Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon.

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin-eosin and immunostaining.

Gene Expression Profiling in Blood and Affected Muscle Tissues Reveals Differential Activation Pathways in Patients with New-onset Juvenile and Adult Dermatomyositis.

OBJECTIVE: To identify shared and differential molecular pathways in blood and affected muscle between adult dermatomyositis (DM) and juvenile DM, and their association with clinical disease activity measures. METHODS: Gene expression of transcription factors and cytokines involved in differentiation and effector function of T cell subsets, regulatory T cells and follicular Th cells, were analyzed in the blood from 21 newly diagnosed adult and 26 juvenile DM subjects and in 15 muscle specimens (7 adult and 8 juvenile DM) using a custom RT2 Profiler PCR Array. Disease activity was determined and measured by established disease activity tools. RESULTS: The most prominent finding was the higher blood expression of Th17-related cytokines [retinoic acid-related orphan receptor-γ, interferon regulatory factor 4, interleukin (IL)-23A, IL-6, IL-17F, and IL-21] in juvenile DM at baseline. In contrast, adult patients with DM showed increased blood levels of STAT3 and BCL6 compared with juvenile DM. In muscle, GATA3, IL-13, and STAT5B were found at higher levels in juvenile patients with DM compared with adult DM. Among 25 patients (11 adult and 14 juvenile DM) who had blood samples at baseline and at 6 months, increased expression of IL-1ß, STAT3, STAT6, STAT5B, and BCL6 was associated with an improvement in global extramuscular disease activity. CONCLUSION: We observed differences in gene expression profiling in blood and muscle between new-onset adult and juvenile DM. Cytokine expression in the blood of juvenile patients with new-onset DM was dominated by Th17-related cytokines compared with adult patients with DM. This may reflect the activation of different Th pathways between muscle and blood.

The type I interferons: Basic concepts and clinical relevance in immune-mediated inflammatory diseases.

There is increasing scientific and clinical interest in elucidating the biology of type I Interferons, which began approximately 60 years ago with the concept of "viral interference", a property that reduces the ability of a virus to infect cells. Although our understanding of the multiple cellular and molecular functions of interferons has advanced significantly, much remains to be learned and type I Interferons remain an active and fascinating area of inquiry. In this review, we cover some general aspects of type I interferon genes, with emphasis on interferon-alpha, and various aspects of molecular mechanisms triggered by type I interferons and toll-like receptor signaling by the Janus activated kinase/signal transducer activation of transcription (JAK-STAT) pathway and interferon regulatory factor pathway. We will also describe the role of type I interferons in autoimmune and inflammatory diseases, and its potential use as therapeutic agent.

Interferon-regulated chemokine score associated with improvement in disease activity in refractory myositis patients treated with rituximab.

OBJECTIVES: The purpose of this study was to investigate whether serum interferon (IFN)-regulated chemokine and distinct cytokine response profiles are associated with clinical improvement in patients with refractory inflammatory myopathy treated with rituximab. METHODS: In a randomised, placebo-phase trial Rituximab in Myositis Trial (RIM), 200 refractory adult and paediatric myositis subjects received rituximab. Following rituximab, clinical response and disease activity were assessed. Serum samples and clinical data were collected at baseline and several time-points after rituximab treatment. Multiplexed sandwich immunoassays quantified serum levels of IFN-regulated chemokines and other pro-inflammatory cytokines. Composite IFN-regulated chemokine and Th1, Th2, Th17 and regulatory cytokine scores were computed. RESULTS: Baseline IFN-regulated chemokine, Th1, Th2, Th17 and regulatory cytokine scores correlated with baseline physician global VAS, whereas the baseline Th1, Th2 and Th17 cytokine scores correlated with baseline muscle VAS. We also found baseline IFN-regulated chemokine scores correlated with specific non-muscular targets such as baseline cutaneous (r=0.29; p=0.002) and pulmonary (r=0.18; p=0.02) VAS scores. Among all cytokine/chemokines examined, the baseline score of IFN-regulated chemokines demonstrated the best correlation with changes in muscle VAS at 8 (r=-0.19; p=0.01) and 16 weeks (r=-0.17; p=0.03) following rituximab and physician global VAS at 16 weeks (r=-0.16; p=0.04). In vitro experiments showed increased levels of IL-8 (p=0.04), MCP-1 (p=0.04), IL-6 (p=0.03), IL-1ß (p=0.04), IL-13 (p=0.04), IL-10 (p=0.02), IL-2 (p=0.04) and IFN-γ (p=0.02) in supernatants of TLR-3 stimulated PBMCs from non-responder compared to patients responders to rituximab. CONCLUSIONS: IFN-regulated chemokines before treatment is associated with improvement in disease activity measures in refractory myositis patients treated with rituximab.

BAFF expression correlates with idiopathic inflammatory myopathy disease activity measures and autoantibodies.

OBJECTIVE: To investigate B cell survival cytokine messenger RNA (mRNA) levels as biomarkers of idiopathic inflammatory myopathies (IIM). METHODS: We measured and compared mRNA levels of B cell survival cytokines by quantitative real-time polymerase chain reaction in 98 patients with IIM, 38 patients with systemic lupus erythematosus, and 21 healthy controls. The cytokines were B cell-activating factor belonging to the tumor necrosis factor family (BAFF); ΔBAFF; and a proliferation-inducing ligand (APRIL); and their receptors BAFF-R, transmembrane activator and calcium modulator and cyclophilin ligand interactor, and B cell maturation antigen (BCMA). We also identified autoantibodies, including anti-Sm, anti-RNP, anti-SSA/Ro, anti-SSB/La, anti-topoisomerase 1, anti-hystidyl-tRNA synthetase, anti-ribosomal P, and anti-chromatin. Clinical disease activity was assessed by the International Myositis Assessment and Clinical Studies core set tool. We examined correlation of mRNA with disease activity, medication use, and autoantibodies. RESULTS: We found a positive correlation of BAFF and ΔBAFF expression with 3 disease activity measures, with ΔBAFF having a stronger correlation. Similarly, anti-SSA/Ro-52 and/or anti-SSA/Ro-60 had a strong positive correlation with mRNA levels of BAFF and ΔBAFF, and with relative ratios of BAFF/APRIL and BCMA/BAFF-R. CONCLUSION: These findings highlight the potential importance of BAFF, ΔBAFF, and BAFF-R in the pathogenesis of IIM, and suggest an important role in the assessment of disease activity.

Increased expression of ADAMTS13 mRNA correlates with ischemic cerebrovascular disease in systemic lupus erythematosus patients.

OBJECTIVE: We investigated ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13) messenger RNA levels as a biomarker of disease features in systemic lupus erythematosus. METHODS: We measured and compared messenger RNA (mRNA) levels of ADAMTS13 in peripheral blood cells in patients with systemic lupus erythematosus and healthy control subjects by whole-genome microarray. We retrospectively analyzed the correlations of ADAMTS13 mRNA expression with clinical features, laboratory parameters, therapeutic features, and disease activity (according to the Systemic Lupus Erythematosus Disease Activity Index). We also examined the association of three single nucleotide polymorphisms (rs4962145, rs2285467, and rs685523) of the ADAMTS13 gene with patient characteristics. RESULTS: In 309 patients, the median ADAMTS13 mRNA expression levels were significantly higher in blood cells of systemic lupus erythematosus patients than in 23 healthy controls (p = .03). Notably, ADAMTS13 mRNA expression levels were significantly higher in systemic lupus erythematosus patients with a history of stroke (p = .02) or transient ischemic attack (p = .02). Among the three single nucleotide polymorphisms analyzed, rs2285467 was significantly associated with stroke (p = .03) and anticardiolipin antibodies (p = .04). CONCLUSIONS: Increased expression of ADAMTS13 mRNA in blood cells is associated with the presence of ischemic cerebrovascular disease in systemic lupus erythematosus patients and suggests a potential role for ADAMTS13 in the pathogenesis of ischemic cerebrovascular disease in these patients.

Extranodal lymphoid microstructures in inflamed muscle and disease severity of new-onset juvenile dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (DM) is an autoimmune disease of childhood characterized by lesions in skin and muscle that are populated by plasmacytoid dendritic cells (PDCs) and lymphocyte infiltrates. We undertook this study to examine the cellular composition, organization, and molecular milieu of the cellular infiltrates in muscle in juvenile DM and to correlate the infiltrates with clinical disease manifestations. METHODS: Since PDCs and lymphocyte foci express CCL19 and CCL21, we investigated for in situ formation of lymphoid microstructures that could be sites of extranodal immune activation. RESULTS: Analyses of muscle biopsy samples from children with new-onset juvenile DM showed 3 categories of lesions: diffuse infiltrates, lymphocytic aggregates lacking follicle-like organization, and follicle-like structures. The last of these exhibited elements of classic lymphoid follicles, including networks of follicular dendritic cells and high endothelial venules. They also expressed high levels of CXCL13 and lymphotoxins known to support lymphoid organogenesis. There were also resident naive CD45RA+ T cells and maternally derived B cells and PDCs. Patients with diffuse infiltrates or lymphocytic aggregates were responsive to standard therapy with steroids and methotrexate, but those with follicle-like structures tended to have severe disease that required additional agents such as intravenous Ig or rituximab. CONCLUSION: These data suggest that lymphoneogenesis is a component of the early disease process in juvenile DM. Ectopic lymphoid structures could indicate a severe course of disease; their early detection could be a tool for disease management.

Involvement of dendritic cells in autoimmune diseases in children.

Dendritic cells (DCs) are professional antigen-presenting cells that are specialized in the uptake of antigens and their transport from peripheral tissues to the lymphoid organs. Over the last decades, the properties of DCs have been intensely studied and much knowledge has been gained about the role of DCs in various diseases and health conditions where the immune system is involved, particularly in cancer and autoimmune disorders. Emerging clues in autoimmune diseases, suggest that dendritic cell dysregulation might be involved in the development of various autoimmune disorders in both adults and children. However, studies investigating a possible contribution of DCs in autoimmune diseases in the pediatric population alone are scanty. The purpose of this review is to give a general overview of the current literature on the relevance of dendritic cells in the most common autoimmune conditions of childhood.

Plasmacytoid dendritic cells in inflamed muscle of patients with juvenile dermatomyositis.

OBJECTIVE: To examine whether dendritic cells (DCs) are constituents of muscle inflammation in juvenile dermatomyositis (DM). METHODS: The types, numbers, and activation state of DC subsets in inflamed muscle tissue from patients with juvenile DM and in noninflamed muscle tissue from control subjects were examined by multicolor immunofluorescence. Chemokine expression of the muscle-infiltrating cells was examined by laser capture microdissection and quantitative polymerase chain reaction. RESULTS: Plasmacytoid DCs were the predominant component of the inflamed muscle tissue from patients with juvenile DM. These cells were identified by coexpression of CD4 and CD123, but not CD11c, and also expressed CD83, indicating maturity of the cells. In contrast, in noninflamed muscle, plasmacytoid DCs were scarce and did not express CD83. Mononuclear cells surrounding the blood vessels of inflamed muscle contained abundant transcripts of CCL19 and CCL21, but very little CCL18 transcripts. In contrast, cells from noninflamed muscle contained negligible amounts of CCL19 and CCL21, but had high amounts of CCL18. Both the inflamed and noninflamed muscle tissue had equivalent levels of CXCL12 transcripts, but inflamed muscle contained more transcripts of the CXCL12 receptor CXCR4. CONCLUSION: These findings are consistent with the idea that plasmacytoid DCs are mediators of muscle inflammation in juvenile DM. The abundance of CD83+ plasmacytoid DCs in perivascular areas and the overexpression of CCL19 and CCL21 in perivascular cellular foci suggest that in situ activation and maturation of resident plasmacytoid DCs are central to the initiation and perpetuation of muscle inflammation in juvenile DM.