once-daily oral filgotinib in 1252 patients with RA randomised 2:1:1:2 to filgotinib 200 mg with MTX (FIL200 +MTX), filgotinib 100 mg with MTX (FIL100 +MTX), filgotinib 200 mg monotherapy (FIL200), or MTX. The primary endpoint was proportion achieving 20% improvement in American College of Rheumatology criteria (ACR20) at week 24. RESULTS: The primary endpoint was achieved by 81% of patients receiving FIL200+ MTX versus 71% receiving MTX (p<0.001). A significantly greater proportion treated with FIL100+ MTX compared with MTX achieved an ACR20 response (80%, p=0.017) at week 24. Significant improvement in Health Assessment Questionnaire-Disability Index was seen at week 24; least-squares mean change from baseline was -1.0 and -0.94 with FIL200+MTX and FIL100+MTX, respectively, versus -0.81 with MTX (p<0.001, p=0.008, respectively). Significantly higher proportions receiving FIL200+MTX (54%) and FIL100+MTX (43%) achieved DAS28(CRP) <2.6 versus MTX (29%) (p<0.001 for both) at week 24. Hierarchical testing stopped for comparison of ACR20 for FIL200 monotherapy (78%) versus MTX (71%) at week 24 (p=0.058). Adverse event rates through week 52 were comparable between all treatments. CONCLUSIONS: FIL200+MTX and FIL100+MTX both significantly improved signs and symptoms and physical function in patients with active RA and limited or no prior MTX exposure; FIL200 monotherapy did not have a superior ACR20 response rate versus MTX. Filgotinib was well tolerated, with acceptable safety compared with MTX.

Role for ZAP-70 Signaling in the Differential Effector Functions of Rituximab and Obinutuzumab (GA101) in Chronic Lymphocytic Leukemia B Cells.

Rituximab (RTX) has been the hallmark anti-CD20 mAb for the treatment of B cell neoplasms, including B cell chronic lymphocytic leukemia (B-CLL). Recently, a novel humanized anti-CD20 mAb obinutuzumab (GA101) has been implemented as first-line CLL therapy. Treatment of CLL patients with RTX is associated with CD20 loss via an FcγR-mediated process, trogocytosis. RTX-induced trogocytosis has been characterized as both the means of resistance to therapy, via loss of cell surface target proteins (antigenic modulation), as well as a process that alters B cell phenotype and function. This study investigates the nature and clinical relevance of GA101-mediated trogocytosis. In this study, we demonstrate that GA101 is a more potent mediator of trogocytosis than RTX in vitro in both normal B cells and B-CLL cells. Qualitative differences in the effector function of these anti-CD20 Abs appear specific to B-CLL cells. GA101-mediated CD19 and CD20 trogocytosis from B-CLL cells is associated with its ability to induce homotypic adhesion (HA). The degree of HA varies between CLL patients and positively correlates with the expression of ZAP-70, a BCR-associated kinase. Deregulation of ZAP-70 using tyrosine kinase inhibitors, gefitinib or ibrutinib, diminishes HA formation and trogocytosis by GA101. Taken together, these findings elucidate the differences in trogocytosis and HA formation mediated by anti-CD20 mAbs RTX and GA101, as well as provide a novel link between ZAP-70 expression and these effector functions.

"NETtling" the host: Breaking of tolerance in chronic inflammation and chronic infection.

How and why we break tolerance to self-proteins still remains a largely unanswered question. Neutrophils have been identified as a rich source of autoantigens in a wide array of autoimmune diseases that arise as a consequence of different environmental and genetic factors, e.g. rheumatoid arthritis (RA), lupus, vasculitis, cystic fibrosis (CF) etc. Specifically, neutrophil extracellular trap (NET) formation has been identified as a link between innate and adaptive immune responses in autoimmunity. Autoantigens including neutrophil granular proteins (targeted by anti-neutrophil cytoplasmic antibodies, ANCA) as well as post-translationally modified proteins, i.e. citrullinated and carbamylated proteins targeted by anti-citrullinated protein antibodies (ACPA) and anti-carbamylated protein antibodies (ACarPA), respectively, localize to the NETs. Moreover, NETs provide stimuli to dendritic cells that potentiate adaptive autoimmune responses. However, while NETs promote inflammation and appear to induce humoral autoreactivity across autoimmune diseases, the antigen specificity of autoantibodies found in these disorders is striking. These unique autoantigen signatures suggest that not all NETs are created equal and that the environment in which NETs arise shapes their disease-specific character. In this review article, we discuss the effects of different stimuli on the mechanism of NET formation as well as how they contribute to antigen specificity in the breaking of immune tolerance. Specifically, we compare and contrast the autoreactive nature of NETs in two settings of chronic airway inflammation: one triggered by smoking, a recognized environmental NET stimulus in RA patients, and one mediated by Pseudomonas aeruginosa, the most prevalent lung pathogen in CF patients. Finally, we draw attention to novel findings that, together with the specific environmental/chemical stimuli, should be taken into account when investigating how and why antigen specificity arises in the context of NET formation.

Efficacy and safety of tabalumab, an anti-B-cell-activating factor monoclonal antibody, in patients with rheumatoid arthritis who had an inadequate response to methotrexate therapy: results from a phase III multicentre, randomised, double-blind study.

OBJECTIVES: Randomised, double-blind, placebo-controlled study to evaluate efficacy and safety of tabalumab in patients with rheumatoid arthritis (RA) with inadequate responses to methotrexate (MTX-IR). METHODS: 1041 patients with moderate-severe RA despite ongoing MTX enrolled in a 52-week study evaluating subcutaneous tabalumab 120 mg every four weeks (120/Q4W) or 90 mg every two weeks (90/Q2W) versus placebo. Primary endpoints were American College of Rheumatology 20% (ACR20) response rate and Health Assessment Questionnaire-Disability Index change from baseline at 24 weeks and modified Total Sharp Score (mTSS) change at 52 weeks. RESULTS: There were no significant differences in ACR20 responses at week 24 or mTSS change from baseline at week 52 among treatment groups. Declines were seen in CD20+ B cells and immunoglobulin levels in tabalumab groups, but not placebo: B cells (-15.0%, -18.8%, 5.3%, in the 120/Q4W, 90/Q2W, and placebo groups, respectively); IgM (-16.3%, -19.4%, -0.1%), IgA (-11.4%, -4.7%, 1.2%) and IgG (-8.6%, -7.8%, 0.1%). Discontinuations due to adverse events were similar between groups. Numerically more serious infections were reported in tabalumab groups (1.7%, 0.6%, 0.3%); numerically more injection-site reactions were reported in the 90/Q2W group (2.3%, 4.3%, 2.3%). CONCLUSIONS: Neither clinical efficacy nor significant safety signals were observed with tabalumab despite evidence of biological activity. This study was terminated early due to insufficient efficacy. TRIAL REGISTRATION NUMBER: NCT01198002.

Rituximab mediates loss of CD19 on B cells in the absence of cell death.

OBJECTIVE: To evaluate loss of the B cell-specific marker CD19 after the addition of rituximab (RTX) to healthy donor blood and to determine the role of complement-mediated cytotoxicity in these cells. METHODS: Whole blood and peripheral blood mononuclear cells (PBMCs) from healthy donors were evaluated for the loss of CD19 in the presence of RTX using flow cytometry. The effect of complement on CD19 loss was examined using serum-free media, C3- and C5-deficient sera, and a C5-blocking antibody. Evidence of B cell death was evaluated by measuring messenger RNA (mRNA) levels as well as by flow cytometry. Transfer of CD19 antigen to monocytes and neutrophils was evaluated by flow cytometry and confocal microscopy. RESULTS: RTX induced a rapid decrease in CD19 count (mean 51%; n = 37) in PBMCs. This reduction occurred in the absence of complement. Despite the decrease in CD19 expression, B cell death did not occur, as evidenced by a lack of change in CD19 or CD20 mRNA levels and a lack of change in CD19 levels determined by intracellular staining and through the use of viability dyes. The CD19 antigen was shown to be transferred to monocytes and neutrophils in an Fc-dependent manner. CONCLUSION: Our findings indicate that the addition of RTX to healthy donor PBMCs in vitro results in complement-independent loss of CD19 without causing B cell death. CD19 is transferred from B cells to monocytes and neutrophils during shaving of the RTX-CD20 complex in an Fc-dependent manner. These data suggest that monitoring the effect of RTX by measuring the CD19+ cell count may be compromised by this activity.

Increased frequency of complement C4B deficiency in rheumatoid arthritis.

OBJECTIVE: To assess the copy number variation of complement C4A and C4B genes in patients with rheumatoid arthritis (RA). METHODS: DNA samples were obtained from 299 patients and controls and analyzed for copy number variation of total complement C4, C4A, and C4B genes. The results were compared by chi-square analysis, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: Chi-square analysis revealed similar distribution patterns of total C4 alleles in RA patients (n = 160), non-RA patients (n = 88), and healthy controls (n = 51). There was no trend toward C4A deficiency as in lupus. Significant differences in C4B distribution were observed in RA patients, in whom an ∼2-fold increase in the frequency of homozygous and/or heterozygous C4B deficiency (0 or 1 allele) (40%) was present relative to non-RA patients or healthy controls (both 21.6%). C4B deficiency was more frequent in seropositive RA patients than in seronegative RA patients (44% versus 31%). The odds of C4B deficiency were 2.99 (95% CI 1.58-5.65) (P = 0.0006) in seropositive RA patients relative to non-RA controls. These findings were confirmed in a larger healthy control cohort, yielding an OR of 1.83 (95% CI 1.21-2.76) (P = 0.0056). The association of the shared epitope with C4B deficiency was significantly greater in seropositive RA patients than in non-seropositive RA controls (96% versus 54.5%) (P < 0.0001), suggesting that C4B deficiency interacts with the shared epitope in the development of seropositive RA. CONCLUSION: Our findings indicate a relationship between C4B copy number variation and RA that approximates that seen between C4A copy number variation and lupus. The concurrence of C4B deficiency and the shared epitope in seropositive RA may have broad implications for our understanding of RA pathogenesis.

The thematic role of extracellular loop of VraG in activation of the membrane sensor GraS in a cystic fibrosis MRSA strain differs in nuance from the CA-MRSA strain JE2.

Patients with cystic fibrosis (CF) often suffer recurrent bronchial bacterial infections that lead to deterioration of lung function over time. The infections in CF patients are often due to S. aureus and P. aeruginosa that colonize the airways. Significantly, methicillin-resistant S. aureus (MRSA) makes it challenging for treatment in CF patients due to its feature of multiple antibiotic resistance. In bronchial airways, cationic antimicrobial peptides are often present in mucosa cells, neutrophils, and macrophages that interfere with bacterial proliferation. The major mechanism for resistance to the bactericidal activity of cationic peptides in S. aureus is mediated by the GraRS two-component system that activates expression of MprF and DltABCD to increase surface positive charge to repel interactions with cationic peptides. We recently found that VraG, a membrane permease component of the VraFG efflux pumps, harbors a long 200-residue extracellular loop (EL) that utilizes K380 to interact with the negatively charged 9-residue extracellular loop of the membrane sensor GraS to control mprF expression in a community-acquired MRSA strain JE2. In this study, we extended this observation to a CF MRSA strain CF32A1 where we affirmed that the EL loop of VraG controls GraS-mediated signal transduction; however, in contrast to community acquired MRSA strain JE2, the CF MRSA strain CF32A1 requires both K380 and K388 in the EL of VraG to properly modulate signal transduction mediated by GraS. This effect was not attributable to the several single nucleotide polymorphisms that exist between VraG and GraS in the two MRSA strains.

Killing three birds with one BPI: Bactericidal, opsonic, and anti-inflammatory functions.

Bactericidal/permeability-increasing protein (BPI) is an anti-microbial protein predominantly expressed in azurophilic granules of neutrophils. BPI has been shown to mediate cytocidal and opsonic activity against Gram-negative bacteria, while also blunting inflammatory activity of lipopolysaccharide (LPS). Despite awareness of these functions in vitro, the magnitude of the contribution of BPI to innate immunity remains unclear, and the nature of the functional role of BPI in vivo has been submitted to limited investigation. Understanding this role takes on particular interest with the recognition that autoimmunity to BPI is tightly linked to a specific infectious trigger like Pseudomonas aeruginosa in chronic lung infection. This has led to the notion that anti-BPI autoantibodies compromise the activity of BPI in innate immunity against P. aeruginosa, which is primarily mediated by neutrophils. In this review, we explore the three main mechanisms in bactericidal, opsonic, and anti-inflammatory of BPI. We address the etiology and the effects of BPI autoreactivity on BPI function. We explore BPI polymorphism and its link to multiple diseases. We summarize BPI therapeutic potential in both animal models and human studies, as well as offer therapeutic approaches to designing a sustainable and promising BPI molecule.

Bactericidal/Permeability-Increasing Protein Preeminently Mediates Clearance of Pseudomonas aeruginosa In Vivo via CD18-Dependent Phagocytosis.

Chronic Pseudomonas aeruginosa infection mysteriously occurs in the airways of patients with cystic fibrosis (CF), bronchiectasis (BE), and chronic obstructive pulmonary disease (COPD) in the absence of neutrophil dysfunction or neutropenia and is strongly associated with autoimmunity to bactericidal permeability-increasing protein (BPI). Here, we define a critical role for BPI in in vivo immunity against P. aeruginosa. Wild type and BPI-deficient (Bpi-/-) mice were infected with P. aeruginosa, and bacterial clearance, cell infiltrates, cytokine production, and in vivo phagocytosis were quantified. Bpi-/- mice exhibited a decreased ability to clear P. aeruginosa in vivo in concert with increased neutrophil counts and cytokine release. Bpi-/- neutrophils displayed decreased phagocytosis that was corrected by exogenous BPI in vitro. Exogenous BPI also enhanced clearance of P. aeruginosa in Bpi-/- mice in vivo by increasing P. aeruginosa uptake by neutrophils in a CD18-dependent manner. These data indicate that BPI plays an essential role in innate immunity against P. aeruginosa through its opsonic activity and suggest that perturbations in BPI levels or function may contribute to chronic lung infection with P. aeruginosa.

An Integrated Analysis of the Safety of Tofacitinib in Psoriatic Arthritis across Phase III and Long-Term Extension Studies with Comparison to Real-World Observational Data.

INTRODUCTION: Tofacitinib is an oral Janus kinase inhibitor for the treatment of psoriatic arthritis (PsA). OBJECTIVE: Our objective was to compare the incidence rates (IRs) of adverse events in tofacitinib clinical trials and real-world observational data for alternative treatments. METHODS: The tofacitinib "dose-comparison cohort" included months 0-12 of two phase III studies (tofacitinib 5 [n = 238] and 10 [n = 236] mg twice daily [BID]); the "all-tofacitinib comparison cohort" (n = 783) included two phase III and one ongoing long-term extension study (data cutoff May 2016). An "observational comparison cohort" (n = 5799) comprised patients initiating a conventional synthetic disease-modifying antirheumatic drug (DMARD), biologic DMARD, or apremilast in the US Truven MarketScan database from 2010 to 2015. IRs for serious infections (SIEs; requiring hospitalization), herpes zoster (HZ), malignancies (excluding non-melanoma skin cancer [NMSC]), NMSC, and major adverse cardiovascular events (MACE) across cohorts were qualitatively compared. RESULTS: IRs (patients with events/100 patient-years) for SIEs were similar between the tofacitinib dose-comparison cohort (5 mg BID: 1.3; 10 mg BID: 2.0) and the observational comparison cohort (1.1-7.9; treatment dependent). The tofacitinib dose-comparison cohort had a higher rate of HZ (5 mg BID: 2.0; 10 mg BID: 2.7) than did the observational comparison cohort (0.8-2.0). IRs for NMSC were generally lower in the all-tofacitinib comparison cohort (0.5) than in the observational comparison cohort (0.4-6.0). IRs for MACE, malignancies excluding NMSC, and NMSC were similar between cohorts. CONCLUSION: In patients with PsA, tofacitinib had a safety profile similar to that of other systemic therapies in real-world settings, except for the risk of HZ, a known risk of tofacitinib. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01877668; NCT01882439; NCT01976364.

Regulation of Pseudomonas aeruginosa-Mediated Neutrophil Extracellular Traps.

Pseudomonas aeruginosa is the most prevalent opportunistic pathogen in the airways of cystic fibrosis (CF) patients. The pulmonary disorder is characterized by recurrent microbial infections and an exaggerated host inflammatory immune response led primarily by influx of neutrophils. Under these conditions, chronic colonization with P. aeruginosa is associated with diminished pulmonary function and increased morbidity and mortality. P. aeruginosa has a wide array of genetic mechanisms that facilitate its persistent colonization of the airway despite extensive innate host immune responses. Loss of function mutations in the quorum sensing regulatory gene lasR have been shown to confer survival advantage and a more pathogenic character to P. aeruginosa in CF patients. However, the strategies used by LasR-deficient P. aeruginosa to modulate neutrophil-mediated bactericidal functions are unknown. We sought to understand the role of LasR in P. aeruginosa-mediated neutrophil extracellular trap (NET) formation, an important anti-microbial mechanism deployed by neutrophils, the first-line responder in the infected airway. We observe mechanistic and phenotypic differences between NETs triggered by LasR-sufficient and LasR-deficient P. aeruginosa strains. We uncover that LasR-deficient P. aeruginosa strains fail to induce robust NET formation in both human and murine neutrophils, independently of bacterial motility or LPS expression. LasR does not mediate NET release via downstream quorum sensing signaling pathways but rather via transcriptional regulation of virulence factors, including, but not restricted to, LasB elastase and LasA protease. Finally, our studies uncover the differential requirements for NADPH oxidase in NET formation triggered by different P. aeruginosa strains.

Triple Positivity for Anti-Citrullinated Protein Autoantibodies, Rheumatoid Factor, and Anti-Carbamylated Protein Antibodies Conferring High Specificity for Rheumatoid Arthritis: Implications for Very Early Identification of At-Risk Individuals.

OBJECTIVE: In rheumatoid arthritis (RA), anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are commonly used to aid in the diagnosis. Although these autoantibodies are mainly found in RA, their specificity is not optimal. It is therefore difficult to identify RA patients, especially in very early disease, based on the presence of ACPAs and RF alone. In addition, anti-carbamylated protein (anti-CarP) antibodies have diagnostic and prognostic value, since their presence is associated with joint damage in RA patients and also associated with the future development of RA in patients with arthralgia. Therefore, the aim of the present study was to investigate the value of combined antibody testing in relation to prediction and diagnosis of (early) RA. METHODS: A literature search resulted in identification of 12 relevant studies, consisting of RA patients, pre-RA individuals, disease controls, healthy first-degree relatives of RA patients, and healthy control subjects, in which data on RF, ACPAs, and anti-CarP antibody status were available. Using these data, random effects meta-analyses were carried out for several antibody combinations. RESULTS: The individual antibodies were highly prevalent in patients with RA (34-80%) compared to the control groups, but were also present in non-RA controls (0-23%). For the classification of most subjects correctly as having RA or as a non-RA control, the combination of ACPAs and/or RF often performed well (specificity 65-100%, sensitivity 59-88%). However, triple positivity for ACPAs, RF, and anti-CarP antibodies resulted in a higher specificity for RA (98-100%), accompanied by a lower sensitivity (11-39%). CONCLUSION: As the rheumatology field is moving toward very early identification of RA and possible screening for individuals at maximum risk of RA in populations with a low pretest probability, an autoantibody profile of triple positivity for ACPAs, RF, and anti-CarP provides interesting information that might help identify individuals at risk of developing RA.

Delineation of a novel pathway that regulates CD154 (CD40 ligand) expression.

The expression of CD154 (CD40 ligand) by activated T lymphocytes plays a central role in humoral and cellular immunity. The fundamental importance of this protein in mounting an immune response has made it an attractive target for immunomodulation. Several studies have demonstrated that CD154 expression is regulated at the level of mRNA turnover in a manner distinct from other cytokine genes. We have purified, sequenced, and characterized the two major proteins that bind the CD154 3' untranslated region (3'UTR) as members of the polypyrimidine tract binding protein (PTB) family. One of these proteins is a previously unreported alternatively spliced PTB isoform, which we call PTB-T. These proteins interact with a polypyrimidine-rich region within the CD154 3'UTR that lacks any known cis-acting instability elements. The polypyrimidine-rich region of the CD154 3'UTR was both necessary and sufficient to mediate changes in reporter gene expression and mRNA accumulation, indicating the presence of a novel cis-acting instability element. The presence of a cis-acting instability element in the polypyrimidine-rich region was confirmed using a tetracycline-responsive reporter gene approach. The function of this cis-acting element appears to be dependent on the relative cytoplasmic levels of PTB and PTB-T. Cotransfection of vectors encoding PTB-T consistently decreased the CD154 3'UTR-dependent luciferase expression. In contrast, transfection of plasmids encoding PTB tended to increase CD154 3'UTR-dependent luciferase expression. Thus, the CD154 3'UTR contains a novel cis-acting element whose function is determined by the binding of PTB and PTB-T. These data identify a specific pathway that regulates CD154 expression that can potentially be selectively targeted for the treatment of autoimmune disease and allograft rejection.

Brief Report: Anti-Carbamylated Protein Antibodies in Rheumatoid Arthritis Patients Are Reactive With Specific Epitopes of the Human Fibrinogen ß-Chain.

OBJECTIVE: Anti-carbamylated protein (anti-CarP) antibodies are associated with the risk and severity of rheumatoid arthritis (RA) and are primarily directed against fibrinogen. The lack of understanding of anti-CarP antibody reactivity has limited analysis of the immunopathogenic associations in RA. To address this shortcoming, we mapped anti-CarP antibody epitope reactivity in RA patient sera. METHODS: Immunoblotting identified a patient serum sample with specific reactivity to the carbamylated human fibrinogen ß-chain. Liquid chromatography mass spectrometry (LC-MS) identified sites of homocitrullines (carbamylated lysines) present in the human fibrinogen ß-chain. The reactivity of an anti-CarP antibody-positive cohort to specific peptides containing carbamylated lysines was determined by enzyme-linked immunosorbent assay, through direct binding (n = 63 sera) and by competition assays (n = 40 sera). RESULTS: Serum with specific reactivity to carbamylated, but not citrullinated, fibrinogen ß-chain was identified in a specimen obtained from an RA patient. LC-MS identified carbamylation of 9 of 34 lysines in the human fibrinogen ß-chain. Mapping of immunoreactivity to tryptic peptide fragments demonstrated several candidate carbamylated epitopes that were confirmed by competition experiments. Peptides containing a homocitrulline at position 83 appeared to be an immunodominant epitope in some RA patient sera, with additional reactivity to peptides containing homocitrullines at positions 52, 264, 351, 367, and 374. CONCLUSION: Anti-CarP antibodies appear to preferentially target specific regions of the human fibrinogen ß-chain that contain homocitrullines. Interestingly, humoral immunoreactivity appears to be relatively restricted in some patients, which may enable detection of specific relationships with disease phenotype.

Review of Routine Laboratory Monitoring for Patients with Rheumatoid Arthritis Receiving Biologic or Nonbiologic DMARDs.

Safety concerns associated with many drugs indicated for the treatment of rheumatoid arthritis (RA) can be attenuated by the early identification of toxicity through routine laboratory monitoring; however, a comprehensive review of the recommended monitoring guidelines for the different available RA therapies is currently unavailable. The aim of this review is to summarize the current guidelines for laboratory monitoring in patients with RA and to provide an overview of the laboratory abnormality profiles associated with each drug indicated for RA. Recommendations for the frequency of laboratory monitoring of serum lipids, liver transaminases, serum creatinine, neutrophil counts, and platelet counts in patients with RA were compiled from a literature search for published recommendations and guidelines as well as the prescribing information for each drug. Laboratory abnormality profiles for each drug were compiled from the prescribing information for each drug and a literature search including meta-analyses and primary clinical trials data.