Transinteractome analysis reveals distinct niche requirements for isotype-based plasma cell subsets in the bone marrow.

Bone marrow (BM) long-lived plasma cells (PCs) are essential for long-term protection against infection, and their persistence within this organ relies on interactions with Cxcl12-expressing stromal cells that are still not clearly identified. Here, using single cell RNAseq and in silico transinteractome analyses, we identified Leptin receptor positive (LepR+ ) mesenchymal cells as the stromal cell subset most likely to interact with PCs within the BM. Moreover, we demonstrated that depending on the isotype they express, PCs may use different sets of integrins and adhesion molecules to interact with these stromal cells. Altogether, our results constitute an unprecedented characterization of PC subset stromal niches and open new avenues for the specific targeting of BM PCs based on their isotype.

Integrated analysis of dermal blister fluid proteomics and genome-wide skin gene expression in systemic sclerosis: an observational study.

Background: Skin fibrosis is a hallmark feature of systemic sclerosis. Skin biopsy transcriptomics and blister fluid proteomics give insight into the local environment of the skin. We have integrated these modalities with the aim of developing a surrogate for the modified Rodnan skin score (mRSS), using candidate genes and proteins from the skin and blister fluid as anchors to identify key analytes in the plasma. Methods: In this single-centre, prospective observational study at the Royal Free Campus, University College London, London, UK, transcriptional and proteomic analyses of blood and skin were performed in a cohort of patients with systemic sclerosis (n=52) and healthy controls (n=16). Weighted gene co-expression network analysis was used to explore the association of skin transcriptomics data, clinical traits, and blister fluid proteomic results. Candidate hub analytes were identified as those present in both blister and skin gene sets (modules), and which correlated with plasma (module membership >0·7 and gene significance >0·6). Hub analytes were confirmed using RNA transcript data obtained from skin biopsy samples from patients with early diffuse cutaneous systemic sclerosis at 12 months. Findings: We identified three modules in the skin, and two in blister fluid, which correlated with a diagnosis of early diffuse cutaneous systemic sclerosis. From these modules, 11 key hub analytes were identified, present in both skin and blister fluid modules, whose transcript and protein levels correlated with plasma protein concentrations, mRSS, and showed statistically significant correlation on repeat transcriptomic samples taken at 12 months. Multivariate analysis identified four plasma analytes as correlates of mRSS (COL4A1, COMP, SPON1, and TNC), which can be used to differentiate disease subtype. Interpretation: This unbiased approach has identified potential biological candidates that might be drivers of local skin pathogenesis in systemic sclerosis. By focusing on measurable analytes in the plasma, we generated a promising composite plasma protein biomarker that could be used for assessment of skin severity, case stratification, and as a potential outcome measure for clinical trials and practice. Once fully validated, the biomarker score could replace a clinical score such as the mRSS, which carries substantial variability. Funding: GlaxoSmithKline and UK Medical Research Council.

Biological and clinical insights from a randomized phase 2 study of an anti-oncostatin M monoclonal antibody in systemic sclerosis.

OBJECTIVES: The cytokine oncostatin M (OSM) is implicated in the pathology of SSc. Inhibiting OSM signalling using GSK2330811 (an anti-OSM monoclonal antibody) in patients with SSc has the potential to slow or stop the disease process. METHODS: This multicentre, randomized, double-blind, placebo-controlled study enrolled participants ≥18 years of age with active dcSSc. Participants were randomized 3:1 (GSK2330811:placebo) in one of two sequential cohorts to receive GSK2330811 (cohort 1: 100 mg; cohort 2: 300 mg) or placebo s.c. every other week for 12 weeks. The primary endpoint was safety; blood and skin biopsy samples were collected to explore mechanistic effects on inflammation and fibrosis. Clinical efficacy was an exploratory endpoint. RESULTS: Thirty-five participants were randomized to placebo (n = 8), GSK2330811 100 mg (n = 3) or GSK2330811 300 mg (n = 24). Proof of mechanism, measured by coordinate effects on biomarkers of inflammation or fibrosis, was not demonstrated following GSK2330811 treatment. There were no meaningful differences between GSK2330811 and placebo for any efficacy endpoints. The safety and tolerability of GSK2330811 were not favourable in the 300 mg group, with on-target, dose-dependent adverse events related to decreases in haemoglobin and platelet count that were not observed in the 100 mg or placebo groups. CONCLUSION: Despite a robust and novel experimental medicine approach and evidence of target engagement, anticipated SSc-related biologic effects of GSK2330811 were not different from placebo and safety was unfavourable, suggesting OSM inhibition may not be a useful therapeutic strategy in SSc. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, NCT03041025; EudraCT, 2016-003417-95.

Molecular basis for clinical diversity between autoantibody subsets in diffuse cutaneous systemic sclerosis.

OBJECTIVES: Clinical heterogeneity is a cardinal feature of systemic sclerosis (SSc). Hallmark SSc autoantibodies are central to diagnosis and associate with distinct patterns of skin-based and organ-based complications. Understanding molecular differences between patients will benefit clinical practice and research and give insight into pathogenesis of the disease. We aimed to improve understanding of the molecular differences between key diffuse cutaneous SSc subgroups as defined by their SSc-specific autoantibodies METHODS: We have used high-dimensional transcriptional and proteomic analysis of blood and the skin in a well-characterised cohort of SSc (n=52) and healthy controls (n=16) to understand the molecular basis of clinical diversity in SSc and explore differences between the hallmark antinuclear autoantibody (ANA) reactivities. RESULTS: Our data define a molecular spectrum of SSc based on skin gene expression and serum protein analysis, reflecting recognised clinical subgroups. Moreover, we show that antitopoisomerase-1 antibodies and anti-RNA polymerase III antibodies specificities associate with remarkably different longitudinal change in serum protein markers of fibrosis and divergent gene expression profiles. Overlapping and distinct disease processes are defined using individual patient pathway analysis. CONCLUSIONS: Our findings provide insight into clinical diversity and imply pathogenetic differences between ANA-based subgroups. This supports stratification of SSc cases by ANA antibody subtype in clinical trials and may explain different outcomes across ANA subgroups in trials targeting specific pathogenic mechanisms.

The role of baseline BLyS levels and type 1 interferon-inducible gene signature status in determining belimumab response in systemic lupus erythematosus: a post hoc meta-analysis.

BACKGROUND: Elevated B lymphocyte stimulator (BLyS) levels in patients with systemic lupus erythematosus (SLE) correlate positively with disease activity; BLyS expression is directly linked to interferon (IFN) pathway activation. This post hoc meta-analysis of BLISS-52 and BLISS-76 explored the relationship between baseline BLyS mRNA/protein levels and/or type 1 IFN-inducible gene signature (IFN-1) and responses to the BLyS-targeting monoclonal antibody belimumab in SLE. METHODS: In BLISS-52 and BLISS-76, patients with autoantibody-positive SLE and a SELENA-SLEDAI score ≥ 6 and receiving stable standard SLE therapy were randomised to intravenous belimumab 10 mg/kg or placebo, plus standard of care (SoC), for 52 or 76 weeks. For this post hoc meta-analysis, patients with an appropriate mRNA sample were stratified by BLyS mRNA expression (tertiles: high/medium/low; revised quantiles: high/low), IFN-1 mRNA expression (high/low) and BLyS protein level (high/low). Co-primary endpoints were correlation between baseline BLyS and IFN-1 mRNA levels and SLE Responder Index (SRI)4 response at week 52 within BLyS/IFN-1 subgroups. Secondary endpoints included time to first severe SELENA-SLEDAI Flare Index (SFI) flare. RESULTS: Of 554 patients included in this analysis, 281 had received belimumab and 273 had received placebo. Baseline BLyS and IFN-1 mRNA levels were highly correlated (Spearman's rank correlation coefficient 0.7799; 95% confidence interval [CI] 0.7451, 0.8106; p < 0.0001). The proportion of SRI4 responders was higher with belimumab versus placebo in all subgroups, but the difference reached statistical significance in the medium BLyS mRNA tertile (odds ratio [OR] 2.17; 95% CI 1.16, 4.04; p = 0.0153), high BLyS mRNA quantile (OR 1.58; 95% CI 1.02, 2.44; p = 0.0402), high IFN-1 mRNA (OR 1.58; 95% CI: 1.08, 2.31; p = 0.0186) and high BLyS protein (OR 3.57; 95% CI 1.63, 7.83; p = 0.0015) subgroups only. The risk of severe SFI flare was significantly lower with belimumab than placebo in the high BLyS mRNA quantile (hazard ratio [HR] 0.59; 95% CI 0.36, 0.97; p = 0.0371) and high BLyS protein (HR 0.39; 95% CI 0.19, 0.79; p = 0.0090) subgroups. CONCLUSIONS: This post hoc meta-analysis demonstrated a tendency towards improved response to add-on intravenous belimumab 10 mg/kg versus SoC alone in patients with high baseline BLyS protein and IFN-1 mRNA levels and medium/high BLyS mRNA levels.

Belimumab in kidney transplantation: an experimental medicine, randomised, placebo-controlled phase 2 trial.

BACKGROUND: B cells produce alloantibodies and activate alloreactive T cells, negatively affecting kidney transplant survival. By contrast, regulatory B cells are associated with transplant tolerance. Immunotherapies are needed that inhibit B-cell effector function, including antibody secretion, while sparing regulators and minimising infection risk. B lymphocyte stimulator (BLyS) is a cytokine that promotes B-cell activation and has not previously been targeted in kidney transplant recipients. We aimed to determine the safety and activity of an anti-BLyS antibody, belimumab, in addition to standard-of-care immunosuppression in adult kidney transplant recipients. We used an experimental medicine study design with multiple secondary and exploratory endpoints to gain further insight into the effect of belimumab on the generation of de-novo IgG and on the regulatory B-cell compartment. METHODS: We undertook a double-blind, randomised, placebo-controlled phase 2 trial of belimumab, in addition to standard-of-care immunosuppression (basiliximab, mycophenolate mofetil, tacrolimus, and prednisolone) at two centres, Addenbrooke's Hospital, Cambridge, UK, and Guy's and St Thomas' Hospital, London, UK. Participants were eligible if they were aged 18-75 years and receiving a kidney transplant and were planned to receive standard-of-care immunosuppression. Participants were randomly assigned (1:1) to receive either intravenous belimumab 10 mg per kg bodyweight or placebo, given at day 0, 14, and 28, and then every 4 weeks for a total of seven infusions. The co-primary endpoints were safety and change in the concentration of naive B cells from baseline to week 24, both of which were analysed in all patients who received a transplant and at least one dose of drug or placebo (the modified intention-to-treat [mITT] population). This trial has been completed and is registered with ClinicalTrials.gov, NCT01536379, and EudraCT, 2011-006215-56. FINDINGS: Between Sept 13, 2013, and Feb 8, 2015, of 303 patients assessed for eligibility, 28 kidney transplant recipients were randomly assigned to receive belimumab (n=14) or placebo (n=14). 25 patients (12 [86%] patients assigned to the belimumab group and 13 [93%] patients assigned to the placebo group) received a transplant and were included in the mITT population. We observed similar proportions of adverse events in the belimumab and placebo groups, including serious infections (one [8%] of 12 in the belimumab group and five [38%] of 13 in the placebo group during the 6-month on-treatment phase; and none in the belimumab group and two [15%] in the placebo group during the 6-month follow-up). In the on-treatment phase, one patient in the placebo group died because of fatal myocardial infarction and acute cardiac failure. The co-primary endpoint of a reduction in naive B cells from baseline to week 24 was not met. Treatment with belimumab did not significantly reduce the number of naive B cells from baseline to week 24 (adjusted mean difference between the belimumab and placebo treatment groups -34·4 cells per µL, 95% CI -109·5 to 40·7). INTERPRETATION: Belimumab might be a useful adjunct to standard-of-care immunosuppression in renal transplantation, with no major increased risk of infection and potential beneficial effects on humoral alloimmunity. FUNDING: GlaxoSmithKline.

NBEAL2 is required for neutrophil and NK cell function and pathogen defense.

Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity.

Type I interferon as a biomarker in autoimmunity and viral infection: a leukocyte subset-specific analysis unveils hidden diagnostic options.

Interferon alpha and its surrogates, including IP-10 and SIGLEC1, paralleled changes of disease activity in systemic lupus erythematosus (SLE). However, the whole blood interferon signature (WBIFNS)-the current standard for type I IFN assessment in SLE-does not correlate with SLE disease activity in individual patients over time. The underlying causes for this apparent contradiction have not been convincingly demonstrated. Using a multicenter dataset of gene expression data from leukocyte subsets in SLE, we identify distinctive subset-specific contributions to the WBIFNS. In a subsequent analysis, the effects of type I interferon on cellular blood composition in patients with SLE and hepatitis B were also studied over time. We found that type I interferon mediates significant alterations in whole blood composition, including a neutropenia and relative lymphocytosis. Given different effects of type 1 interferon on different leukocyte subsets, these shifts confound measurement of a type 1 interferon signature in whole blood. To minimize and overcome these limitations of the WBIFNS, we suggest to measure IFN-induced transcripts or proteins in a specific leukocyte subset to improve clinical impact of interferon biomarkers. KEY MESSAGES: Myeloid cells contribute more to the WBIFNS in SLE than their lymphocytic counterpart. Very similar leukocyte subsets reveal distinctive IFN signatures. IFN alpha mixes up composition of blood and leads to a preferential neutropenia, yielding relative lymphocytosis.

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity.

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91phox and p22phox subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene bc017643, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91phox and p22phox Consequently, Eros-deficient mice quickly succumb to infection. Eros also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. Eros is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.

Histological and Extended Clinical Outcomes After ABO-Incompatible Renal Transplantation Without Splenectomy or Rituximab.

BACKGROUND: Excellent short-term results have been reported in ABO-incompatible (ABOi) renal transplant recipients managed solely with antibody removal and conventional immunosuppression. However, long-term clinical outcomes with this regimen and predictive information from protocol biopsies are lacking. METHODS: We compared outcome data in ABOi and ABO-compatible (ABOc) recipients receiving this regimen approximately 4 years posttransplant, and histology from biopsies approximately 12 months posttransplant. RESULTS: Patient and graft survivals among 54 ABOi recipients were 98.1% and 90.7%, respectively, at 4 years. Graft function was similar between ABOi (creatinine, 140.3 µmol/L) and ABOc recipients (creatinine, 140.2 µmol/L) (P = 0.99), with no significant change over the study period in either group (Δcreatinine, -0.83 vs 6.6 µmol/L) (P = 0.59). There was no transplant glomerulopathy in biopsies from either group. Interstitial fibrosis (IF) and tubular atrophy (TA) was present in 7 (28%) of 25 ABOi compared with 7 (20.6%) of 34 ABOc (P = 0.52). Progression of IF/TA from implantation was noted in 6 (24%) of 25 ABOi and 6 (17.6%) of 34 ABOc, respectively. C4d staining without antibody-mediated rejection was present in 13 (52%) 25 early posttransplant biopsies from ABOi recipients by immunohistochemistry, but in only 4 (16%) of 25 at 12 months. CONCLUSIONS: ABO-incompatible renal transplant performed with antibody removal and conventional immunosuppression continues to provide excellent patient and graft survival, and stable renal function over 4 years. Coupled with absent transplant glomerulopathy and low rates of progressive IF/TA on earlier biopsies, this suggests that ABOi with conventional immunosuppression and antibody removal, without rituximab or splenectomy, can achieve long-term outcomes comparable to ABO-compatible transplantation.

Glucocorticoid-Induced Leucine Zipper Protein Controls Macropinocytosis in Dendritic Cells.

Ag sampling is a key process in dendritic cell (DC) biology. DCs use constitutive macropinocytosis, receptor-mediated endocytosis, and phagocytosis to capture exogenous Ags for presentation to T cells. We investigated the mechanisms that regulate Ag uptake by DCs in the steady-state and after a short-term LPS exposure in vitro and in vivo. We show that the glucocorticoid-induced leucine zipper protein (GILZ), already known to regulate effector versus regulatory T cell activation by DCs, selectively limits macropinocytosis, but not receptor-mediated phagocytosis, in immature and recently activated DCs. In vivo, the GILZ-mediated inhibition of Ag uptake is restricted to the CD8α+ DC subset, which expresses the highest GILZ level among splenic DC subsets. In recently activated DCs, we further establish that GILZ limits p38 MAPK phosphorylation, providing a possible mechanism for GILZ-mediated macropinocytosis control. Finally, our results demonstrate that the modulation of Ag uptake by GILZ does not result in altered Ag presentation to CD4 T cells but impacts the efficiency of cross-presentation to CD8 T cells. Altogether, our results identify GILZ as an endogenous inhibitor of macropinocytosis in DCs, the action of which contributes to the fine-tuning of Ag cross-presentation.

Leucocyte subset-specific type 1 interferon signatures in SLE and other immune-mediated diseases.

OBJECTIVES: Type 1 interferons (IFN-1) are implicated in the pathogenesis of systemic lupus erythematosus (SLE), but most studies have only reported the effect of IFN-1 on mixed cell populations. We aimed to define modules of IFN-1-associated genes in purified leucocyte populations and use these as a basis for a detailed comparative analysis. METHODS: CD4+ and CD8+ T cells, monocytes and neutrophils were purified from patients with SLE, other immune-mediated diseases and healthy volunteers and gene expression then determined by microarray. Modules of IFN-1-associated genes were defined using weighted gene coexpression network analysis. The composition and expression of these modules was analysed. RESULTS: 1150 of 1288 IFN-1-associated genes were specific to myeloid subsets, compared with 11 genes unique to T cells. IFN-1 genes were more highly expressed in myeloid subsets compared with T cells. A subset of neutrophil samples from healthy volunteers (HV) and conditions not classically associated with IFN-1 signatures displayed increased IFN-1 gene expression, whereas upregulation of IFN-1-associated genes in T cells was restricted to SLE. CONCLUSIONS: Given the broad upregulation of IFN-1 genes in neutrophils including in some HV, investigators reporting IFN-1 signatures on the basis of whole blood samples should be cautious about interpreting this as evidence of bona fide IFN-1-mediated pathology. Instead, specific upregulation of IFN-1-associated genes in T cells may be a useful biomarker and a further mechanism by which elevated IFN-1 contributes to autoimmunity in SLE.

Peripheral Immune Cell Populations Associated with Cognitive Deficits and Negative Symptoms of Treatment-Resistant Schizophrenia.

BACKGROUND: Hypothetically, psychotic disorders could be caused or conditioned by immunological mechanisms. If so, one might expect there to be peripheral immune system phenotypes that are measurable in blood cells as biomarkers of psychotic states. METHODS: We used multi-parameter flow cytometry of venous blood to quantify and determine the activation state of 73 immune cell subsets for 18 patients with chronic schizophrenia (17 treated with clozapine), and 18 healthy volunteers matched for age, sex, BMI and smoking. We used multivariate methods (partial least squares) to reduce dimensionality and define populations of differentially co-expressed cell counts in the cases compared to controls. RESULTS: Schizophrenia cases had increased relative numbers of NK cells, naïve B cells, CXCR5+ memory T cells and classical monocytes; and decreased numbers of dendritic cells (DC), HLA-DR+ regulatory T-cells (Tregs), and CD4+ memory T cells. Likewise, within the patient group, more severe negative and cognitive symptoms were associated with decreased relative numbers of dendritic cells, HLA-DR+ Tregs, and CD4+ memory T cells. Motivated by the importance of central nervous system dopamine signalling for psychosis, we measured dopamine receptor gene expression in separated CD4+ cells. Expression of the dopamine D3 (DRD3) receptor was significantly increased in clozapine-treated schizophrenia and covaried significantly with differentiated T cell classes in the CD4+ lineage. CONCLUSIONS: Peripheral immune cell populations and dopaminergic signalling are disrupted in clozapine-treated schizophrenia. Immuno-phenotypes may provide peripherally accessible and mechanistically specific biomarkers of residual cognitive and negative symptoms in this treatment-resistant subgroup of patients.

A distinct plasmablast and naïve B-cell phenotype in primary immune thrombocytopenia.

Primary immune thrombocytopenia is an autoimmune disorder in which platelet destruction is a consequence of both B- and T-cell dysregulation. Flow cytometry was used to further characterize the B- and T-cell compartments in a cross-sectional cohort of 26 immune thrombocytopenia patients including antiplatelet antibody positive (n=14) and negative (n=12) patients exposed to a range of therapies, and a cohort of matched healthy volunteers. Markers for B-cell activating factor and its receptors, relevant B-cell activation markers (CD95 and CD21) and markers for CD4(+) T-cell subsets, including circulating T-follicular helper-like cells, were included. Our results indicate that an expanded population of CD95(+) naïve B cells correlated with disease activity in immune thrombocytopenia patients regardless of treatment status. A population of CD21-naïve B cells was specifically expanded in autoantibody-positive immune thrombocytopenia patients. Furthermore, the B-cell maturation antigen, a receptor for B-cell activating factor, was consistently and strongly up-regulated on plasmablasts from immune thrombocytopenia patients. These observations have parallels in other autoantibody-mediated diseases and suggest that loss of peripheral tolerance in naïve B cells may be an important component of immune thrombocytopenia pathogenesis. Moreover, the B-cell maturation antigen represents a potential target for plasma cell directed therapies in immune thrombocytopenia.

The Contribution of Transcriptomics to Biomarker Development in Systemic Vasculitis and SLE.

A small but increasing number of gene expression based biomarkers are becoming available for routine clinical use, principally in oncology and transplantation. These underscore the potential of gene expression arrays and RNA sequencing for biomarker development, but this potential has not yet been fully realized and most candidates do not progress beyond the initial report. The first part of this review examines the process of gene expression- based biomarker development, highlighting how systematic biases and confounding can significantly skew study outcomes. Adequate validation in an independent cohort remains the single best means of protecting against these concerns. The second part considers gene-expression based biomarkers in Systemic Lupus Erythematosus (SLE) and systemic vasculitis. The type 1 interferon inducible gene signature remains by far the most studied in autoimmune rheumatic disease. While initially presented as an objective, blood-based biomarker of active SLE, subsequent research has shown that it is not specific to SLE and that its association with disease activity is considerably more nuanced than first thought. Nonetheless, it is currently under evaluation in ongoing trials of anti-interferon therapy. Other candidate markers of note include a prognostic CD8+ T-cell gene signature validated in SLE and ANCA-associated vasculitis, and a disease activity biomarker for SLE derived from modules of tightly correlated genes.

Emerging concepts in the pathogenesis of antineutrophil cytoplasmic antibody-associated vasculitis.

PURPOSE OF REVIEW: Antineutrophil cytoplasmic antibodies (ANCAs) remain central to our current understanding of the pathogenesis of ANCA-associated vasculitis (AAV), and this review considers recent developments in the context of four key questions: are there targets for ANCA beyond myeloperoxidase (MPO) and proteinase 3 (PR3); are all ANCA pathogenic; how are ANCAs generated; and how do ANCA cause disease? RECENT FINDINGS: B-cell epitope mapping raises the possibility that only a subset of ANCA may be pathogenic. Anti-lysosomal-associated membrane protein 2 autoantibodies have recently emerged as a novel form of ANCA and can be found in anti-MPO and anti-PR3 negative disease. These also provide recent evidence for molecular mimicry in the pathogenesis of AAV, but a definitive proof in human AAV remains elusive. Neutrophil extracellular traps may represent an important mechanism by which MPO and PR3 are taken up by dendritic cells for presentation to the adaptive immune system, and the role of the alternative pathway of complement in AAV has recently been emphasized, with therapeutic implications. SUMMARY: Our current understanding of the pathogenesis of AAV not only reinforces the central role of neutrophils but also provides a sound rationale for B-cell and complement-directed therapies.

Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation.

BACKGROUND: Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study. RESULTS: Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this "gold-standard" comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues. CONCLUSIONS: Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.

A type I interferon transcriptional signature precedes autoimmunity in children genetically at risk for type 1 diabetes.

Diagnosis of the autoimmune disease type 1 diabetes (T1D) is preceded by the appearance of circulating autoantibodies to pancreatic islets. However, almost nothing is known about events leading to this islet autoimmunity. Previous epidemiological and genetic data have associated viral infections and antiviral type I interferon (IFN) immune response genes with T1D. Here, we first used DNA microarray analysis to identify IFN-ß-inducible genes in vitro and then used this set of genes to define an IFN-inducible transcriptional signature in peripheral blood mononuclear cells from a group of active systemic lupus erythematosus patients (n = 25). Using this predefined set of 225 IFN signature genes, we investigated the expression of the signature in cohorts of healthy controls (n = 87), patients with T1D (n = 64), and a large longitudinal birth cohort of children genetically predisposed to T1D (n = 109; 454 microarrayed samples). Expression of the IFN signature was increased in genetically predisposed children before the development of autoantibodies (P = 0.0012) but not in patients with established T1D. Upregulation of IFN-inducible genes was transient, temporally associated with a recent history of upper respiratory tract infections (P = 0.0064), and marked by increased expression of SIGLEC-1 (CD169), a lectin-like receptor expressed on CD14(+) monocytes. DNA variation in IFN-inducible genes altered T1D risk (P = 0.007), as exemplified by IFIH1, one of the genes in our IFN signature for which increased expression is a known risk factor for disease. These findings identify transient increased expression of type I IFN genes in preclinical diabetes as a risk factor for autoimmunity in children with a genetic predisposition to T1D.