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Rheumatoid arthritis (RA) is a systemic and incurable autoimmune disease characterized by chronic inflammation in synovial lining of joints. To identify the signaling pathways involved in RA, its disease activity, and treatment response, we adapted a systems immunology approach to simultaneously quantify 42 signaling nodes in 21 immune cell subsets (e.g., IFNα→p-STAT5 in B cells) in peripheral blood mononuclear cells (PBMC) from 194 patients with longstanding RA (including 98 patients before and after treatment), and 41 healthy controls (HC). We found multiple differences between patients with RA compared to HC, predominantly in cytokine-induced Jak/STAT signaling in many immune cell subsets, suggesting pathways that may be associated with susceptibility to RA. We also found that high RA disease activity, compared to low disease activity, was associated with decreased (e.g., IFNα→p-STAT5, IL-10→p-STAT1) or increased (e.g., IL-6→STAT3) response to stimuli in multiple cell subsets. Finally, we compared signaling in patients with established, refractory RA before and six months after initiation of methotrexate (MTX) or TNF inhibitors (TNFi). We noted significant changes from pre-treatment to post-treatment in IFNα→p-STAT5 signaling and IL-10→p-STAT1 signaling in multiple cell subsets; these changes brought the aberrant RA signaling profiles toward those of HC. This large, comprehensive functional signaling pathway study provides novel insights into the pathogenesis of RA and shows the potential of quantification of cytokine-induced signaling as a biomarker of disease activity or treatment response.

Rheumatoid arthritis (RA), a chronic autoimmune disease characterized by circulating autoantibodies, involves many cytokine-mediated signaling pathways in multiple immune cell subsets. Most studies of immune cells in RA have limitations, such as analysis of a small number of cell subsets or pathways, and limited longitudinal data on patient phenotypes. In this study, we used an innovative systems immunology approach to simultaneously quantify up to 882 signaling nodes (Jak/STAT signaling readouts modulated by cytokines and other stimuli) in 21 immune cell subsets. We studied 194 RA patients and 41 controls, including 146 well-characterized RA patients prior to, and 6 months after, initiation of methotrexate or biologic agents from the Treatment Efficacy and Toxicity in RA Database and Repository (TETRAD). There was strikingly attenuated signaling capacity in RA patients in IFNα stimulation followed by measurement of phosphorylated STAT1 [IFNα→p-STAT1] in six immune cell subsets. Multiple nodes showed negative association with disease activity, including IFNα→STAT5 signaling in naive and memory B cells. In contrast, IL-6-induced STAT1 and STAT3 activation in central memory CD4-negative T cells showed a positive association with disease activity. Multiple nodes were associated with treatment response, including IFNα→STAT1 in monocytes and IL-6→STAT3 in CD4+ naive T cells. Decision tree analysis identified a model combining these two nodes as a high-performing classifier of treatment response to TNF inhibitors. Our study provides novel information on RA disease mechanisms and serves as a framework for the discovery and validation of biomarkers of treatment response in RA.

The 620W allelic variant of the intracellular tyrosine phosphatase, PTPN22, is associated with a number of different autoimmune disorders, and this provides direct evidence for common mechanisms underlying many of these diseases. The associated allele appears to influence thresholds for T cell receptor signaling, and a variety of disease models involving both central and peripheral tolerance can be proposed. However, given the fact that PTPN22 is expressed in a variety of immunologically relevant cell types, the precise mechanisms for these associations remain unclear. In general, the PTPN22 620W allele appears to play a role in autoimmune disorders that have a prominent humoral component, suggesting that further investigation of PTPN22 activity in B cells will be useful. From a genetic perspective, the data highlights the genetic heterogeneity underlying autoimmunity in different ethnic groups.

We have completed a genome wide linkage scan using >5700 informative single-nucleotide polymorphism (SNP) markers (Illumina IV SNP linkage panel) in 642 Caucasian families containing affected sibling pairs with rheumatoid arthritis (RA), ascertained by the North American Rheumatoid Arthritis Consortium. The results show striking new evidence of linkage at chromosomes 2q33 and 11p12 with logarithm of odds (LOD) scores of 3.52 and 3.09, respectively. In addition to a strong and broad linkage interval surrounding the major histocompatibility complex (LOD>16), regions with LOD>2.5 were observed on chromosomes 5 and 10. Additional linkage evidence (LOD scores between 1.46 and 2.35) was also observed on chromosomes 4, 7, 12, 16 and 18. This new evidence for multiple regions of genetic linkage is partly explained by the significantly increased information content of the Illumina IV SNP linkage panel (75.6%) compared with a standard microsatellite linkage panel utilized previously (mean 52.6%). Stratified analyses according to whether or not the sibling pair members showed elevated anticyclic citrullinated peptide titers indicates significant variation in evidence for linkage among strata on chromosomes 4, 5, 6 and 7. Overall, these new linkage data should reinvigorate efforts to utilize positional information to identify susceptibility genes for RA.

We carried out gene expression profiling of peripheral blood mononuclear cells (PBMCs) in 29 patients with active rheumatoid arthritis (RA) and 21 control subjects using Affymetrix U95Av2 arrays. Using cluster analysis, we observed a significant alteration in the expression pattern of 81 genes (P

Objective To examine the association between HLA–DRB1 alleles and the production of anti–cyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF) autoantibodies in patients with rheumatoid arthritis (RA). Methods We studied 1,723 Caucasian RA patients enrolled in the North American Rheumatoid Arthritis Consortium (NARAC) family cohort and the Study of New Onset Rheumatoid Arthritis (SONORA) cohort. All patients were tested for anti-CCP antibodies (by enzyme-linked immunosorbent assay), RF (by nephelometry), and HLA–DR genotype (by polymerase chain reaction and sequence-specific oligonucleotide hybridization). Results When controlled for the presence of RF, anti-CCP positivity was strongly associated with the HLA–DRB1 shared epitope (SE). In RF+ patients, the presence of the SE was very significantly associated with anti-CCP positivity, with an odds ratio (OR) of 5.8 and a 95% confidence interval (95% CI) of 4.1–8.3. This relationship was also seen in RF– patients (OR 3.1 [95% CI 1.8–5.3]). In contrast, RF positivity was not significantly associated with presence of the SE independently of anti-CCP antibodies. Strikingly, HLA–DRB1*03 was strongly associated with reduced anti-CCP titers, even after controlling for the presence of the SE and restricting the analysis to anti-CCP+ patients. HLA–DR3 was also associated with anti-CCP– RA in our population. Conclusion The HLA–DRB1 SE is strongly associated with the production of anti-CCP antibodies, but not RF. In contrast, HLA–DR3 alleles are associated with anti-CCP– disease and with lower levels of anti-CCP antibodies, even when controlling for the SE. These data emphasize the complexity of the genetic effects of the major histocompatibility complex on the RA phenotype.

Monitoring of gene and protein expression in peripheral blood cells has significant potential for improving the diagnosis and therapy of many human diseases. As genomic-scale microarray and proteomic technologies are applied to peripheral blood, it is important to consider the variables that may affect interpretation of data. Here we report experiments performed to identify genes that are particularly sensitive to ex vivo handling prior to RNA extraction for gene expression microarrays or quantitative real-time RT-PCR assays. We examined Affymetrix gene expression in samples from eight normal individuals where blood was processed for RNA either immediately after blood draw or the next day following overnight incubation. These studies identified hundreds of genes that are sensitive to ex vivo handling of blood, and suggest that this is an important variable to consider when designing and interpreting human PBMC experiments.

High mobility group box chromosomal protein 1 (HMGB-1), a DNA binding protein, is an intracellular protein that facilitates DNA bending, stabilizes nucleosome formation, and modulates the interactions of regulatory molecules with their targets. Recent evidence has revealed that HMGB-1 is also a cytokine, because it 1) is released from activated monocyte/macrophages, 2) mediates delayed endotoxin lethality, 3) activates macrophages, 4) stimulates inflammation in vivo, and 5) is a therapeutic target in diverse animal models of inflammatory disease (1–4). Our review accompanies the report by Taniguchi and colleagues (5), in this issue of Arthritis & Rheumatism, of a new study of HMGB-1 in the pathogenesis of arthritis. We summarize here the history of research on HMGB-1 as an intracellular protein and discuss in more detail a rapidly growing body of evidence that implicates HMGB-1 as a cytokine that can mediate inflammation and arthritis. Some important new findings are highlighted, and a series of observations that warrant additional study are raised. The molecular basis of the cytokine activity of HMGB-1 and the biochemistry of this potentially important therapeutic target are discussed.

Systemic lupus erythematosus (SLE) is a complex, inflammatory autoimmune disease that affects multiple organ systems. We used global gene expression profiling of peripheral blood mononuclear cells to identify distinct patterns of gene expression that distinguish most SLE patients from healthy controls. Strikingly, about half of the patients studied showed dysregulated expression of genes in the IFN pathway. Furthermore, this IFN gene expression “signature” served as a marker for more severe disease involving the kidneys, hematopoetic cells, and/or the central nervous system. These results provide insights into the genetic pathways underlying SLE, and identify a subgroup of patients who may benefit from therapies targeting the IFN pathway.

We have previously reported that CD8(+)CD28(-) T cells have relatively shorter telomeres compared with CD8(+)CD28(+) T cells. Oligoclonal expansion is a common feature of CD8(+) T cells in human peripheral blood, and these expansions predominantly occur in the CD57(+)/CD28(-) population. We studied the telomere length in subsets of CD8(+) T cells using quantitative fluorescence in situ hybridization and flow cytometry (flow FISH). Our results confirm that CD8(+)CD28(-) T cells have shorter telomeres as compared with their CD28(+) counterpart cells. In addition, the oligoclonally expanded cells within the CD8(+)CD28(-) T cell subset generally have even shorter telomeres than the CD28(-) subset as a whole. We conclude that the presence of clonal expansions in the CD8(+)CD28(-) T cell population largely explain the shorter telomeres in this subset. These clonally expanded CD8(+)CD28(-) T cells generally have characteristics of terminally differentiated effector cells. Nevertheless, there is considerable individual variation in the degree of telomere shortening in these cells, which may reflect host genetic factors as well as the type and timing of the antigenic exposure.

The development of effective adjuvant therapies for the treatment of high-risk melanoma patients is critical for the prevention of metastatic disease and improvement of patient survival. Active specific immunotherapy has been tested as an adjuvant treatment in numerous clinical trials with overall limited, but occasionally promising, success rates. Newcastle disease virus (NDV) oncolysate has been utilized as an adjunctive immunotherapeutic agent in the postsurgical management of these patients. A phase II study initiated in 1975 using adjuvant vaccine therapy composed of allogeneic and autologous human melanoma cells infected with live NDV (NDV oncolysate) in patients with AJCC stage III melanoma following therapeutic lymph node dissection has shown >60% survival rate at 10 years with no adverse effects. Continued long-term analysis of trials with promising early results as well as assessment of immunologic responses generated in these patients may result in improved therapeutic decisions for clinical trials in the future. We analyzed the 15-year survival of patients treated postsurgically with NDV oncolysate in the phase II study described above. In an attempt to understand the immunological effects of this treatment, we have also carried out a comprehensive analysis of the peripheral blood T cell repertoire in these patients. The overall 15-year survival of this group of patients is 55%. Previous studies have suggested that improved outcome in patients undergoing immunotherapy is correlated with increased numbers of CD8+CD57+ cells. In surviving patients, we observed a striking oligoclonality in the CD8+ T cell population in peripheral blood, which reflects clonal expansions in the CD8+CD57+ subset. The data suggest that adjuvant vaccination with NDV oncolysates is associated with prolonged survival of patients with lymph node-positive malignant melanoma and that CD8+ T cells may be an important component of therapeutic efficacy.

To elucidate endogenous mechanisms underlying cerebral damage during ischemia, brain polyamine oxidase activity was measured in rats subjected to permanent occlusion of the middle cerebral artery. Brain polyamine oxidase activity was increased significantly within 2 h after the onset of ischemia in brain homogenates (15.8 ± 0.9 nmol/h/mg protein) as compared with homogenates prepared from the normally perfused contralateral side (7.4 ± 0.5 nmol/h/mg protein) (P

(Auto)antigen engagement by the B-cell receptor (BCR) and possibly the sites where this occurs influence the outcome of chronic lymphocytic leukemia (CLL). To test if selection for autoreactivity leads to increased aggressiveness and if this selection plays out equally in primary and secondary tissues, we used T-cell leukemia (TCL)1 cells reactive with the autoantigen phosphatidylcholine (PtC). After repeated transfers of splenic lymphocytes from a single mouse with oligoclonal PtC-reactive cells, outgrowth of cells expressing a single IGHV-D-J rearrangement and superior PtC-binding and disease virulence occurred. In secondary tissues, increased PtC-binding correlated with enhanced BCR signaling and cell proliferation, whereas reduced signaling and division of cells from the same clone was documented in cells residing in the bone marrow, blood, and peritoneum, even though cells from the last site had highest surface membrane IgM density. Gene-expression analyses revealed reciprocal changes of genes involved in BCR-, CD40-, and PI3K-signaling between splenic and peritoneal cells. Our results suggest autoantigen-stimulated BCR signaling in secondary tissues promotes selection, expansion, and disease progression by activating pro-oncogenic signaling pathways, and that--outside secondary lymphoid tissues--clonal evolution is retarded by diminished BCR-signaling. This transferrable, antigenic-specific murine B-cell clone (TCL1-192) provides a platform to study the types and sites of antigen-BCR interactions and genetic alterations that result and may have relevance to patients.

Oligoclonality of the CD8+ T cell subset is a common and characteristic feature of the normal human peripheral T cell repertoire. These clonally expanded populations are predominantly found in a CD57+ or CD28- CD8+ T cell subset. While CD8 oligoclonality is somewhat more common in the older age group, it is also very prevalent in young to middle-aged adults. Recent experiments have also demonstrated that the clonally expanded populations may actually occur in two distinct subpopulations of CD8+ CD28- cells, distinguished by the expression of the CD57 surface marker. A major difficulty with studies involving CD8+ CD28- CD57+ T cells is their relative lack of proliferative capacity. We have recently investigated the possibility that this phenotype may be due to a state of "replicative senescence" in some cases. In this regard, we have demonstrated that the telomere lengths of CD8+ CD28- T cells are generally shorter than that of their CD8+ CD28+ counterparts, consistent with a distinct replicative history for the CD8+ CD28- population. Additional studies of the normal biology of clonally expanded CD8+ T cells are likely to yield important insights into immune function in health and disease.

Griffin et al., respond: In January 2011, we reported the unique CD20+CD27+CD43+ phenotype of human B1 cells (Griffin et al., 2011a). In December 2011, we reported that the human B1 cell pool consists of functionally distinct CD11b− and CD11b+ populations, with the former particularly proficient at antibody secretion and the latter particularly efficient in stimulating T cells (Griffin and Rothstein, 2011). We also noted that the frequency of CD11b+ B1 cells is increased in the circulation of patients with lupus. As part of this work, we included microarray data that pointed to transcriptional differences between CD11b− and CD11b+ B1 cells. We have read with great interest the letter by Reynaud and Weill, who have mined the microarray data we deposited in the National Center for Biotechnology Information (NCBI) repository and drawn inferences, incorrectly as it turns out, regarding the nature and origin of CD11b− and CD11b+ B1 cells. Reynaud and Weill speculated that CD11b− B1 cells represent B cell–T cell doublets because this population, in aggregate, expressed genes characteristic of T cells along with genes characteristic of B cells. The potential issue of B cell–T cell doublets, raised previously by these same authors (Descatoire et al., 2011), is a topic we have already addressed by demonstrating that our sort-purified B1 cells exist as singlets (Griffin and Rothstein, 2011; Griffin et al., 2011b). At the same time, we identified procedures that can be used to circumvent the potential problem of doublet events (Griffin et al., 2011b). In our published work, we reported that B1 cells sort-purified without CD3− gating contained an estimated 1–3% CD3+ events (Griffin and Rothstein, 2011; Griffin et al., 2011b). We have now sort-purified CD11b− B1 cells again, exactly as was done to generate our published microarray results, and have analyzed three independent postsort samples for CD3 expression, along with purified T cells (Fig. 1 A). We again find a very small proportion of CD3+ events (1.9 ± 0.66%; mean ± SEM) in the CD11b− B1 cell population. This very low level of CD3 positivity is likely attributable to a minor degree of T cell contamination that could account for the appearance of very low levels of some characteristically T cell transcripts among CD11b− B1 cells. In keeping with this, flow cytometric analysis of unsorted CD19-enriched PBMCs demonstrates a small degree of CD2, CD7, CD8, and CD3 staining among CD11b− B1 cells that is eliminated by CD3 exclusion and subsequent analysis of CD3−CD20+CD27+CD43+CD11b− B1 cells (Fig. 1, B and C). Importantly, when CD3 exclusion is combined with sort purification, expression of T cell genes such as CD7, CD8, and granzyme A is essentially eliminated (to

Objective Anti–tumor necrosis factor α (anti-TNF) therapy is a mainstay of treatment in rheumatoid arthritis (RA). The aim of the present study was to test established RA genetic risk factors to determine whether the same alleles also influence the response to anti-TNF therapy. Methods A total of 1,283 RA patients receiving etanercept, infliximab, or adalimumab therapy were studied from among an international collaborative consortium of 9 different RA cohorts. The primary end point compared RA patients with a good treatment response according to the European League Against Rheumatism (EULAR) response criteria (n = 505) with RA patients considered to be nonresponders (n = 316). The secondary end point was the change from baseline in the level of disease activity according to the Disease Activity Score in 28 joints (▵DAS28). Clinical factors such as age, sex, and concomitant medications were tested as possible correlates of treatment response. Thirty-one single-nucleotide polymorphisms (SNPs) associated with the risk of RA were genotyped and tested for any association with treatment response, using univariate and multivariate logistic regression models. Results Of the 31 RA-associated risk alleles, a SNP at the PTPRC (also known as CD45) gene locus (rs10919563) was associated with the primary end point, a EULAR good response versus no response (odds ratio [OR] 0.55, P = 0.0001 in the multivariate model). Similar results were obtained using the secondary end point, the ▵DAS28 (P = 0.0002). There was suggestive evidence of a stronger association in autoantibody-positive patients with RA (OR 0.55, 95% confidence interval [95% CI] 0.39–0.76) as compared with autoantibody-negative patients (OR 0.90, 95% CI 0.41–1.99). Conclusion Statistically significant associations were observed between the response to anti-TNF therapy and an RA risk allele at the PTPRC gene locus. Additional studies will be required to replicate this finding in additional patient collections.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by unpredictable flares of disease activity and irreversible damage to multiple organ systems. An earlier study showed that SLE patients carrying an interferon (IFN) gene expression signature in blood have elevated serum levels of IFN-regulated chemokines. These chemokines were associated with more-severe and active disease and showed promise as SLE disease activity biomarkers. This study was designed to validate IFN-regulated chemokines as biomarkers of SLE disease activity in 267 SLE patients followed up longitudinally.To validate the potential utility of serum chemokine levels as biomarkers of disease activity, we measured serum levels of CXCL10 (IFNgamma-inducible 10-kd protein), CCL2 (monocyte chemotactic protein 1), and CCL19 (macrophage inflammatory protein 3beta) in an independent cohort of 267 SLE patients followed up longitudinally over 1 year (1,166 total clinic visits).Serum chemokine levels correlated with lupus activity at the current visit (P = 2 x 10(-10)), rising at the time of SLE flare (P = 2 x 10(-3)) and decreasing as disease remitted (P = 1 x 10(-3)); they also performed better than the currently available laboratory tests. Chemokine levels measured at a single baseline visit in patients with a Systemic Lupus Erythematosus Disease Activity Index ofor =4 were predictive of lupus flare over the ensuing year (P = 1 x 10(-4)).Monitoring serum chemokine levels in SLE may improve the assessment of current disease activity, the prediction of future disease flares, and the overall clinical decision-making.

Rheumatoid arthritis is a chronic inflammatory disease with a substantial genetic component. Susceptibility to disease has been linked with a region on chromosome 2q.We tested single-nucleotide polymorphisms (SNPs) in and around 13 candidate genes within the previously linked chromosome 2q region for association with rheumatoid arthritis. We then performed fine mapping of the STAT1-STAT4 region in a total of 1620 case patients with established rheumatoid arthritis and 2635 controls, all from North America. Implicated SNPs were further tested in an independent case-control series of 1529 patients with early rheumatoid arthritis and 881 controls, all from Sweden, and in a total of 1039 case patients and 1248 controls from three series of patients with systemic lupus erythematosus.A SNP haplotype in the third intron of STAT4 was associated with susceptibility to both rheumatoid arthritis and systemic lupus erythematosus. The minor alleles of the haplotype-defining SNPs were present in 27% of chromosomes of patients with established rheumatoid arthritis, as compared with 22% of those of controls (for the SNP rs7574865, P=2.81x10(-7); odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.32). The association was replicated in Swedish patients with recent-onset rheumatoid arthritis (P=0.02) and matched controls. The haplotype marked by rs7574865 was strongly associated with lupus, being present on 31% of chromosomes of case patients and 22% of those of controls (P=1.87x10(-9); odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.55). Homozygosity of the risk allele, as compared with absence of the allele, was associated with a more than doubled risk for lupus and a 60% increased risk for rheumatoid arthritis.A haplotype of STAT4 is associated with increased risk for both rheumatoid arthritis and systemic lupus erythematosus, suggesting a shared pathway for these illnesses.

Objective Recently, Swedish members of the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) provided evidence that smoking may trigger RA-specific immune reactions to citrullinated protein in carriers of HLA–DR shared epitope alleles. In an effort to confirm this interaction between smoking and shared epitope alleles, we performed a case-only analysis of 3 North American RA cohorts. Methods A total of 2,476 white patients with RA were studied, 1,105 from the North American Rheumatoid Arthritis Consortium (NARAC) family collection, 753 from the National Inception Cohort of Rheumatoid Arthritis Patients (Inception Cohort), and 618 from the Study of New Onset Rheumatoid Arthritis (SONORA). All patients were HLA–DRB1 typed, and tested for anti–cyclic citrullinated peptide (anti-CCP) and rheumatoid factor. Information about smoking history was obtained by questionnaire. Results A significant association was found between smoking and the presence of anti-CCP in the NARAC and the Inception Cohort, but not in the SONORA. The shared epitope alleles consistently correlated with anti-CCP in all 3 populations. Using multiple logistic regression analyses, shared epitope alleles were still the most significant risk factor for anti-CCP positivity. Weak evidence of gene–environment interaction between smoking and shared epitope alleles for anti-CCP formation was found only in the NARAC. Conclusion Unlike the EIRA data, we could not confirm a major gene–environment interaction for anti-CCP formation between shared epitope alleles and smoking in 3 North American RA cohorts. Our data indicate a need for further studies to address the full range of environmental factors other than smoking that may be associated with citrullination and RA.

Background: The risk of venous thromboembolism (VTE) in hospitalized medical patients remains high and pharmacologic thromboprophylaxis remains suboptimal. Current national guidelines call for physicians to assess an individual patientÕs VTE and bleeding risk, prior to administering VTE prophylaxis. Therefore, Risk Assessment Models (RAMs) are needed to identify those patients at high risk for a bleed. The International Medical Prevention Registry on Venous Thromboembolism (IMPROVE) bleed RAM remains the only evidence-derived and weighted bleed risk model in this population using 11 clinical and laboratory factors (Table 1). However, this RAM has not undergone external validation. The aim of our study is to validate the IMPROVE bleed RAM for hospitalized medical patients in a large tertiary health system. Methods A retrospective chart review was conducted, utilizing data from our electronic health records (EHR) and administrative data systems, between October 1st 2012 and July 31st 2014. After applying the inclusion and exclusion criteria as outlined in the IMPROVE study, and accounting for missing EHR data, we identified 12082 subjects. Missing data included INR, platelet count, accurate weights and gender. Within this population we identified each of the eleven IMPROVE RAM risk factors utilizing administrative (ICD9) and EMR data. We identified outcomes, major (MB) or non-major bleeds (NMB), using ICD9 billing codes. To validate the IMPROVE bleed RAM, we then applied the point scoring system to compute risk scores for each patient in the validation sample. Lastly, we dichotomized the patients into those with a score =7 (high risk), as outlined in the original study, and compared the frequencies/rates of bleeding in the derivation and validation samples at these two different levels. All analyses were carried out in SAS Version 9.3 (Cary, NC). Results: Among the 12082 subjects, 2.6% had a bleed within 14 days (compared with 2.1% in the original IMPROVE inception cohort). In our population, overall bleeding rates were 2.2% in those with a score =7. NMB occurred in 1.29% of patients with a score =7, p=7, (p Conclusion: This study represents the first external validation of an evidence-derived and weighted bleed RAM, the IMPROVE bleed RAM, in a hospitalized medically ill patient population. A cut-point score of 7 or above was able to identify a high-risk patient group for MB and NMB, with a doubling of the bleed rates, consistent with the original derivation score. The high NPV for any bleed and MB are evidence that the RAM is also useful in the clinical setting to predict those patients at low risk of a bleed. However, a limitation of this study was the retrospective study design and reliance on claims data, likely resulting in an underestimation of NMB and the magnitude of effect the score has on bleed risk. The IMPROVE bleed RAM has the potential to allow for more tailored approaches to pharmacologic thromboprophylaxis in hospitalized medical patients. Table 1. IMPROVE Bleed Score Risk Factors Points Moderate Renal Failure (GFR 30-59 vs. >= 60 mL/min/m^2) 1 Male vs. Female 1 Age, 40-84 vs. = 60 mL/min/m^2) 2.5 Hepatic Failure (INR>1.5) 2.5 Age >=85 vs. Disclosures Spyropoulos: Daiichi Sankyo, Inc.: Consultancy.

The current methods for molecular typing of HLA-DR alleles incur a substantial financial burden when performing large population studies. In the current study, we aimed to provide much less expensive typing approach with high predictability for DRB1 genotype. We have used a panel of three microsatellite markers in the class II region (D6S2666, D6S2665 and D6S2446) for genotyping and haplotype reconstruction in a total of 1687 Caucasian (1313 RA patients and 374 controls) and 1364 Korean individuals (744 RA patients and 620 controls), all of whom were previously genotyped for DRB1. We found that a total of 88.4 and 87.4% of all observed three-marker haplotypes could determine the DR type with a positive predictive value >0.8 with high sensitivity and specificity. There was a high degree of haplotype conservation when comparing Caucasian and Asian populations. Interestingly, we found that the majority of DRB1*09 and DRB1*10 alleles share a common three-marker haplotype in both Caucasian and Asian populations. This is unexpected, since these two alleles are found on very different haplotype families. In addition, these two alleles are both associated with rheumatoid arthritis, making the elucidation of these haplotype relationships potentially important for understanding disease susceptibility.

Human autoimmune diseases are well suited for the application of gene expression profiling. Sampling of blood cells and target tissues has already revealed many important pathways contributing to this spectrum of disorders, and many commonalities are emerging. For instance, clinically distinct diseases such as systemic lupus erythematosus, Sjogren's syndrome, dermatomyositis, and psoriasis all show evidence for dysregulation of the type I interferon pathway. These data suggest that autoimmune diseases will eventually be categorized at the level of gene expression. This work has led to advances in our understanding of disease pathogenesis and in the future promises to facilitate assessments of disease activity and improve targeting of therapies. Here, we review the literature on gene profiling in human autoimmune diseases and provide perspective on the current state of the art.

Background Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disorder that affects multiple organ systems and is characterized by unpredictable flares of disease. Recent evidence indicates a role for type I interferon (IFN) in SLE pathogenesis; however, the downstream effects of IFN pathway activation are not well understood. Here we test the hypothesis that type I IFN-regulated proteins are present in the serum of SLE patients and correlate with disease activity. Methods and Findings We performed a comprehensive survey of the serologic proteome in human SLE and identified dysregulated levels of 30 cytokines, chemokines, growth factors, and soluble receptors. Particularly striking was the highly coordinated up-regulation of 12 inflammatory and/or homeostatic chemokines, molecules that direct the movement of leukocytes in the body. Most of the identified chemokines were inducible by type I IFN, and their levels correlated strongly with clinical and laboratory measures of disease activity. Conclusions These data suggest that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed in human SLE. Furthermore, the levels of serum chemokines may serve as convenient biomarkers for disease activity in lupus., A comprehensive survey of the serologic proteome in human SLE suggests that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed., Editors' Summary Background. The term “lupus,” meaning wolf in Latin, is often used as an abbreviation for the disease systemic lupus erythematosus (SLE). The name may have been given because some people with SLE have a rash that slightly resembles a wolf's face. The condition affects around 50 to 100 people per 100,000, and is much more common in women than men. SLE is a complicated disease that comes about when antibodies inappropriately attack the body's own connective tissues, although it is not known why this happens. Symptoms vary between different people; the disease may get better and then worse, without explanation; and can affect many different organs including the skin, joints, kidneys, blood cells, and brain and nervous system. SLE is difficult for doctors to diagnose. Although the disease cannot be cured, patients who are diagnosed with SLE can be treated for their symptoms, and the right management can slow progress of the disease. One area of SLE research focuses on finding “molecular markers” (e.g., proteins or other compounds) that could be tested for in the blood. Researchers hope this would help doctors to more accurately diagnose SLE initially, and then also help to track progress in a patient's condition. Why Was This Study Done? “Gene expression” is a term meaning the process by which a gene's DNA sequence is converted into the structures and functions of a cell. These investigators had found in previous studies that certain genes were more “highly expressed” in the blood cells of patients with SLE. Some of these genes were already known to be regulated by interferons (a group of proteins, produced by certain blood cells, that are important in helping to defend against viral infections). The investigators performing this study wanted to understand more clearly the role of interferon in SLE and to see whether the genes that are more highly expressed in patients with SLE go on to produce higher levels of protein, which might then provide useful markers for monitoring the condition. What Did the Researchers Do and Find? This research project was a “case-control” study, in which the researchers compared the levels of certain proteins in the blood of people who had SLE with the levels in people who did not have the condition. Thirty people were recruited as cases, from a group of patients with SLE who have been under evaluation at Johns Hopkins School of Medicine since 1987. Fifteen controls were recruited from a group of healthy people of similar age and sex as the patients with SLE; everyone involved in the study gave their consent to take part. Blood samples were taken from each individual, and the serum (liquid component of blood) was separated out. The serum levels of 160 different blood proteins were then measured. When comparing levels of blood proteins between the groups, the researchers found that 30 specific proteins were present at higher or lower levels in the SLE-affected patients. Many of these proteins are cytokines, which are regulated by interferons and are involved in the process of “signaling” within the immune system. A few proteins were found at lower levels. Levels of the interferon-regulated proteins were, on average, seen at higher levels in people whose condition was more severe. What Do These Findings Mean? These results suggest that patients with SLE are likely to have a very different pattern of regulation of certain proteins within the blood, particularly the proteins involved in signaling within the immune system. The authors propose that these proteins may be involved in the progression of the disease. There is also the possibility that some of these proteins may prove useful in diagnostic tests, or in tests for monitoring how the disease progresses. However, before any such tests could be used in clinical practice, they would need to be further developed and then thoroughly tested in clinical trials. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030491 Patient information from the UK National Health Service on systemic lupus erythematosus Patient handout from the US National Institutes of Health MedlinePLUS encyclopedia entry on lupus Information on lupus from the UK Arthritis Research Campaign

Psoriatic arthritis (PsA) is a chronic and erosive form of arthritis of unknown cause. We aimed to characterize the PsA phenotype using gene expression profiling and comparing it with healthy control subjects and patients rheumatoid arthritis (RA). Peripheral blood cells (PBCs) of 19 patients with active PsA and 19 age- and sex-matched control subjects were used in the analyses of PsA, with blood samples collected in PaxGene tubes. A significant alteration in the pattern of expression of 313 genes was noted in the PBCs of PsA patients on Affymetrix U133A arrays: 257 genes were expressed at reduced levels in PsA, and 56 genes were expressed at increased levels, compared with controls. Downregulated genes tended to cluster to certain chromosomal regions, including those containing the psoriasis susceptibility loci PSORS1 and PSORS2. Among the genes with the most significantly reduced expression were those involved in downregulation or suppression of innate and acquired immune responses, such as SIGIRR, STAT3, SHP1, IKBKB, IL-11RA, and TCF7, suggesting inappropriate control that favors proin-flammatory responses. Several members of the MAPK signaling pathway and tumor suppressor genes showed reduced expression. Three proinflammatory genes--S100A8, S100A12, and thioredoxin--showed increased expression. Logistic regression and recursive partitioning analysis determined that one gene, nucleoporin 62 kDa, could correctly classify all controls and 94.7% of the PsA patients. Using a dataset of 48 RA samples for comparison, the combination of two genes, MAP3K3 followed by CACNA1S, was enough to correctly classify all RA and PsA patients. Thus, PBC gene expression profiling identified a gene expression signature that differentiated PsA from RA, and PsA from controls. Several novel genes were differentially expressed in PsA and may prove to be diagnostic biomarkers or serve as new targets for the development of therapies.

We genotyped 525 independent North American white individuals with systemic lupus erythematosus (SLE) for the PTPN22 R620W polymorphism and compared the results with data generated from 1,961 white control individuals. The R620W SNP was associated with SLE (genotypic P=.00009), with estimated minor (T) allele frequencies of 12.67% in SLE cases and 8.64% in controls. A single copy of the T allele (W620) increases risk of SLE (odds ratio [OR]=1.37; 95% confidence interval [CI] 1.07–1.75), and two copies of the allele more than double this risk (OR=4.37; 95% CI 1.98–9.65). Together with recent evidence showing association of this SNP with type 1 diabetes and rheumatoid arthritis, these data provide compelling evidence that PTPN22 plays a fundamental role in regulating the immune system and the development of autoimmunity.

Patients with B-cell chronic lymphocytic leukemia (B-CLL) segregate into subgroups with very different survival times. Because clinical observations suggest that leukemic cells accumulate at different rates, we measured telomere length and telomerase activity in B-CLL cells to distinguish differences in cellular replication. Our data indicate that the telomeres of B-CLL cells are shorter than telomeres of B cells from healthy subjects, indicating that the leukemic cells have a prolonged proliferative history. Leukemic cells of the immunoglobulin V gene mutation subgroups differ in telomere length and telomerase activity. B lymphocytes from the subgroup with poor outcome and with limited IgV gene mutations have uniformly shorter telomeres and more telomerase activity than those from the subgroup with better outcome and with considerable mutations. Differences in telomere length appear to largely reflect the proliferative histories of precursors of the leukemic cells, although differences in cell division, masked by the action of telomerase, cannot be excluded. These results may provide insight into the stages of maturation and the activation pathways of the cells that give rise to B-CLL. In addition, they reinforce the concept that B-CLL is not simply an accumulative disease of slowly dividing B lymphocytes but possibly one of B cells with extensive proliferative histories.

Telomere Flow FISH is a recently developed method which allows the measurement of telomere length in purified subsets of cells using flow cytometry. However, the harsh conditions required for flow FISH have precluded its use with conventional cell surface staining, thus limiting its utility for large scale clinical studies. We have now developed a method which permits simultaneous analysis of cell surface markers along with telomere length estimation by flow cytometry. This new assay employs the covalent crosslinking of monoclonal antibodies conjugated with a heat stable fluorochrome to the cell surface prior to flow FISH. Using this technique we have confirmed that human germinal center B cells (IgD(-)/CD38(+)) have dramatically longer telomeres than pre-germinal center founder B cells (IgD(+)/CD38(+)). This approach simplifies the analysis of complex cell populations and will facilitate widespread investigation of telomere length in health and disease states.

CD8-positive T cells are thought to play an important role in the control of infection by human immunodeficiency virus (HIV) as a result of their cytotoxic activity and by releasing soluble factors. In AIDS patients, the absolute number of CD8+ T lymphocytes is decreased in peripheral blood and their turnover rate is increased, suggesting that there is more cell renewal and cell death occurring. Anti-retroviral therapy raises CD8+ T-cell counts in HIV-infected patients. Here we report that the death rate of CD8+ T cells by apoptosis increased markedly during HIV infection of peripheral blood mononuclear cells in vitro. Apoptosis is induced in a dose-dependent manner by recombinant envelope glycoprotein gp120 from HIV strain X4, or by stromal-derived factor-1 (SDF-1), the physiological ligand of the chemokine receptor CXCR4. Apoptosis is mediated by the interaction between tumour-necrosis factor-alpha bound to the membrane of macrophages (mbTNF) and a receptor on CD8+ T cells (TNF-receptor II, or TNFRII). The expression of both of these cell-surface proteins is upregulated by HIV infection or by treatment with recombinant gp120 or SDF-1. Apoptosis of CD8+ T cells isolated from HIV-infected patients is also mediated by macrophages through the interaction between mbTNF and TNFRII. These results indicate that the increased turnover of CD8+ T cells in HIV-infected subjects is mediated by the HIV envelope protein through the CXCR4 chemokine receptor.

Introduction Rheumatoid arthritis (RA) is associated with hypogalactosylation of immunoglobulin G (IgG). We examined whether a proxy measure for galactosylation of IgG N-glycans could predict response to therapy or was differentially affected by methotrexate (MTX) or TNF blockade. Methods Using a previously defined normal phase high-performance liquid chromatography approach, we ascertained the galactosylation status of whole serum N-glycans in two well-defined RA clinical cohorts: the Autoimmune Biomarkers Collaborative Network (n = 98) and Nested I (n = 64). The ratio of agalactosylated to monogalactosylated N-glycans in serum (sG0/G1) was determined before and during therapy with MTX or TNF inhibition and correlated with anticitrullinated peptide antibody (ACPA) status and clinical response as assessed by 28-joint Disease Activity Score utilizing C-reactive peptide and European League Against Rheumatism response criteria. Results RA patients from both cohorts exhibited elevation of sG0/G1 at baseline. Improvement in clinical scores correlated with a reduction in sG0/G1 (Spearman's ρ = 0.31 to 0.37; P < 0.05 for each cohort). However, pretreatment sG0/G1 was not predictive of clinical response. Changes in sG0/G1 were similar in the MTX and TNF inhibitor groups. Corrected for disease activity, ACPA positivity correlated with higher sG0/G1. Conclusions Baseline serum N-glycan hypogalactosylation, an index previously correlated with hypogalactosylation of IgG N-glycans, did not distinguish patients with rheumatoid arthritis who were likely to experience a favorable clinical response to MTX or TNF blockade. Clinical improvement was associated with partial glycan normalization. ACPA-positive patients demonstrated enhanced N-glycan aberrancy compared with ACPA-negative patients.

Abstract 373 Based on the relatively unique structural and antigen-binding features of the B-cell receptors (BCRs) expressed on chronic lymphocytic leukemia (CLL) cells, it seems likely that the disease derives from antigen-selected B lymphocytes that have undergone chronic autoantigenic stimulation. In the support to this hypothesis, TCL1 transgenic (Tg) mice that develop many features of human CLL have very similar BCR rearrangements, some of which are autoreactive and closely resemble murine autoantibodies. In a previous report from our laboratory, it was shown that 4 out of 20 tested TCL1 Tg mice had BCRs resemble to autoantibodies reactive with phosphatidylcholine (PtC), a major component in biological membranes. BCR reactive to PtC is abundant in the murine B1 population that produces natural autoantibodies. An IgM crossreactive with PtC can be found in normal human serum, patients with autoimmune hemolytic anemia, systemic lupus erythematosus, as well as B-CLL. In this study, we aimed to characterize CLL cells that express BCRs with PtC antigen reactivity, and to evaluate the promotion of CLL through such BCRs. We found that, as previously reported, certain TCL1 Tg CLL murine cells produced IgMs crossreactive with PtC, and these Abs were coded by VH11 and VH12 genes. The binding of PtC was BCR-dependent, and cells with higher levels of surface IgM bound PtC better. Unselected splenocytes from a TCL1 mouse with B-cell clones expressing VH11 and VH12 IGHVs were then transferred into SCID mice. After SCID recipients developed CLL, splenocytes were again injected into another SCID mouse. Such adoptive transfers promoted CLL with accelerated kinetics at each transfer (time to developing CLL: at 1st transfer, 6 months; at 2nd transfer, 2 months; at 3rd transfer, 5 weeks). And while disease progression was accelerated during serial transfers, the PtC-binding population underwent relatively preferential expansion and eventually became the major clone in both spleen and peritoneum (from the average of 15% at the 1st transfer, to 48–60% at the 2nd transfer, and 48–71% at the 3rd transfer). The clonal expansion of PtC+ CLL cells in the accelerated CLL model suggests that clones stimulated by autoantigen have advantage of growth and survival. To further investigate this hypothesis, a million of sorted PtC+ or PtC- CLL cells were injected into different SCID mice. SCID mice injected with PtC+ cells developed larger spleens containing more cells in a significantly accelerated manner than mice given PtC- cells. Furthermore, in vitro PtC+ splenocytes showed better proliferation than PtC- cells when cultured with PtC stimulation. Microarray analysis performed on sorted PtC+ and PtC- CLL cells obtained from peritoneum washout or spleens from three TCL1 mice also showed distinct genetic signatures that suggested better survival and proliferation of PtC+ cells. Finally, we sought key factors involved in the evolution of normal PtC-binding B lymphocytes to CLL cells. Therefore, normal B-1 and TCL1 Tg CLL cells from the peritoneal cavity were sorted for PtC binding, and the positive and negative binding populations were analyzed for genome-wide gene expression. After eliminating TCL1-specific changes by subtracting gene differences identified between normal and TCL1 PtC- cells, 41 significantly differentially expressed genes were identified. Of interest, the abnormal downregulated genes found in CLL PtC+ cells include the negative regulators of NF-kappa-B and WNT signaling pathways. In conclusion, B-cell clones expressing VH11 and VH12 genes that bind PtC were found in the normal TCL1 Tg repertoire. After serial adoptive transfers, these cells were selected for PtC-binding subclones and accelerated CLL. This promotion of CLL can be explained by survival and growth advantages for PtC+ cells in vivo, analogous to what was found in vitro. In contrast, cells with extremely low levels of surface IgM and minimal PtC binding that receive suboptimal BCR signals have retarded growth and may be tolerized. Thus, our data suggest that chronic stimulation of BCRs by autoantigens leads to an accumulated activation of oncogenic pathways and finally transformation to CLL. Disclosures: No relevant conflicts of interest to declare.

Introduction Anti-TNF therapies have revolutionized the treatment of rheumatoid arthritis (RA), a common systemic autoimmune disease involving destruction of the synovial joints. However, in the practice of rheumatology approximately one-third of patients demonstrate no clinical improvement in response to treatment with anti-TNF therapies, while another third demonstrate a partial response, and one-third an excellent and sustained response. Since no clinical or laboratory tests are available to predict response to anti-TNF therapies, great need exists for predictive biomarkers. Methods Here we present a multi-step proteomics approach using arthritis antigen arrays, a multiplex cytokine assay, and conventional ELISA, with the objective to identify a biomarker signature in three ethnically diverse cohorts of RA patients treated with the anti-TNF therapy etanercept. Results We identified a 24-biomarker signature that enabled prediction of a positive clinical response to etanercept in all three cohorts (positive predictive values 58 to 72%; negative predictive values 63 to 78%). Conclusions We identified a multi-parameter protein biomarker that enables pretreatment classification and prediction of etanercept responders, and tested this biomarker using three independent cohorts of RA patients. Although further validation in prospective and larger cohorts is needed, our observations demonstrate that multiplex characterization of autoantibodies and cytokines provides clinical utility for predicting response to the anti-TNF therapy etanercept in RA patients.

Introduction There is increasing evidence that autoantibodies and immune complexes (ICs) contribute to synovitis in rheumatoid arthritis (RA), yet the autoantigens incorporated in ICs in RA remain incompletely characterised. Methods We used the C1q protein to capture ICs from plasma derived from human RA and control patients. Antibodies specific for immunoglobulin were used to detect ICs, and fibrinogen antibodies were used to detect fibrinogen-containing ICs. RA and control plasma were separated by liquid chromatography, and fractions then characterised by ELISA, immunoblotting and mass spectrometry. Immunohistochemical staining was performed on rheumatoid synovial tissue. Results C1q-immunoassays demonstrated increased levels of IgG (p = 0.01) and IgM (p = 0.0002) ICs in plasma derived from RA patients possessing anti-cyclic citrullinated peptide (CCP+) autoantibodies as compared with healthy controls. About one-half of the anti-CCP+ RA possessed circulating ICs containing fibrinogen (p = 0.0004). Fractionation of whole RA plasma revealed citrullinated fibrinogen in the high molecular weight fractions that contained ICs. Positive correlations were observed between fibrinogen-containing ICs and anti-citrullinated fibrinogen autoantibodies, anti-CCP antibody, rheumatoid factor and certain clinical characteristics. Immunohistochemical staining demonstrated co-localisation of fibrinogen, immunoglobulin and complement component C3 in RA pannus tissue. Mass spectrometry analysis of immune complexes immunoprecipitated from RA pannus tissue lysates demonstrated the presence of citrullinated fibrinogen. Conclusion Circulating ICs containing citrullinated fibrinogen are present in one-half of anti-CCP+ RA patients, and these ICs co-localise with C3 in the rheumatoid synovium suggesting that they contribute to synovitis in a subset of RA patients.

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease, affecting approximately 1% of the adult population worldwide, with an estimated heritability of 60%. To identify genes involved in RA susceptibility, we investigated the association between putative functional single-nucleotide polymorphisms (SNPs) and RA among white individuals by use of a case-control study design; a second sample was tested for replication. Here we report the association of RA susceptibility with the minor allele of a missense SNP in PTPN22 (discovery-study allelic P=6.6 x 10(-4); replication-study allelic P=5.6 x 10(-8)), which encodes a hematopoietic-specific protein tyrosine phosphatase also known as "Lyp." We show that the risk allele, which is present in approximately 17% of white individuals from the general population and in approximately 28% of white individuals with RA, disrupts the P1 proline-rich motif that is important for interaction with Csk, potentially altering these proteins' normal function as negative regulators of T-cell activation. The minor allele of this SNP recently was implicated in type 1 diabetes, suggesting that the variant phosphatase may increase overall reactivity of the immune system and may heighten an individual carrier's risk for autoimmune disease.

To the editor: We recently reported that human B-1 cells express the phenotype CD20+CD27+CD43+CD70−.[1][1],[2][2] Covens et al[3][3] now assert that B cells with this phenotype resemble plasmablasts and thus represent preplasmablasts. Their conclusion rests to a large extent on microarray data