Regulation of neutrophil myeloperoxidase inhibitor SPIN by the small RNA Teg49 in Staphylococcus aureus.

Teg49 is a Staphylococcus aureus trans-acting regulatory sRNA derived from cleavage of the sarA P3 transcript. We showed by RNA-Seq here that the 5' trident-like structure in Teg49 regulates transcriptionally (direct and indirect) 22 genes distinct from sarA. Among these, Teg49 was noted to repress spn, encoding a 102 residue preprotein which yields the mature 73 residue peptide which inhibits the catalytic activity of myeloperoxidase in human neutrophils. Teg49 was found to regulate spn mRNA post-transcriptionally in strain SH1000 through 9-nt base-pairing between hairpin loop 2 of Teg49 and an exposed bulge of the spn mRNA. Mutations of the Teg49 binding site disrupted the repression of spn, leading to reduced degradation, and increased half-life of spn mRNA in the Teg49 mutant. The spn-Teg49 interaction was also confirmed with a synonymous spn mutation to yield enhanced spn expression in the mutant vs. the parent. The Teg49 mutant with increased spn expression exhibited enhanced resistance to MPO activity in vitro. Killing assays with human neutrophils showed that the Teg49 mutant was more resistant to killing after phagocytosis. Altogether, this study shows that Teg49 in S. aureus has a distinct and important regulatory profile whereby this sRNA modulates resistance to myeloperoxidase-mediated killing by human neutrophils.

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.

Upadacitinib as monotherapy in patients with active rheumatoid arthritis and inadequate response to methotrexate (SELECT-MONOTHERAPY): a randomised, placebo-controlled, double-blind phase 3 study.

BACKGROUND: Upadacitinib, an oral Janus kinase (JAK)1-selective inhibitor, showed efficacy in combination with stable background conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) in patients with rheumatoid arthritis who had an inadequate response to DMARDs. We aimed to evaluate the safety and efficacy of upadacitinib monotherapy after switching from methotrexate versus continuing methotrexate in patients with inadequate response to methotrexate. METHODS: SELECT-MONOTHERAPY was conducted at 138 sites in 24 countries. The study enrolled adults (≥18 years) who fulfilled the 2010 American College of Rheumatology (ACR)-European League Against Rheumatism (EULAR) classification criteria for rheumatoid arthritis. Patients with active rheumatoid arthritis despite stable methotrexate were randomly assigned 2:2:1:1 to switch to once-daily monotherapy of of upadacitinib or to continue methotrexate at their existing dose as blinded study drug; starting from week 14, patients assigned to continue methotrexate were switched to 15 mg or 30 mg once-daily upadacitinib per prespecified random assignment at baseline. The primary endpoints in this report are proportion of patients achieving 20% improvement in the ACR criteria (ACR20) at week 14, and proportion achieving low disease activity defined as 28-joint Disease Activity Score using C-reactive protein (DAS28[CRP]) of 3·2 or lower, both with non-responder imputation at week 14. Outcomes were assessed in patients who received at least one dose of study drug. This study is active but not recruiting and is registered with ClinicalTrials.gov, number NCT02706951. FINDINGS: Patients were screened between Feb 23, 2016, and May 19, 2017 and 648 were randomly assigned to treatment. 598 (92%) completed week 14. At week 14, an ACR20 response was achieved by 89 (41%) of 216 patients (95% CI 35-48) in the continued methotrexate group, 147 (68%) of 217 patients (62-74) receiving upadacitinib 15 mg, and 153 (71%) of 215 patients (65-77) receiving upadacitinib 30 mg (p<0·0001 for both doses vs continued methotrexate). DAS28(CRP) 3·2 or lower was met by 42 (19%) of 216 (95% CI 14-25) in the continued methotrexate group, 97 (45%) of 217 (38-51) receiving upadacitinib 15 mg, and 114 (53%) of 215 (46-60) receiving upadacitinib 30 mg (p<0·0001 for both doses vs continued methotrexate). Adverse events were reported in 102 patients (47%) on continued methotrexate, 103 (47%) on upadacitinib 15 mg, and 105 (49%) on upadacitinib 30 mg. Herpes zoster was reported by one (<1%) patient on continued methotrexate, three (1%) on upadacitinib 15 mg, and six (3%) on upadacitinib 30 mg. Three malignancies (one [<1%] on continued methotrexate, two [1%] on upadacitinib 15 mg), three adjudicated major adverse cardiovascular events (one [<1%] on upadacitinib 15 mg, two [<1%] on upadacitinib 30 mg), one adjudicated pulmonary embolism (<1%; upadacitinib 15 mg), and one death (<1%; upadacitinib 15 mg, haemorrhagic stroke [ruptured aneurysm]) were reported in the study. INTERPRETATION: Upadacitinib monotherapy showed statistically significant improvements in clinical and functional outcomes versus continuing methotrexate in this methotrexate inadequate-responder population. Safety observations were similar to those in previous upadacitinib rheumatoid arthritis studies. FUNDING: AbbVie Inc, USA.

Tofacitinib for Psoriatic Arthritis in Patients with an Inadequate Response to TNF Inhibitors.

BACKGROUND: Tofacitinib is an oral Janus kinase inhibitor that is under investigation for the treatment of psoriatic arthritis. We evaluated tofacitinib in patients with active psoriatic arthritis who had previously had an inadequate response to tumor necrosis factor (TNF) inhibitors. METHODS: In this 6-month randomized, placebo-controlled, double-blind, phase 3 trial, we randomly assigned 395 patients, in a 2:2:1:1 ratio, to four regimens: 5 mg of tofacitinib administered orally twice daily (132 patients); 10 mg of tofacitinib twice daily (132 patients); placebo, with a switch to 5 mg of tofacitinib twice daily at 3 months (66 patients); or placebo, with a switch to 10 mg of tofacitinib twice daily at 3 months (65 patients). Data from the patients who received placebo during the first 3 months of the trial were pooled. The primary end points were the percentage of patients who had at least 20% improvement according to the criteria of the American College of Rheumatology (ACR20 response) and the change from baseline score on the Health Assessment Questionnaire-Disability Index (HAQ-DI; scores range from 0 to 3, with higher scores indicating greater disability) at the month 3 analysis. RESULTS: At 3 months, the rates of ACR20 response were 50% with the 5-mg dose of tofacitinib and 47% with the 10-mg dose, as compared with 24% with placebo (P<0.001 for both comparisons); the corresponding mean changes from baseline in HAQ-DI score were -0.39 and -0.35, as compared with -0.14 (P<0.001 for both comparisons). Serious adverse events occurred in 4% of the patients who received the 5-mg dose of tofacitinib continuously and in 6% who received the 10-mg dose continuously. Over the course of 6 months, there were four serious infections, three herpes zoster infections, one myocardial infarction, and one ischemic stroke among the patients who received tofacitinib continuously. Elevations of aspartate and alanine aminotransferase concentrations of three or more times the upper limit of the normal range occurred in more patients who received tofacitinib continuously than in patients who received placebo followed by tofacitinib. CONCLUSIONS: In this trial involving patients with active psoriatic arthritis who had had an inadequate response to TNF inhibitors, tofacitinib was more effective than placebo over 3 months in reducing disease activity. Adverse events were more frequent with tofacitinib than with placebo. (Funded by Pfizer; OPAL Beyond ClinicalTrials.gov number, NCT01882439 .).

Kikuchi-Fujimoto disease preceded by lupus erythematosus panniculitis: do these findings together herald the onset of systemic lupus erythematosus?

Kikuchi-Fujimoto disease (KFD), also known as histiocytic necrotizing lymphadenitis, is a rare disorder that must be distinguished from systemic lupus erythematosus (SLE). Although a minority of patients with KFD develop SLE, most patients have a self-limited disease. Importantly, KFD can have skin manifestations resembling cutaneous lupus. Therefore, the diagnosis of SLE should be predicated on a complete rheumatologic workup and not on the constellation of skin disease and lymphadenitis. Nonetheless, as our exceedingly rare case illustrates, patients who do not initially meet diagnostic criteria for SLE require dermatologic follow-up. We present a young adult woman who had a remote history of KFD and later presented with combined features of discoid lupus and lupus erythematosus panniculitis (LEP). On subsequent rheumatologic workup, she fulfilled criteria for SLE. We discuss the differential diagnosis of both KFD and LEP and emphasize how strong communication among dermatologists and other healthcare providers is essential in the management of patients with KFD.

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.

Tocilizumab combination therapy or monotherapy or methotrexate monotherapy in methotrexate-naive patients with early rheumatoid arthritis: 2-year clinical and radiographic results from the randomised, placebo-controlled FUNCTION trial.

OBJECTIVE: Investigate whether the efficacy and safety of intravenous tocilizumab (TCZ) demonstrated at week 52 in patients with early rheumatoid arthritis (RA) are maintained to week 104. METHODS: Methotrexate (MTX)-naive patients with early progressive RA were randomly assigned to double-blind 4 mg/kg TCZ+MTX, 8 mg/kg TCZ+MTX, 8 mg/kg TCZ+placebo or placebo+MTX for 104 weeks. Patients not receiving 8 mg/kg TCZ and not achieving Disease Activity Score-28 joints (DAS28-erythrocyte sedimentation rate (ESR)) ≤3.2 at week 52 switched to escape therapy (8 mg/kg TCZ+MTX). Analyses were exploratory. RESULTS: Intent-to-treat and safety populations included 1157 and 1153 patients, respectively. DAS28-ESR remission (<2.6) rates were maintained from weeks 52 to 104 (eg, 8 mg/kg TCZ+MTX, 49.3% to 47.6%). Placebo+MTX and 4 mg/kg TCZ+MTX escape patients' week 104 response rates were 51.4% and 30.5%, respectively. Inhibition of radiographic progression was maintained with 8 mg/kg TCZ (eg, 8 mg/kg TCZ+MTX mean (SD) change from baseline in modified total Sharp score: 0.13 (1.28), week 52; 0.19 (2.08), week 104). The safety profile of TCZ was consistent with that of previous reports. CONCLUSIONS: Patients with early RA treated with TCZ monotherapy or TCZ+MTX maintained clinical benefits during their second year of treatment with no new safety signals. TRIAL REGISTRATION NUMBER: NCT01007435; Results.

Effect of tocilizumab on neutrophils in adult patients with rheumatoid arthritis: pooled analysis of data from phase 3 and 4 clinical trials.

Objectives: To investigate changes in neutrophil count and occurrences of infection in RA patients treated with the IL-6 receptor-α inhibitor tocilizumab (TCZ). Methods: Data were pooled from patients who received i.v. TCZ (4 mg/kg + MTX, 8 mg/kg ± DMARDs, 10 mg/kg) or placebo + DMARDs in phase 3/4 clinical trials, long-term extensions or a pharmacology study. Neutrophil counts were measured routinely according to the Common Toxicity Criteria for Adverse Events grades; TCZ dosing was adjusted if necessary. Covariates associated with decreased neutrophil counts were assessed with multivariate regression analysis. Infection rates within 30 days of neutrophil count changes were calculated per 100 patient-years of TCZ exposure. Results: In placebo-controlled parts of trials, more TCZ-treated than placebo-treated patients had grade 1/2 or 3/4 neutrophil counts (TCZ: 28.2%/3.1%; placebo: 8.9%/0.2%). In placebo-controlled trials + long-term extensions, 4171 patients provided 16204.8 patient-years of TCZ exposure. Neutrophil counts decreased through week 6 from baseline [mean ( s . d .) change, -2.17 (2.16) × 10 9 /l) and remained stable thereafter. Rates (95% CI) of serious infections within 30 days of normal [4.66 (4.31, 5.03)], grade 1/2 [2.48 (1.79, 3.34)] and 3/4 [2.77 (0.34, 10.01)] neutrophil counts were similar. Baseline neutrophil count <2 × 10 9 /l and female gender were associated with grade 3/4 neutrophil counts [odds ratio (OR) (95% CI): 19.02 (6.76, 53.52), 2.55 (1.40, 4.66)]. Patients who stopped TCZ in response to decreased neutrophil count returned more quickly to normal levels than patients who reduced or continued their dose. Conclusion: Decreases in neutrophil counts in patients taking TCZ do not appear to be associated with serious infections and are normalized by current risk mitigation guidelines.

Tocilizumab in early progressive rheumatoid arthritis: FUNCTION, a randomised controlled trial.

OBJECTIVES: The efficacy of tocilizumab (TCZ), an anti-interleukin-6 receptor antibody, has not previously been evaluated in a population consisting exclusively of patients with early rheumatoid arthritis (RA). METHODS: In a double-blind randomised controlled trial (FUNCTION), 1162 methotrexate (MTX)-naive patients with early progressive RA were randomly assigned (1:1:1:1) to one of four treatment groups: 4 mg/kg TCZ+MTX, 8 mg/kg TCZ+MTX, 8 mg/kg TCZ+placebo and placebo+MTX (comparator group). The primary outcome was remission according to Disease Activity Score using 28 joints (DAS28-erythrocyte sedimentation rate (ESR) <2.6) at week 24. Radiographic and physical function outcomes were also evaluated. We report results through week 52. RESULTS: The intent-to-treat population included 1157 patients. Significantly more patients receiving 8 mg/kg TCZ+MTX and 8 mg/kg TCZ+placebo than receiving placebo+MTX achieved DAS28-ESR remission at week 24 (45% and 39% vs 15%; p<0.0001). The 8 mg/kg TCZ+MTX group also achieved significantly greater improvement in radiographic disease progression and physical function at week 52 than did patients treated with placebo+MTX (mean change from baseline in van der Heijde-modified total Sharp score, 0.08 vs 1.14 (p=0.0001); mean reduction in Health Assessment Disability Index, -0.81 vs -0.64 (p=0.0024)). In addition, the 8 mg/kg TCZ+placebo and 4 mg/kg TCZ+MTX groups demonstrated clinical efficacy that was at least as effective as MTX for these key secondary endpoints. Serious adverse events were similar among treatment groups. Adverse events resulting in premature withdrawal occurred in 20% of patients in the 8 mg/kg TCZ+MTX group. CONCLUSIONS: TCZ is effective in combination with MTX and as monotherapy for the treatment of patients with early RA. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, number NCT01007435.

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.

Safety of infusing rituximab at a more rapid rate in patients with rheumatoid arthritis: results from the RATE-RA study.

BACKGROUND: As recommended in the current prescribing information, rituximab infusions in patients with rheumatoid arthritis (RA) take 4.25 hours for the first infusion and 3.25 hours for subsequent infusions, which is a burden on patients and the health care system. We therefore evaluated the safety of infusing rituximab at a faster rate for an infusion period of 2 hours in patients with RA. METHODS: Patients with an inadequate response to anti-TNF who were rituximab-naive or -experienced received 2 courses of rituximab: Infusion 1 (Day 1) was administered over the standard 4.25 hours, and Infusions 2 (Day 15), 3 (Day 168) and 4 (Day 182) were administered over a faster 2-hour period. The primary endpoint was incidence of infusion-related reactions (IRRs) associated with Infusion 2. RESULTS: Of the 351 patients enrolled, 87% and 13% were rituximab-naive and -experienced, respectively. The incidence (95% CI) of IRRs associated with Infusion 1 was 16.2% (12.5%, 20.5%) and consistent with weighted historical incidence of 20.7% (19.4%, 22.1%). The incidence (95% CI) of IRRs associated with Infusions 2, 3, and 4 compared with respective weighted historical incidences at the standard infusion rate was 6.5% (4.1%, 9.7%) vs 8.1% (7.2%, 9.1%); 5.9% (3.5%, 9.3%) vs 11.5% (10.3%, 12.8%); and 0.7 (0.1%, 2.6%) vs 5.0% (4.2%, 6.0%), respectively. All IRRs were grade 1 or 2, except for 3 grade 3 IRRs associated with Infusion 1 and 2 grade 3 IRRs associated with Infusion 2. Four patients experienced a total of 5 grade 3 IRRs; 3 of these patients continued on to received subsequent infusions at the faster rate. There were no serious IRRs. CONCLUSION: This study demonstrated that rituximab can be administered at the faster infusion rate at the second and subsequent infusions without increasing the rate or severity of IRRs.

Depletion of hnRNP A2/B1 overrides the nuclear retention of the HIV-1 genomic RNA.

hnRNP A2 is a cellular protein that is important for nucleocytoplasmic and cytosolic trafficking of the HIV-1 genomic RNA. Both hnRNP A2's interaction with HIV-1 RNA and its expression levels influence the activities of Rev in mediating nucleocytoplasmic export of the HIV-1 genomic RNA. While the lack of Rev expression during HIV-1 gene expression results in nuclear retention of HIV-1 genomic RNA, we show here by fluorescence in situ hybridization and fractionation studies that the genomic RNA translocates to the cytoplasm when hnRNP A2/B1 are depleted from cells. Polyribosome analyses revealed that the genomic RNA was shunted into a cytoplasmic, dense polyribosomal fraction. This fraction contained several RNA-binding proteins involved in viral gene expression and RNA trafficking but did not contain the translation initiation factor, eIF4G1. Amino acid incorporation into nascent polypeptides in this fraction was also greatly reduced, demonstrating that this fraction contains mRNAs that are poorly translated. These results demonstrate that hnRNP A2/B1 expression plays roles in the nuclear retention of the HIV-1 genomic RNA in the absence of Rev and in the release of the genomic RNA from translationally inactive, cytoplasmic RNP complexes.

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.

Sustained inhibition of progressive joint damage with rituximab plus methotrexate in early active rheumatoid arthritis: 2-year results from the randomised controlled trial IMAGE.

BACKGROUND: In the IMAGEstudy, rituximab plus methotrexate (MTX) inhibited joint damage and improved clinical outcomes at 1 year in MTX-naïve patients with early active rheumatoid arthritis. OBJECTIVE: The aim of this study was to assess joint damage progression and clinical outcomes over 2 years. METHODS: Patients (n=755) were randomised to receive rituximab 2×500 mg+MTX, 2×1000 mg+MTX or placebo+MTX. The placebo-controlled period continued to week 104. Two-year end points were defined as secondary or exploratory and included change in total Genant-modified Sharp score (mTSS), total erosion score and joint space narrowing score from baseline to week 104. Clinical efficacy and physical function end points were also assessed. RESULTS: At 2 years, rituximab 2×1000 mg+MTX maintained inhibition of progressive joint damage versus MTX alone (mTSS change 0.41 vs 1.95; p<0.0001 (79% inhibition)), and a higher proportion of patients receiving rituximab 2×1000 mg+MTX had no radiographic progression over 2 years compared with those receiving MTX alone (57% vs 37%; p<0.0001). Contrary to 1-year results, exploratory analysis of rituximab 2×500 mg+MTX at 2 years showed that progressive joint damage was slowed by ∼61% versus placebo+MTX (mTSS, exploratory p=0.0041). Improvements in clinical signs and symptoms and physical function seen after 1 year in rituximab-treated patients versus those receiving placebo were maintained at year 2. Safety profiles were similar between groups. CONCLUSIONS: Treatment with rituximab 2×1000 mg+MTX was associated with sustained improvements in radiographic, clinical and functional outcomes over 2 years. Clinical trials.gov identifier NCT00299104.