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OBJECTIVES: SSc is associated with increased health-care resource utilization and economic burden. The Collaborative National Quality and Efficacy Registry (CONQUER) is a US-based collaborative that collects longitudinal follow-up data on SSc patients with

OBJECTIVE: To assess the predictive significance of blood neutrophil count and the ratio between neutrophil and lymphocyte count (neutrophil-to-lymphocyte ratio [NLR]) for disease severity and mortality in systemic sclerosis (SSc). METHODS: Neutrophil and lymphocyte counts were prospectively measured in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) and the Scleroderma Lung Study II (SLS II). Forced vital capacity percent predicted (FVC%) and modified Rodnan skin thickness score (MRSS) were used as surrogate measures for disease severity. Longitudinal analyses were performed using generalized linear mixed models. Cox proportional hazards models evaluated the predictive significance of these cell counts for mortality. RESULTS: Of the 447 SSc patients in the GENISOS cohort at the time of analysis, 377 (84.3%) had available baseline blood neutrophil and lymphocyte counts. Higher baseline neutrophil count and NLR predicted lower serially obtained FVC% (b = -4.74, P = 0.009 and b = -2.68, P = 0.028, respectively) and higher serially obtained MRSS (b = 4.07, P

Objectives Polymorphism in a coding region of deoxyribonuclease I-like III (DNASE1L3), causing amino acid substitution of Arg-206 to Cys (R206C), is a robustly replicated heritable risk factor for SSc and other autoimmune diseases. DNASE1L3 is secreted into the circulation, where it can digest genomic DNA (gDNA) in apoptosis-derived membrane vesicles (AdMVs). We sought to determine the impact of DNASE1L3 R206C on digestion of circulating gDNA in SSc patients and healthy controls (HCs). Methods The ability of DNASE1L3 to digest AdMV-associated gDNA was tested in vitro. The effect of R206C substitution on extracellular secretion of DNASE1L3 was determined using a transfected cell line and primary monocyte-derived dendritic cells from SSc patients. Plasma samples from SSc patients and HCs with DNASE1L3 R206C or R206 wild type were compared for their ability to digest AdMV-associated gDNA. The digestion status of endogenous gDNA in plasma samples from 123 SSc patients and 74 HCs was determined by measuring the proportion of relatively long to short gDNA fragments. Results The unique ability of DNASE1L3 to digest AdMV-associated gDNA was confirmed. Extracellular secretion of DNASE1L3 R206C was impaired. Plasma from individuals with DNASE1L3 R206C had reduced ability to digest AdMV-associated gDNA. The ratio of long: short gDNA fragments was increased in plasma from SSc patients with DNASE1L3 R206C, and this ratio correlated inversely with DNase activity. Conclusion Our results confirm that circulating gDNA is a physiological DNASE1L3 substrate and show that its digestion is reduced in SSc patients with the DNASE1L3 R206C variant. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.09.033 Byline: Brian Skaug (1), Zhijian J. Chen (1)(2) Abstract: Cells express a plethora of interferon-stimulated genes (ISGs) in response to viral infection. Among these is ISG15, a ubiquitin-like protein (UBL) that can be covalently attached to both host and viral proteins. Here we review recent advances toward understanding the role and mechanism of ISG15 modification in antiviral defense. Author Affiliation: (1) Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA (2) Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148, USA

Objective: Although a high‐resolution computed tomography (HRCT) scan of the chest is the gold standard test for the detection of interstitial lung disease (ILD), there is no consensus among rheumatologists regarding the use of HRCT to screen for ILD in their patients with systemic sclerosis (SSc). The aims of this study were to describe the HRCT ordering practices at SSc centers in the United States and to determine which patient characteristics are associated with HRCT performance. Methods: We performed a prospective cohort study of patients with SSc enrolled in the US‐based Collaborative National Quality and Efficacy Registry (CONQUER). We performed univariate logistic regression followed by multivariable logistic regression to determine which patient characteristics were associated with HRCT performance. Results: Of the 356 patients with SSc enrolled in CONQUER, 286 (80.3%) underwent HRCT at some point during their disease course. On multivariable analyses, missing total lung capacity percent predicted (odds ratio [OR] 3.26, 95% confidence interval [CI]: 1.53‐7.41, P = 0.007) was positively associated with ever having undergone HRCT, whereas a positive anti‐centromere antibody (OR 0.27, 95% CI: 0.12‐0.61, P = 0.008) and missing forced vital capacity percent predicted (OR 0.29, 95% CI: 0.10‐0.80, P = 0.005) were negatively associated with ever having undergone HRCT. There was a trend toward a positive association between crackles on pulmonary exam and ever having undergone HRCT (OR 2.28, 95% CI: 0.97‐6.05, P = 0.058), although this relationship did not reach statistical significance. Conclusion: The majority of patients with SSc enrolled in CONQUER underwent HRCT. A positive anti‐centromere antibody was the key clinical variable inversely associated with performance of HRCT. [ABSTRACT FROM AUTHOR]

Objectives: Determine relationships between skin gene expression and systemic sclerosis (SSc) clinical disease features, and changes in skin gene expression over time.Methods: A total of 339 forearm skin biopsies were obtained from 113 SSc patients and 44 matched healthy controls. 105 SSc patients had a second biopsy, and 76 had a third biopsy. Global gene expression profiling was performed, and differentially expressed genes and cell type-specific signatures in SSc were evaluated for relationships to modified Rodnan Skin Score (mRSS) and other clinical variables. Changes in skin gene expression over time were analysed by mixed effects models and principal component analysis. Immunohistochemical staining was performed to validate conclusions.Results: Gene expression dysregulation was greater in SSc patients with affected skin than in those with unaffected skin. Immune cell and fibroblast signatures positively correlated with mRSS. High baseline immune cell and fibroblast signatures predicted higher mRSS over time, but were not independently predictive of longitudinal mRSS after adjustment for baseline mRSS. In early diffuse cutaneous SSc, immune cell and fibroblast signatures declined over time, and overall skin gene expression trended towards normalisation. On immunohistochemical staining, most early diffuse cutaneous SSc patients with high baseline T cell and macrophage numbers had declines in these numbers at follow-up.Conclusions: Skin thickness in SSc is related to dysregulated immune cell and fibroblast gene expression. Skin gene expression changes over time in early diffuse SSc, with a tendency towards normalisation. These observations are relevant for understanding SSc pathogenesis and could inform treatment strategies and clinical trial design. [ABSTRACT FROM AUTHOR]

Objectives: Determine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease.Methods: Skin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic Sclerosis (PRESS) cohort (mean disease duration 1.3 years) and 33 matched healthy controls was examined by next-generation RNA sequencing. Data were analysed for cell type-specific signatures and compared with similarly obtained data from 55 previously biopsied patients in Genetics versus Environment in Scleroderma Outcomes Study cohort with longer disease duration (mean 7.4 years) and their matched controls. Correlations with histological features and clinical course were also evaluated.Results: SSc patients in PRESS had a high prevalence of M2 (96%) and M1 (94%) macrophage and CD8 T cell (65%), CD4 T cell (60%) and B cell (69%) signatures. Immunohistochemical staining of immune cell markers correlated with the gene expression-based immune cell signatures. The prevalence of immune cell signatures in early diffuse SSc patients was higher than in patients with longer disease duration. In the multivariable model, adaptive immune cell signatures were significantly associated with shorter disease duration, while fibroblast and macrophage cell type signatures were associated with higher modified Rodnan Skin Score (mRSS). Immune cell signatures also correlated with skin thickness progression rate prior to biopsy, but did not predict subsequent mRSS progression.Conclusions: Skin in early diffuse SSc has prominent innate and adaptive immune cell signatures. As a prominently affected end organ, these signatures reflect the preceding rate of disease progression. These findings could have implications in understanding SSc pathogenesis and clinical trial design. [ABSTRACT FROM AUTHOR]

The identification and characterization of ubiquitination and deubiquitination machinery that has regulated nuclear factor kappa enhancer binding protein (NF-[kappa]B) is discussed. Proteosome-independent functions of ubiquitin, its role in the activation of protein kinase complexes and in coordination of cell survival and apoptosis signals downstream of tumor necrosis factor [alpha] (TNF[alpha]) is described.

Objectives: There is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has been recognised as a master regulator of type I IFN signalling. The objective of this study was to elucidate the role of IRF7 in dermal fibrosis and SSc pathogenesis.Methods: SSc and healthy control skin biopsies were investigated to determine IRF7 expression and activation. The role of IRF7 in fibrosis was investigated using IRF7 knockout (KO) mice in the bleomycin-induced and TSK/+mouse models. In vitro experiments with dermal fibroblasts from patients with SSc and healthy controls were performed.Results: IRF7 expression was significantly upregulated and activated in SSc skin tissue and explanted SSc dermal fibroblasts compared with unaffected, matched controls. Moreover, IRF7 expression was stimulated by IFN-α in dermal fibroblasts. Importantly, IRF7 co-immunoprecipitated with Smad3, a key mediator of transforming growth factor (TGF)-β signalling, and IRF7 knockdown reduced profibrotic factors in SSc fibroblasts. IRF7 KO mice demonstrated attenuated dermal fibrosis and inflammation compared with wild-type mice in response to bleomycin. Specifically, hydroxyproline content, dermal thickness as well as Col1a2, ACTA2 and interleukin-6 mRNA levels were significantly attenuated in IRF7 KO mice skin tissue. Furthermore, IRF7 KO in TSK/+mice attenuated hydroxyproline content, subcutaneous hypodermal thickness, Col1a2 mRNA as well as α-smooth muscle actin and fibronectin expression.Conclusions: IRF7 is upregulated in SSc skin, interacts with Smad3 and potentiates TGF-β-mediated fibrosis, and therefore may represent a promising therapeutic target in SSc. [ABSTRACT FROM AUTHOR]

Tight control of B cell differentiation into plasma cells (PCs) is critical for proper immune responses and the prevention of autoimmunity. The Etsl transcription factor acts in B cells to prevent PC differentiation. Etsl-/- mice accumulate PCs and produce autoantibodies. Etsl expression is downregulated upon B cell activation through the BCR and TLRs and is maintained by the inhibitory signaling pathway mediated by Lyn, CD22 and SiglecG, and SHP-1. In the absence of these inhibitory components, Etsl levels are reduced in B cells in a Btk-dependent manner. This leads to increased PCs, autoantibodies, and an autoimmune phenotype similar to that of Etsl-/- mice. Defects in inhibitory signaling molecules, including Lyn and Etsl, are associated with human lupus, although the effects are more subtle than the complete deficiency that occurs in knockout mice. In this study, we explore the effect of partial disruption of the Lyn/Etsl pathway on B cell tolerance and find that Lyn+/- Etsl+/- mice demonstrate greater and earlier production of IgM, but not IgG, autoantibodies compared with Lyn+/- or Etsl+/- mice. We also show that Btkdependent downregulation of Etsl is important for normal PC homeostasis when inhibitory signaling is intact. Etsl deficiency restores the decrease in steady state PCs and Ab levels observed in Btk-/- mice. Thus, depending on the balance of activating and inhibitory signals to Etsl, there is a continuum of effects on autoantibody production and PC maintenance. This ranges from fullblown autoimmunity with complete loss of Etsl-maintaining signals to reduced PC and Ab levels with impaired Etsl downregulation. [ABSTRACT FROM AUTHOR]

Summary: A20 is a potent anti-inflammatory protein that inhibits NF-κB, and A20 dysfunction is associated with autoimmunity and B cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20''s seventh zinc-finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a noncatalytic mechanism of IKK inhibition by A20 and a means by which polyubiquitin chains can specify a signaling outcome. [ABSTRACT FROM AUTHOR]

Summary: Lysine-63 (K63)-linked polyubiquitination has emerged as a mechanism regulating diverse cellular functions, including activation of the protein kinase IKK in the NF-κB pathways. However, genetic evidence for a key role of K63 polyubiquitination in IKK activation is lacking. Here, we devise a tetracycline-inducible RNAi strategy to replace endogenous ubiquitin with a K63R mutant in a human cell line. We demonstrate that K63 of ubiquitin and the catalytic activity of Ubc13, an E2 that catalyzes K63 polyubiquitination, are required for IKK activation by IL-1β, but surprisingly, not by TNFα. We further show that IKK activation by TNFα requires Ubc5, which functions with the E3 cIAP1 to catalyze polyubiquitination of RIP1 not restricted to K63 of ubiquitin. These results indicate that distinct ubiquitin-dependent mechanisms are employed for IKK activation by different pathways. The ubiquitin replacement methodology described here provides a means to investigate the function of polyubiquitin topology in various cellular processes. [Copyright &y& Elsevier]

Nuclear factor kappa enhancer binding protein (NF-KB) regulates diverse biological processes including immunity, inflammation, and apoptosis. A vast array of cellular stimuli converges on NF-KB, and ubiquitination plays an essential role in the coordination of these signals to regulate NF-κB activity. At least three steps in NF-κB activation directly involve ubiquitination: proteasomal degradation of inhibitor of NF-κB (IκB), processing of NF-κB precursors, and activation of the transforming growth factor (TGF)-β-activated kinase (TAK1) and IκB kinase (IKK) complexes. In this review, we discuss recent advances in the identification and characterization of ubiquitination and deubiquitination machinery that regulate NF-κB. Particular emphasis is given to proteasome-independent functions of ubiquitin, specifically its role in the activation of protein kinase complexes and in coordination of cell survival and apoptosis signals downstream of minor necrosis factor α (TNFα). [ABSTRACT FROM AUTHOR]

Systemic Sclerosis (Scleroderma, SSc) is a multifaceted disease characterized by autoimmunity, vasculopathy, and fibrosis affecting the skin and internal organs. Despite advances in the understanding and treatment of SSc in recent years, SSc continues to cause reduced quality of life and premature mortality. Type I interferons (IFNs), a family of cytokines with essential roles in the immune response to microbial infection, play a pathogenic role in certain autoimmune diseases (reviewed elsewhere in this edition). Polymorphisms in interferon-regulatory factors confer an increased risk of SSc, and IFN excess is evident in the blood and skin of a large percentage of SSc patients. Here we describe the evidence of Type I IFN dysregulation in SSc, revealed predominately by genetics and gene expression profiling. We also discuss evidence regarding mechanisms by which Type I IFN might contribute to SSc pathogenesis, mechanisms driving excess Type I IFN production in SSc, and the potential roles of Type I IFNs as biomarkers and therapeutic targets in SSc. [ABSTRACT FROM AUTHOR]

To gain a broader insight into the role of Bcl-2 proteins in apoptosis induced after mitotic arrest, we investigated the subcellular location, oligomeric structure, and protein interactions of Bax, Bcl-2, and Bcl-xL in vinblastine-treated KB-3 cells. Vinblastine induced the translocation of Bax from the cytosol to the mitochon- dna, which was accompanied by conformational activation and oligomerization of Bax. Bcl-2 was located in the mitochondria, underwent multisite phosphorylation after vinblastine treatment, and was strictly monomeric under all conditions. In contrast, in control cells, Bcl-xL existed in both monomeric (30 kDa) and ohigomeric (150 kDa) forms. Treatment with agents that induced Bcl-xL phosphorylation (microtubule inhibitors) caused loss of the 150-kDa form, but this species was unaffected by apoptotic stimuli that did not stimulate phosphorylation. Vinblastine also promoted Bax activation and Bax oligomerization in HCT116 colon cancer cells. Both wild-type and Bax-deficient HCT116 cells expressed the 150-kDa form of Bcl-xL, which was depleted similarly in both cell lines upon vinblastine treatment. Co-immunoprecipitation studies revealed that in untreated KB-3 cells inactive cytosolic Bax interacted with Bcl-xL, whereas in vinblastine-treated cells, activated mitochondrial Bax did not interact with Bcl-xL. Interaction of Bcl-2 with Bax was not observed under any condition. Overexpression of Bcl-xL inhibited vinbiastine-induced Bax activation and Bax dimerization and in parallel inhibited apoptosis. The results indicate that vinblastine-induced apoptosis requires translocation, activation, and oligomerization of Bax and is associated with specific changes in the oligomeric properties of Bcl-xL, which occur independently of Bax. [ABSTRACT FROM AUTHOR]

Central tolerance checkpoints are critical for the elimination of autoreactive B cells and the prevention of autoimmunity. When autoreactive B cells encounter their Ag at the immature B cell stage, BCR cross-linking induces receptor editing, followed by apoptosis if edited cells remain autoreactive. Although the transcription factor Foxo1 is known to promote receptor editing, the role of the related factor Foxo3 in central B cell tolerance is poorly understood.We find that BCR-stimulated immature B cells fromFoxo3-deficient mice demonstrate reduced apoptosis compared with wild type cells. Despite this, Foxo3-/- mice do not develop increased autoantibodies. This suggests that the increased survival of Foxo3-/- immature B cells allows additional rounds of receptor editing, resulting in more cells "redeeming" themselves by becoming nonautoreactive. Indeed, increased Igl usage and increased recombining sequence recombination among Igλ-expressing cells were observed in Foxo3-/- mice, indicative of increased receptor editing. We also observed that deletion of high-affinity autoreactive cells was intact in the absence of Foxo3 in the anti--hen egg lysozyme (HEL)/membrane-bound HEL model. However, Foxo3 levels in B cells from systemic lupus erythematosus (SLE) patients were inversely correlated with disease activity and reduced in patients with elevated anti-dsDNA Abs. Although this is likely due in part to increased B cell activation in these SLE patients, it is also possible that low-affinity B cells that remain autoreactive after editing may survive inappropriately in the absence of Foxo3 and become activated to secrete autoantibodies in the context of other SLE-associated defects. [ABSTRACT FROM AUTHOR]

Summary: In response to viral infection, RIG-I-like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type I interferons. We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here, we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils that are capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade. [ABSTRACT FROM AUTHOR]

Summary: Cardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However, the mechanisms underlying vascular “tubulogenesis” are only beginning to be unraveled. We show that endothelial tubulogenesis requires the Ras interacting protein 1, Rasip1, and its binding partner, the RhoGAP Arhgap29. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Similarly, depletion of either Rasip1 or Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton, and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. This study identifies Rasip1 as a unique, endothelial-specific regulator of Rho GTPase signaling, which is essential for blood vessel morphogenesis. [Copyright &y& Elsevier]

Dermal fibrosis is a cardinal feature of systemic sclerosis (SSc) for which there are limited treatment strategies. This is in part due to our fragmented understanding of how dermal white adipose tissue (DWAT) contributes to skin fibrosis. We identified elevated sine oculis homeobox homolog 1 (SIX1) expression in SSc skin samples from the GENISOS and PRESS cohorts, the expression of which correlated with adipose-associated genes and molecular pathways. SIX1 localization studies identified increased signals in the DWAT area in SSc and in experimental models of skin fibrosis. Global and adipocyte specific Six1 deletion abrogated end-stage fibrotic gene expression and dermal adipocyte shrinkage induced by SQ bleomycin treatment. Further studies revealed a link between elevated SIX1 and increased expression of SERPINE1 and its protein PAI-1 which are known pro-fibrotic mediators. However, SIX1 deletion did not appear to affect cellular trans differentiation. Taken together these results point at SIX1 as a potential target for dermal fibrosis in SSc.

Objectives: Patients with clinically evident autoimmune disease are at increased risk for premature cardiovascular disease (CVD). Markers of serological autoimmunity such as anti-nuclear antibodies (ANA) are found in approximately 25% of the general population. Yet, the vast majority will not develop clinical autoimmune disease. Serological autoimmunity is a risk factor for CVD death in individuals without autoimmune disease; however, the mechanisms mediating this excess CVD risk have not been elucidated., Methods: We examined associations of ANA with traditional cardiovascular risk factors, inflammatory mediators, and vascular biomarkers in the Dallas Heart Study - a large, representative multiethnic population-based cohort. Plasma ANA were measured by enzyme linked immunosorbent assay in 3,488 Dallas Heart Study participants aged 30 to 65 years who do not have known rheumatologic disease. Associations of ANA with demographic characteristics, cardiovascular risk factors, and biomarkers were assessed using univariable and multivariable linear regression., Results: Factors independently associated with higher ANA include female sex, African-American race/ethnicity, soluble intracellular adhesion molecule-1, soluble CD40 ligand, chemokine CXCL-2, and Cystatin C (p<0.05 for each). ANA was not associated with traditional cardiovascular risk factors, high sensitivity C-reactive protein, coronary artery calcium scores, or aortic wall thickness., Conclusion: ANA are associated with inflammatory mediators and biomarkers of vascular activation, but not with traditional cardiovascular risk factors in a multiethnic population-based cohort. These findings suggest that the cardiovascular risk associated with ANA may involve pathways distinct from traditional risk factors and include dysregulation of endothelial cells and the immune system.

The early development of vertebrate embryos is characterized by rapid cell proliferation necessary to support the embryo's growth. During this period, the embryo must maintain a balance between ongoing cell proliferation and mechanisms that arrest or delay the cell cycle to repair oxidative damage and other genotoxic stresses. The ataxia-telangiectasia mutated (ATM) kinase is a critical regulator of the response to DNA damage, acting through downstream effectors, such as p53 and checkpoint kinases (CHK) to mediate cell-cycle checkpoints in the presence of DNA damage. Mice and humans with inactivating mutations in ATM are viable but have increased susceptibility to cancers. The possible role of ATM in limiting cell proliferation in early embryos has not been fully defined. One target of ATM and CHKs is the Cdc25 phosphatase, which facilitates cell-cycle progression by removing inhibitory phosphates from cyclin-dependent kinases (CDK). We have identified a zebrafish mutant, standstill, with an inactivating mutation in cdc25a. Loss of cdc25a in the zebrafish leads to accumulation of cells in late G(2) phase. We find that the novel family member cdc25d is essential for early development in the absence of cdc25a, establishing for the first time that cdc25d is active in vivo in zebrafish. Surprisingly, we find that cell-cycle progression in cdc25a mutants can be rescued by chemical or genetic inhibition of ATM. Checkpoint activation in cdc25a mutants occurs despite the absence of increased DNA damage, highlighting the role of Cdc25 proteins to balance constitutive ATM activity during early embryonic development.