Lupus-Associated Immune Complexes Activate Human Neutrophils in an FcγRIIA-Dependent but TLR-Independent Response.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of autoantibodies against nucleic acids and nucleoproteins. Anti-dsDNA Abs are considered a hallmark of SLE, and previous studies have indicated that nucleic acid-containing immune complexes (ICs) induce B cell and dendritic cell activation in a TLR-dependent process. How ICs containing nucleic acids affect neutrophil function has not been well investigated. In this study, we report that nucleic acid-containing ICs derived from the sera of SLE patients induce human and mouse neutrophil activation through TLR-independent mechanisms. Soluble ICs containing Sm/RNP, an RNA Ag, activate human neutrophils to produce reactive oxygen species (ROS) and IL-8. In contrast, ICs containing DNA have to be immobilized to efficiently activate neutrophils. We found that deleting TLR7 or TLR9, the receptors for RNA and DNA, had no effect on mouse neutrophil activation induced by RNA-containing and immobilized DNA-containing ICs. Binding of ICs are mediated through FcγRIIA and FcγRIIIB. However, neutrophil activation induced by RNA- and DNA-containing ICs requires FcγRIIA, as blocking FcγRIIA inhibited ROS release from neutrophils. RNA-containing ICs induce calcium flux, whereas TLR7/8 ligand R848 do not. Surprisingly, chloroquine inhibits calcium flux induced by RNA-containing ICs, suggesting that this lesser known function of chloroquine is involved in the neutrophil activation induced by ICs. These data indicate the SLE-derived ICs activate neutrophils to release ROS and chemokines in an FcγRIIA-dependent and TLR7- and TLR9-independent manner that likely contributes to local tissue inflammation and damage.

Proteome-wide analysis and CXCL4 as a biomarker in systemic sclerosis.

BACKGROUND: Plasmacytoid dendritic cells have been implicated in the pathogenesis of systemic sclerosis through mechanisms beyond the previously suggested production of type I interferon. METHODS: We isolated plasmacytoid dendritic cells from healthy persons and from patients with systemic sclerosis who had distinct clinical phenotypes. We then performed proteome-wide analysis and validated these observations in five large cohorts of patients with systemic sclerosis. Next, we compared the results with those in patients with systemic lupus erythematosus, ankylosing spondylitis, and hepatic fibrosis. We correlated plasma levels of CXCL4 protein with features of systemic sclerosis and studied the direct effects of CXCL4 in vitro and in vivo. RESULTS: Proteome-wide analysis and validation showed that CXCL4 is the predominant protein secreted by plasmacytoid dendritic cells in systemic sclerosis, both in circulation and in skin. The mean (±SD) level of CXCL4 in patients with systemic sclerosis was 25,624±2652 pg per milliliter, which was significantly higher than the level in controls (92.5±77.9 pg per milliliter) and than the level in patients with systemic lupus erythematosus (1346±1011 pg per milliliter), ankylosing spondylitis (1368±1162 pg per milliliter), or liver fibrosis (1668±1263 pg per milliliter). CXCL4 levels correlated with skin and lung fibrosis and with pulmonary arterial hypertension. Among chemokines, only CXCL4 predicted the risk and progression of systemic sclerosis. In vitro, CXCL4 down-regulated expression of transcription factor FLI1, induced markers of endothelial-cell activation, and potentiated responses of toll-like receptors. In vivo, CXCL4 induced the influx of inflammatory cells and skin transcriptome changes, as in systemic sclerosis. CONCLUSIONS: Levels of CXCL4 were elevated in patients with systemic sclerosis and correlated with the presence and progression of complications, such as lung fibrosis and pulmonary arterial hypertension. (Funded by the Dutch Arthritis Association and others.).

Ultrasound assessment of subcutaneous compressibility: a potential adjunctive diagnostic tool in eosinophilic fasciitis.

BACKGROUND: Eosinophilic fasciitis (EF) is an autoimmune, fibrotic disorder described initially with scleroderma-like skin changes where deep soft tissue sampling that includes fascia is frequently felt to be necessary to confirm the diagnosis. OBJECTIVE: The objective of this study was to distinguish forearm involvement by EF from other fibrosing diseases and from control subjects with normal skin and fascia using B-mode ultrasound. METHODS: A cross-sectional study over a 4-year period in which clinically involved forearm skin of consecutive patients with EF (n = 12), diabetic cheiroarthropathy (n = 8), diffuse systemic sclerosis (n = 23), and control subjects (n = 8) was evaluated by 12-MHz, B-mode ultrasound for degree of subcutaneous tissue compressibility, and this finding was compared with the criterion standard of clinical diagnostic criteria for each disease process. RESULTS: Subcutaneous compressibility in EF was significantly reduced when compared with diffuse systemic sclerosis and with control subjects. Subcutaneous thinning was observed in some patients with EF (4/12), diabetic cheiroarthropathy (4/8), and diffuse systemic sclerosis (6/23), but not in control subjects. Diabetic cheiroarthropathy and diffuse systemic sclerosis patients with subcutaneous thinning had less than 20% subcutaneous compressibility, whereas only 1 of 12 EF patients had compressibility of more than 20% regardless of subcutaneous thinning. CONCLUSIONS: A 12-MHz, B-mode ultrasound may be used to measure subcutaneous compressibility, thereby serving as an adjunct tool in distinguishing EF from diffuse systemic sclerosis, especially when tissue sampling is less feasible or when the result of tissue sampling is equivocal.

Understanding Stakeholders' Perspectives to Increase COVID-19 Vaccine and Booster Uptake Among Black Individuals With Rheumatic Conditions.

OBJECTIVE: Disparities in COVID-19 vaccine and booster uptake persist. This study aimed to obtain perspectives from community and physician stakeholders on COVID-19 vaccine and booster hesitancy and strategies to promote vaccine uptake among Black individuals with rheumatic and musculoskeletal conditions. METHODS: We invited community leaders and physicians in greater Boston and Chicago to participate in semi-structured interviews using a moderator guide developed a priori. Participants were queried about how to best address vaccine hesitancy, strategies to target high-risk populations, and factors to identify future community leaders. Interviews were audio recorded, transcribed verbatim, and analyzed thematically using Dedoose. RESULTS: A total of 8 physicians and 12 community leaders participated in this study between November 2021 and October 2022. Qualitative analyses revealed misinformation/mixed messaging and mistrust, with subthemes including conspiracy theories, concerns regarding vaccine development and function, racism and historical injustices, and general mistrust of health care systems as the top cited reasons for COVID-19 vaccine hesitancy. Participants also shared demographic-specific differences, such as race, ethnicity, age, and gender that influenced the identified themes, with emphasis on COVID-19 vaccine access and apathy. Strategies for community-based vaccine-related information dissemination included personal storytelling with an iterative and empathetic approach, while recognizing the importance of protecting community leader well-being. CONCLUSION: To increase vaccine uptake among Black individuals with rheumatic conditions, strategies should acknowledge and respond to racial/ethnic and socioeconomic injustices that engender vaccine hesitancy. Messaging should be compassionate, individually tailored, and recognize heterogeneity in experiences and opinions. Results from these analyses will inform a planned community-based intervention in Boston and Chicago.

Pulmonary hypertension: screening and evaluation in scleroderma.

PURPOSE OF REVIEW: The review provides an update of the epidemiology, pathogenesis, risk factors, screening and treatment of pulmonary arterial hypertension in systemic sclerosis. RECENT FINDINGS: Several recent studies have investigated the utility of several noninvasive screening methods and the propagation of new treatments promise the clinician better outcomes than the current median survival time of 1 year for patients with scleroderma-related pulmonary arterial hypertension. SUMMARY: Pulmonary hypertension is a frequent cause of morbidity and mortality in patients with systemic sclerosis. This review discusses the recent changes in the classification of pulmonary hypertension, especially the significance for the rheumatologist. A high clinical suspicion should be maintained, even in early scleroderma. Despite progress in echocardiography and biomarkers, right heart catheterization remains the only test that can diagnose pulmonary hypertension and differentiate pulmonary veno-occlusive disease from pulmonary arterial hypertension. The differentiation of these causes of pulmonary hypertension in the scleroderma patient is essential because the initiation of pulmonary vasodilators in veno-occlusive disease often leads to increased mortality. The role of screening with serum biomarkers and noninvasive testing remains controversial, and in this review we discuss the controversies and new recommendations in detail.

dsRNA activation of endothelin-1 and markers of vascular activation in endothelial cells and fibroblasts.

BACKGROUND: In patients with systemic sclerosis (SSc), the relationship between innate immune activation, represented by increased expression of interferon (IFN)-regulated genes, and vascular injury/activation, manifest by increased endothelin-1 (ET-1), endothelin converting enzyme-1 (ECE1) and intercellular adhesion molecule-1, is uncertain. OBJECTIVE: To investigate the potential roles of innate immune ligands in both these pathogenic pathways. METHODS: The effect of known Toll-like receptor (TLR) ligands was tested in vitro on dermal microvascular and pulmonary arterial endothelial cells, and on dermal fibroblasts cultured from healthy controls and patients with SSc. To test the effect of double-stranded RNA (dsRNA) on vascular activation/injury in vivo, polyinosinic/polycytidylic acid (poly(I:C)) was administered continuously over 7 days by subcutaneous osmotic pump. RESULTS: dsRNA/poly(I:C), but not other TLR ligands, highly stimulated ET-1 protein and mRNA (EDN1), as well as intercellular adhesion molecule-1 (ICAM-1) and IFN-regulated MX2, by endothelial cells and dermal fibroblasts. Poly(I:C) induced EDN1, ECE1, and ICAM-1 mRNA expression in poly(I:C) treated skin. Poly(I:C)-induced EDN1, ECE1 and MX2 was not blocked in mice with the type I IFN receptor deleted. However, poly(I:C)-induced EDN1 and ECE1, but not poly(I:C)-induced ICAM-1 expression was blocked in mice with the TLR3 signalling protein TRIF/TICAM-1 deleted. CONCLUSION: Together these data show that the dsRNA can regulate genes associated with vascular activation, as seen in SSc, that type I IFNs do not mediate these effects, and that EDN1 and ECE1 but not ICAM-1 activation is mediated by TLR3.

Innate Immune Modulation Induced by EBV Lytic Infection Promotes Endothelial Cell Inflammation and Vascular Injury in Scleroderma.

Microvascular injury is considered an initial event in the pathogenesis of scleroderma and endothelial cells are suspected of being the target of the autoimmune process seen in the disease. EBV has long been proposed as a trigger for autoimmune diseases, including scleroderma. Nevertheless, its contribution to the pathogenic process remains poorly understood. In this study, we report that EBV lytic antigens are detected in scleroderma dermal vessels, suggesting that endothelial cells might represent a target for EBV infection in scleroderma skin. We show that EBV DNA load is remarkably increased in peripheral blood, plasma and circulating monocytes from scleroderma patients compared to healthy EBV carriers, and that monocytes represent the prominent subsets of EBV-infected cells in scleroderma. Given that monocytes have the capacity to adhere to the endothelium, we then investigated whether monocyte-associated EBV could infect primary human endothelial cells. We demonstrated that endothelial cells are infectable by EBV, using human monocytes bound to recombinant EBV as a shuttle, even though cell-free virus failed to infect them. We show that EBV induces activation of TLR9 innate immune response and markers of vascular injury in infected endothelial cells and that up-regulation is associated with the expression of EBV lytic genes in infected cells. EBV innate immune modulation suggests a novel mechanism mediating inflammation, by which EBV triggers endothelial cell and vascular injury in scleroderma. In addition, our data point to up-regulation of EBV DNA loads as potential biomarker in developing vasculopathy in scleroderma. These findings provide the framework for the development of novel therapeutic interventions to shift the scleroderma treatment paradigm towards antiviral therapies.

Innate immunity and inflammation in systemic sclerosis.

PURPOSE OF REVIEW: Recent advances in our understanding of innate immunity and inflammation have direct bearing on how we understand autoimmunity, fibrosis and how innate immune sensors might stimulate both of these key features of systemic sclerosis (SSc) RECENT FINDINGS: Nucleic acid containing immune complexes activate toll-like receptors (TLRs) and induce expression of interferon responsive genes (IRGs) and autoantibodies in systemic lupus erythematosus (SLE). Recent studies indicate that increased SSc expression of IRGs may also be mediated by nucleic acid containing immune complexes. An expanding array of non-TLR innate immune pathways has recently been discovered. In particular, nalp3 mediated inflammasome activation of caspase-1 and conversion of pro-IL-1 to IL-1 play a key role in silica-mediated and bleomycin-mediated pulmonary fibrosis. TLR activation stimulates other inflammatory mediators, such as IL-1, IL-6 and TNFa in macrophages and dendritic cells. Activation of these and other inflammatory mediators, through TLR and non-TLR sensors, may cooperate to upregulate fibrotic mediators such as TGFbeta and IL-13. SUMMARY: These observations provide a new paradigm for understanding the relationship between immunity/inflammation and fibrosis. New therapeutics, including TLR agonists and antagonists, and IFN inhibitors are currently under investigation. Further understandings of inflammasome-mediated fibrosis may provide further insights into SSc pathogenesis.

Epstein-Barr virus lytic infection promotes activation of Toll-like receptor 8 innate immune response in systemic sclerosis monocytes.

BACKGROUND: Monocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc. METHODS: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells. RESULTS: Infectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes. CONCLUSION: This study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells.

Comment on perceptual defense in anxiety disorders.

This paper is intended to lend support to the 2003 findings of Poloni, Riquier, Zimmermann, and Borgeat. Further, some extensions of these and other findings with respect to perceptual defense are suggested.

Fresolimumab treatment decreases biomarkers and improves clinical symptoms in systemic sclerosis patients.

BACKGROUND: TGF-ß has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-ß-regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF-ß may benefit these patients. METHODS: Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF-ß isoforms. Seven patients received two 1 mg/kg doses of fresolimumab, and eight patients received one 5 mg/kg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-ß-regulated biomarker genes thrombospondin-1 (THBS1) and cartilage oligomeric protein (COMP) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS). RESULTS: In patient skin, THBS1 expression rapidly declined after fresolimumab treatment in both groups (P = 0.0313 at 7 weeks and P = 0.0156 at 3 weeks), and skin expression of COMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased (P < 0.001 at all time points). Expression levels of other TGF-ß-regulated genes, including SERPINE1 and CTGF, declined (P = 0.049 and P = 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment (P = 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab (P < 0.05). Baseline levels of THBS1 were predictive of reduced THBS1 expression and improved MRSS after fresolimumab treatment. CONCLUSION: The rapid inhibition of TGF-ß-regulated gene expression in response to fresolimumab strongly implicates TGF-ß in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis. TRIAL REGISTRATION: Clinicaltrials.gov NCT01284322.

Safety evaluation of a novel whitening gel, containing 6% hydrogen peroxide and a commercially available whitening gel containing 18% carbamide peroxide in an exaggerated use clinical study.

OBJECTIVES: The aim of this study was to compare the effect on oral soft tissue of a self-applied tooth whitening gel containing 6% hydrogen peroxide (Xtra White) with a marketed paint-on whitening gel containing 18% carbamide peroxide (Colgate Simply White) after 2-weeks of using products four times daily. METHODS: A 2-week, examiner-blind, stratified, parallel design clinical trial was conducted. Twenty subjects were divided into two groups, balanced according to age and gender. Subjects followed a 2-week, twice-daily regimen of brushing with standard fluoride toothpaste and applied gel product to facial aspects of six upper and six lower incisors/canines, twice in succession with 30 min between applications. Soft tissue examinations were performed on Day 1, before the first application of the test products, and on Day 2, 5, 8 and 15. RESULTS: During the 2-week treatment period, twelve adverse reactions were recorded as potentially attributable to the study products, evenly split between the two test groups. All reports were mild in symptoms and resolved without the need of medical intervention. None of the subjects experiencing an adverse event requested to be withdrawn from the study. CONCLUSIONS: Under the exaggerated use conditions of this test, there was no evidence to suggest that either of the whitening gels produced irritation that was building or developing during the course of the study. It is concluded that both products are safe for their intended use.

Epstein-Barr virus infection induces aberrant TLR activation pathway and fibroblast-myofibroblast conversion in scleroderma.

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein-Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV-Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-ß1 (TGFß1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Novel insights on the role of the innate immune system in systemic sclerosis.

Over the last several years the involvement of the innate immune system in the pathogenesis of autoimmune diseases such as systemic lupus erythematosus (SLE) has become well established. As systemic sclerosis (SSc; scleroderma) shares clinical features and autoantibodies with SLE, investigation has recently focused on the role of innate immunity in SSc. This has been supported by recent genetic studies. However, unlike SLE and other related autoimmune diseases, SSc patients suffer from pathologic fibrosis of skin and internal organs. The fibrotic component of SSc shares several features with syndromes following environmental exposures to agents such as organic solvents, silica dust and bleomycin. Recent work in SSc and these related fibrotic diseases have identified several areas in which innate immunity can stimulate inflammation as well as fibrosis. This article will focus on the recent discoveries identifying a prominent role of cells of the innate immune system, pattern recognition receptors, and activation of dendritic cells in the pathogenesis of SSc.

Poly(I:C) drives type I IFN- and TGFß-mediated inflammation and dermal fibrosis simulating altered gene expression in systemic sclerosis.

Immune activation of fibrosis likely has a crucial role in the pathogenesis of systemic sclerosis (SSc). The aim of this study was to better understand the innate immune regulation and associated IFN- and transforming growth factor-ß (TGFß)-responsive gene expression in SSc skin and dermal fibroblasts, in particular the effect of different Toll-like receptor (TLR) ligands. To better understand the relationship between inflammation and fibrosis in vivo, we developed a murine model for chronic innate immune stimulation. We found that expression of both IFN- and TGFß-responsive genes is increased in SSc skin and SSc fibroblasts when stimulated by TLR ligands. In contrast, cutaneous lupus skin showed much more highly upregulated IFN-responsive and much less highly upregulated TGFß-responsive gene expression. Of the TLRs ligands tested, the TLR3 ligand, polyinosinic/polycytidylic acid (Poly(I:C)), most highly increased fibroblast expression of both IFN- and TGFß-responsive genes as well as TLR3. Chronic subcutaneous immune stimulation by Poly(I:C) stimulated inflammation, and IFN- and TGFß-responsive gene expression. However, in this model, type I IFNs had no apparent role in regulating TGFß activity in the skin. These results suggest that TLR agonists may be important stimuli of dermal fibrosis, which is potentially mediated by TLR3 or other innate immune receptors.

B cell depletion with rituximab in patients with diffuse cutaneous systemic sclerosis.

OBJECTIVE: To determine the safety of rituximab, to provide preliminary data regarding the potential efficacy of rituximab, and to investigate the effects of rituximab on autoimmunity and fibrosis in patients with diffuse cutaneous systemic sclerosis (dcSSc). METHODS: Fifteen patients with dcSSc, all of whom experienced their first non-Raynaud's disease-associated disease manifestation within 18 months of trial entry, were recruited to receive 2 intravenous doses of rituximab (1,000 mg), administered 2 weeks apart. Safety, clinical, and exploratory outcomes were evaluated at baseline and at 6 months. The primary outcome was the change in the modified Rodnan skin thickness score (MRSS) at 6 months compared with baseline. RESULTS: Adverse events included frequent infusion reactions and rare infections (urinary tract infection and dental abscess occurred in 1 patient each). The mean change in the MRSS between baseline and 6 months was not significant. Results of pulmonary function tests and other measures of major organ involvement were stable. The modest B cell infiltrates that were present in most skin biopsy specimens at baseline were completely depleted at 6 months in most patients. Autoantibody titers showed only modest and variable changes after treatment. CONCLUSION: In this pilot study, treatment with rituximab appeared to be safe and well tolerated among patients with dcSSc. Rituximab treatment resulted in both depletion of circulating B cells and depletion of dermal B cells but had little effect on the levels of SSc-associated autoantibodies. Rituximab treatment did not appear to result in a significant beneficial effect on skin disease. The potential efficacy of rituximab in other organs such as the lung could not be clearly evaluated in this small open-label trial.

A macrophage marker, Siglec-1, is increased on circulating monocytes in patients with systemic sclerosis and induced by type I interferons and toll-like receptor agonists.

OBJECTIVE: Microarray analyses of peripheral blood leukocytes have shown that patients with systemic lupus erythematosus express increased levels of type I interferon (IFN)-regulated genes. In this study we examined gene expression by peripheral blood mononuclear cells (PBMCs) from patients with systemic sclerosis (SSc) to better understand the dysregulation of the immune system in this disease. METHODS: PBMC gene expression was analyzed by microarray and confirmed by real-time polymerase chain reaction (PCR). Surface protein expression of Siglec-1 was analyzed by flow cytometry in PBMCs from healthy control subjects and patients with SSc, and in control PBMCs that were cultured in vitro with Toll-like receptor (TLR) agonists. RESULTS: SSc patients showed increased expression of a cluster of IFN-regulated genes, including Siglec-1 (CD169, sialoadhesin). This result was verified and extended by real-time PCR, showing that a subset of the SSc patients expressed strikingly increased levels of Siglec-1 messenger RNA (mRNA). Flow cytometry of PBMCs from SSc patients and healthy controls showed increased Siglec-1 surface protein expression, which was restricted to CD14+ monocytes. In vitro studies showed that type I IFN and certain TLR agonists, including TLR-7 and TLR-9, induced Siglec-1 mRNA and protein expression. Moreover, TLR induction of surface Siglec-1 was shown to be type I IFN-dependent. Increased numbers of Siglec-1+ cells were observed by immunohistochemistry in the skin of SSc patients compared with healthy controls. CONCLUSION: Increased expression of Siglec-1 in circulating SSc monocytes and tissue macrophages suggests that type I IFN-mediated activation of monocytes occurs in SSc, possibly through TLR activation of IFN secretion. These observations indicate a potential role for type I IFN-activated monocyte/macrophages in the pathogenesis of SSc.

Recent diuretic use and the risk of recurrent gout attacks: the online case-crossover gout study.

OBJECTIVE: To assess several putative risk factors, including thiazide and loop diuretics use, thought to trigger recurrent gout attacks. METHODS: We conducted an internet-based case-crossover study involving subjects who had a gout attack within the past year. Patients were recruited online and asked to provide access to medical records. Data were obtained on specific diuretic use on each day over the 2-day period prior to an acute gout attack (hazard period) and on each day of 2 days during the intercritical period (control period). We examined the relation of all diuretic use and use of specific diuretics, i.e., thiazide and loop, to the risk of recurrent gout attacks using a conditional logistic regression model adjusting for alcohol consumption and purine intake. RESULTS: One hundred ninety-seven subjects completed both control and hazard period questionnaires. Participants were predominantly male (80%) and over half had a college education. The median time between onset of gout attack and logging on to the website was 2 days. Adjusting for alcohol consumption and purine intake, the odds ratio (OR) for recurrent gout attacks from all diuretic use over the last 48 h was 3.6 (95% confidence interval 1.4-9.7). OR of recurrent gout attacks were 3.2 and 3.8 for use of thiazide and loop, respectively. CONCLUSION: Recent use of diuretics is associated with a significantly increased risk for recurrent gouty arthritis. The increased risk of gout attacks from either thiazide or possibly loop diuretic therapies represents an important modifiable risk factor in patients with gout.

Determination of skin irritation potential in the human 4-h patch test.

Recently adopted legislation in Europe has increased the focus that must be placed on the development of in vitro alternatives to the traditional toxicology tests employed to identify the human health hazards associated with chemicals. Included in these is the rabbit skin-irritation test which is used to discriminate those substances which possess significant acute skin irritation potential from those which are of more limited irritation potential. So far, the considerable efforts to replace this assay with in vitro alternatives have not been successful, which may in part be due to the relatively poor quality of the existing in vivo dataset. To help address this problem, we have elected to present our complete database of information on the skin irritation potential of some 65 substances, all of which have been tested in a standard human 4-h patch test. These provide a high quality dataset, generated in man (the goal of toxicologists' health protection-related activities). The data are presented in the context of results with a concurrent positive skin irritation control to ensure that results from individual experiments can be correlated. Consequently, in vitro or in silico alternatives which can identify the significant acute human skin irritants in this group may well represent suitable alternatives to the rabbit.