Association of anti-TPM4 autoantibodies with vasculopathic cutaneous manifestations in juvenile dermatomyositis.

OBJECTIVES: AECAs are detected in multiple forms of vasculitis or vasculopathy, including JDM. High levels of tropomyosin alpha-4 chain (TPM4) gene expression in cutaneous lesions and TPM4 protein expression in some endothelial cells (ECs) have been proven. Furthermore, the presence of autoantibodies to tropomyosin proteins have been discovered in DM. We therefore investigated whether anti-TPM4 autoantibodies are an AECA in JDM and are correlated with clinical features of JDM. METHODS: The expression of TPM4 protein in cultured normal human dermal microvascular ECs was investigated by Western blotting. Plasma samples from 63 children with JDM, 50 children with polyarticular JIA (pJIA) and 40 healthy children (HC) were tested for the presence of anti-TPM4 autoantibodies using an ELISA. Clinical features were compared between JDM patients with and without anti-TPM4 autoantibodies. RESULTS: Autoantibodies to TPM4 were detected in the plasma of 30% of JDM, 2% of pJIA (P < 0.0001) and 0% of HC (P < 0.0001). In JDM, anti-TPM4 autoantibodies were associated with the presence of cutaneous ulcers (53%; P = 0.02), shawl sign rash (47%; P = 0.03), mucous membrane lesions (84%; P = 0.04) and subcutaneous edema (42%; P < 0.05). Anti-TPM4 autoantibodies significantly correlated with the use of intravenous steroids and IVIG therapy in JDM (both P = 0.01). The total number of medications received was higher in patients with anti-TPM4 autoantibodies (P = 0.02). CONCLUSION: Anti-TPM4 autoantibodies are detected frequently in children with JDM and are novel myositis-associated autoantibodies. Their presence correlates with vasculopathic and other cutaneous manifestations of JDM that may be indicative of more refractory disease.

Association of anti-HSC70 autoantibodies with cutaneous ulceration and severe disease in juvenile dermatomyositis.

OBJECTIVES: JDM is an inflammatory myopathy characterized by prominent vasculopathy. AECAs are frequently detected in inflammatory and autoimmune diseases. We sought to determine whether AECAs correlate with clinical features of JDM, and thus serve as biomarkers to guide therapy or predict outcome. METHODS: Plasma samples from 63 patients with JDM, 49 patients with polyarticular JIA and 40 juvenile healthy controls were used to detect anti-heat shock cognate 71 kDa protein (HSC70) autoantibodies, a newly identified AECA, in ELISA assays. Clinical features were compared between JDM patients with and without anti-HSC70 autoantibodies. RESULTS: Anti-HSC70 autoantibodies were detected in 35% of patients with JDM, in 0% of patients with JIA (P < 0.0001) and in 0% of healthy donors (P < 0.0001). Both the presence of cutaneous ulcers (59% vs 17%, P < 0.002) and the use of wheelchairs and/or assistive devices (64% vs 27%, P < 0.007) were strongly associated with anti-HSC70 autoantibodies in JDM. High scores on the severity of myositis damage measures at the time of measurement of anti-HSC70 autoantibodies and an increased number of hospitalizations were also associated with anti-HSC70 autoantibodies. Intravenous immunoglobulin therapy was used more often in anti-HSC70 autoantibody-positive patients. CONCLUSION: Anti-HCS70 autoantibodies are detected frequently in children with JDM and are novel myositis-associated autoantibodies correlating with disease severity.

Using the tools of proteomics to understand the pathogenesis of idiopathic inflammatory myopathies.

PURPOSE OF REVIEW: One of the most important advances in medical research over the past 20 years has been the emergence of technologies to assess complex biological processes on a global scale. Although a great deal of attention has been given to genome-scale genetics and genomics technologies, the utility of studying the proteome in a comprehensive way is sometimes under-appreciated. In this review, we discuss recent advances in proteomics as applied to dermatomyositis/polymyositis as well as findings from other inflammatory diseases that may enlighten our understanding of dermatomyositis/polymyositis. RECENT FINDINGS: Proteomic approaches have been used to investigate basic mechanisms contributing to lung and skin disease in dermatomyositis/polymyositis as well as to the muscle disease itself. In addition, proteomic approaches have been used to identify autoantibodies targeting the endothelium in juvenile dermatomyositis. Studies from other inflammatory diseases have shown the promise of using proteomics to characterize the composition of immune complexes and the protein cargoes of exosomes. SUMMARY: There are many relevant scientific and clinical questions in dermatomyositis/polymyositis that can be addressed using proteomics approaches. Careful attention to both methodology and analytic approaches are required to obtain useful and reproducible data.

Plasma exosomes from children with juvenile dermatomyositis are taken up by human aortic endothelial cells and are associated with altered gene expression in those cells.

BACKGROUND: The pathology of juvenile dermatomyositis (JDM) is characterized by prominent vessel wall and perivascular inflammation. This feature of the disease has remained unexplained and under-investigated. We have hypothesized that plasma exosomes, which play an important role in inter-cellular communication, may play a role in the vascular injury associated with JDM. OBJECTIVE: To characterize the circulating exosomes of children with JDM and determine whether the small RNA cargoes within those exosomes are capable of altering transcriptional programs within endothelial cells. DESIGN/METHODS: We purified exosomes from plasma samples of children with active, untreated JDM (n = 6) and healthy controls (n = 9). We characterized the small RNA cargoes in JDM and control exosomes by RNA sequencing using the Illumina HiSeq 2500 platform. We then incubated isolated exosomes from healthy controls and children with JDM with cultured human aortic endothelial cells (HAEC) for 24 h. Fluorescence microscopy was used to confirm that both control and JDM exosomes were taken up by HAEC. RNA was then purified from HAEC that had been incubated with either control or JDM exosomes and sequenced on the Illumina platform. Differential expression of mRNAs from HAEC incubated with control or JDM exosomes was ascertained using standard computational methods. Finally, we assessed the degree to which differential gene expression in HAEC could be attributed to the different small RNA cargoes in JDM vs control exosomes using conventional and novel analytic methods. RESULTS: We identified 10 small RNA molecules that showed differential abundance when we compared JDM and healthy control exosomes. Fluorescence microscopy of labeled exosomes confirmed that both JDM and control exosomes were taken up by HAEC. Differential gene expression analysis revealed 59 genes that showed differential expression between HAEC incubated with JDM exosomes vs HAEC incubated with exosomes from controls. Statistical analysis of gene expression data demonstrated that multiple miRNAs exerted transcriptional control on multiple genes with HAEC. CONCLUSIONS: Plasma exosomes from children with active, untreated JDM are taken up by HAEC and are associated with alterations in gene expression in those cells. These findings provide new insight into potential mechanisms leading to the targeting of vascular tissue by the immune system in JDM.

Multiple target autoantigens on endothelial cells identified in juvenile dermatomyositis using proteomics.

Objective: Although generally classified within the group of inflammatory myopathies, JDM displays many pathological features of vasculitis. Previous work has shown that AECA are abundant in other forms of vasculitis. We therefore investigated whether such antibodies might also be detected in JDM. Methods: We screened plasma from children with JDM for the presence of AECA by western blotting and 2D gel electrophoresis (2DE) using proteins extracted from human aortic endothelial cells as the substrate. We performed mass spectrometry to identify candidate antigens from 2DE gels and used ELISA to confirm the presence of specific antibodies. Results: We identified 22 candidate target autoantigens for AECA probed with JDM plasma. Interestingly, 17 of these 22 target antigens were proteins associated with antigen processing and protein trafficking. ELISA confirmed the presence of antibodies to heat shock cognate 71 kDa protein in JDM plasma, particularly in children with active, untreated disease. Conclusion: Children with JDM express antibodies to autoantigens in endothelial cells. The clinical and pathological significance of such autoantibodies require further investigation.

Using proteomic and genomic methods to understand JDM.

Juvenile dermatomyositis is a complex illness characterized by vascular/perivascular inflammation, primarily in the skin and muscles. In this review, we discuss how proteomic and genomic technologies have expanded our understanding of the immune pathogenesis of this disease. We will also discuss further directions that the field may take to use existing and developing technologies to further our understanding of this often-perplexing disease.

Age-related Decrease of Sirtuin 2 Protein in Human Peripheral Blood Mononuclear Cells.

Sirtuin 2, which is mainly present in cytoplasm, plays an important role in mammalian development, caloric restriction, metabolic regulation cellular antioxidant potential and the regulation of aging. We found that the protein level of sirtuin 2 in human peripheral blood mononuclear cells (PBMCs) decreases with an advance in donor age in men and women. Our data suggest that sirtuin 2 level in PBMCs decreases with age in both men and women and may have a potential as a useful biomarker monitoring health conditions and aging.

The DNA repair enzyme apurinic/apyrimidinic endonuclease (Apex nuclease) 2 has the potential to protect against down-regulation of chondrocyte activity in osteoarthritis.

Apurinic/apyrimidinic endonuclease 2 (Apex 2) plays a critical role in DNA repair caused by oxidative damage in a variety of human somatic cells. We speculated that chondrocyte Apex 2 may protect against the catabolic process of articular cartilage in osteoarthritis (OA). Higher levels of Apex 2 expression were histologically observed in severely compared with mildly degenerated OA cartilage from STR/OrtCrlj mice, an experimental model which spontaneously develops OA. The immunopositivity of Apex 2 was significantly correlated with the degree of cartilage degeneration. Moreover, the OA-related catabolic factor interleukin-1ß induced the expression of Apex 2 in chondrocytes, while Apex 2 silencing using small interfering RNA reduced chondrocyte activity in vitro. The expression of Apex 2 in chondrocytes therefore appears to be associated with the degeneration of articular cartilage and could be induced by an OA-related catabolic factor to protect against the catabolic process of articular cartilage. Our findings suggest that Apex 2 may have the potential to prevent the catabolic stress-mediated down-regulation of chondrocyte activity in OA.

A novel anti-peroxiredoxin autoantibody in patients with Kawasaki disease.

Antibodies to the anti-oxidative peroxiredoxin (Prx) enzymes occur in both systemic autoimmune disease and vasculitis in adulthood. Because increased oxidative stress induces vasculitis in Kawasaki disease (KD), autoimmunity to Prxs in patients with KD was investigated. The presence of antibodies to Prx 1, 2 and 4 was analyzed by ELISA and Western blot. Of 30 patients with KD, 13 (43.3%) possessed antibodies to Prx 2, whereas these antibodies were present in only 1 of 10 patients (10.0%) with sepsis (4 with purulent meningitis and 6 with septicemia). In contrast, antibodies to Prx 1 and 4 were not detected in either group. There was no significant correlation among the titers of the three antibodies. Clinical parameters were compared between anti-Prx 2-positive and -negative patients. The presence of anti-Prx 2 antibodies correlated with a longer period of fever and poor response to high-dose γ-globulin therapy in patients with KD. Anti-Prx 2-positive patients had significantly greater excretion of urinary 8-isoprostaglandin than did anti-Prx 2-negative patients. These results provide the first evidence for an antibody to Prx 2 in patients with KD. They also suggest that this antibody might serve as a marker of disease severity and be involved in the pathophysiology of vasculitis in some patients with KD.

Anti-endothelial cell antibodies (AECA) in patients with systemic vasculitis: our research using proteomics.

IMPORTANCE OF THE FIELD: Anti-endothelial cell antibodies (AECA) may cause damage to endothelial cell (EC) functions and therefore may be of a pathophysiological role in systemic vasculitis. The pathophysiological role of AECA, however, is still uncertain. AREAS COVERED IN THIS REVIEW: To clarify the detailed roles of AECA, various methods for identification of target proteins of AECA have been developed, such as expression libraries and proteomic approaches combining two-dimensional electrophoresis and immunoblotting. WHAT THE READER WILL GAIN: Advances, including our research, have been made in defining the target antigens of AECA, which we summarize in this review. Furthermore, we discuss the possible significance of AECA in the pathophysiology of vascular damage and the value of AECA in systemic vasculitis. TAKE HOME MESSAGE: To identify target antigens of AECA and to establish a standardized method for measuring AECA would be helpful in the search for a possible pathophysiological role of AECA in systemic vasculitis.

Statin prevents chondrocyte aging and degeneration of articular cartilage in osteoarthritis (OA).

Recent reports have shown that statin (HMG-CoA reductase inhibitors) may have the potential to inhibit inflammatory arthritis. More recently, the idea that chondrocyte aging is closely associated with the progression of cartilage degeneration has been promulgated. Here, we demonstrate the potential of statin as protective agents against chondrocyte aging and degeneration of articular cartilage during the progression of osteoarthritis (OA), both in vitro and in vivo. The OA-related catabolic factor, IL-1ß induced marked downregulation of cellular activity, expression of a senescent biomarker, specific senescence-associated ß-galactosidase activity and shortening of the cellular lifespan in chondrocytes. In contrast, treatment with statin inhibited the IL-1ß-induced production of cartilage matrix degrading .enzymes (metalloprotease-1 and -13) and cellular senescence in of chondrocytes in vitro. In addition, this statin accelerated the production of cartilage matrix proteoglycan in chondrocytes. The in vivo study was performed on the STR/OrtCrlj mouse, an experimental model which spontaneously develops an osteoarthritic process. In this mouse model, treatment with statin significantly reduced the degeneration of articular cartilage, while the control knee joints showed progressive cartilage degeneration over time. These findings suggest that statin may have the potential to prevent the catabolic stress-induced chondrocyte disability and aging observed in articular cartilage. Our results indicate that statin are potential therapeutic agents for protection of articular cartilage against the progression of OA.

[Detection of specific markers: our research on the marker in patients with Kawasaki disease].

Anti-endothelial cell antibodies (AECA) were antibodies targeting the antigens expressed on the endothelial cell surface. It has been reported that AECA were detected frequently in patients with vasculitis and were associated with disease activity and vasculitis symptoms. Consequently, AECA are thought to be involved in pathophysiology of vasculitis, including Kawasaki disease (KD); however, the role of AECA is not clear yet. One of the causes is that target proteins of AECA have been poorly identified. Therefore, we try to detect new target proteins of AECA in patients with vasculitis using proteomics. We have identified 63 proteins out of about 150 endothelial cell-specific candidate target proteins of AECA in patients with vasculitis so far. One of the identified proteins was peroxiredoxin2 (Prx2), an antioxidant enzyme. Our research suggests that the anti-Prx2 antibodies are detected frequently in patients with vasculitis and may have pathogenic roles in vasculitis via inflammatory cytokines/chemokines production and inhibition of anti-oxidative activity of Prx2. In this paper, we overview our study of the autoantigens detected by AECA in patients with vasculitis, and will provide some data on clinical significance of autoantibodies to Prx2, a target protein of AECA, in patients with KD.

Water-soluble fullerene (C60) inhibits the development of arthritis in the rat model of arthritis.

Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the development of inflammation and osteoclastogenesis, suggesting the implication of oxygen free radicals in the pathogenesis of arthritis. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against synovitis in arthritis, both in vitro and in vivo. In the presence or absence of C60 (0.1, 1.0, 10.0 muM), human synovial fibroblasts, synovial infiltrating lymphocytes or macrophages were incubated with tumor necrosis factor-alpha (TNF-alpha) (10.0 ng/mL), and the production of proinflammatory cytokines by the individual cells were analyzed. C60 significantly suppressed the TNF-alpha-induced production of proinflammatory cytokines in synovial fibroblasts, synovial infiltrating lymphocytes and macrophages in vitro. Adjuvant induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 10.0 muM. The left ankle joint was injected intraarticularly with water-soluble C60 (20 mul) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 mul), once weekly for eight weeks. Ankle joint tissues were prepared for histological analysis. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced synovitis and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of synovitis and joint destruction with time. These findings indicate that C60 is a potential therapeutic agent for inhibition of arthritis.

Water-soluble fullerene (C60) inhibits the osteoclast differentiation and bone destruction in arthritis.

Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the receptor activator NFkappaB (RANK) signal pathway required for osteoclast differentiation. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against the RANK-induced osteoclastogenesis and osteoclastic bone destruction in arthritis, both in vitro and in vivo. The effects of C60 on the RANK-induced osteoclastogenesis and osteoclastic bone resorption were examined in vitro. Adjuvant-induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 1.0 microM. The left ankle joint was injected intra-articularly with water-soluble C60 (20 microl) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 microl) once weekly for eight weeks. Ankle joint tissues were prepared for histologic analysis. C60 significantly inhibited the responses of osteoclast precursor cells to RANK ligand, including osteoclast differentiation and osteoclastic bone resorption in vitro. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced the number of osteoclasts and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of joint destruction with time. These findings indicate that C60 downregulates the RANK-induced osteoclast differentiation and is a potential therapeutic agent for inhibition of osteoclastic bone destruction in arthritis.

Angiogenic growth factors inhibit chondrocyte ageing in osteoarthritis: potential involvement of catabolic stress-induced overexpression of caveolin-1 in cellular ageing.

OBJECTIVE: Recently, attention has been attracted by the finding that overexpression of caveolin-1 induces cellular senescence in age-related diseases. We aimed to ascertain whether angiogenic growth factors (AGFs) can inhibit interleukin (IL)-1beta-induced senescence in human chondrocytes by downregulation of caveolin-1. METHODS: We investigated the intracellular signalling pathways involved in chondrocyte ageing. Human chondrocytes were isolated from the articular cartilage of patients undergoing arthroplastic knee surgery in osteoarthritis (OA). Chondrocytes were stimulated with or without IL-1beta (10 ng/mL) in the presence or absence of vascular endothelial growth factor, basic fibroblast growth factor or hepatocyte growth factor (20 ng/mL). After 72-h incubation, we observed the expression of caveolin-1 in human chondrocytes by immunohistochemistry, and analysed the protein levels of caveolin-1 by Western blot. We examined the time-course of phosphorylation patterns of mammalian mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K) by Western blot, and used several specific protein kinase inhibitors to evaluate the involvement of the intracellular signalling pathways. Also, chondrocyte replicative lifespan was analyzed in the presence or absence of AGFs. RESULTS: Treatment with AGFs inhibited IL-1beta-induced overexpression of caveolin-1 in human OA chondrocytes. Treatment with AGFs all down-regulated protein levels of IL-1beta-accelerated expression of caveolin-1 in chondrocytes. IL-1beta significantly decreased the cellular replicative lifespan in chondrocytes. Treatment with AGFs prevented the IL-1beta-induced shortening of chondrocyte replicative lifespan. The specific inhibitors for MAPK/extracellular signal-regulated kinase and PI3-K cancelled the AGF-induced downregulation of overexpression of caveolin-1. CONCLUSION: Our results suggest that AGFs downregulated IL-1beta-induced chondrocyte ageing and overexpression of caveolin-1 in human chondrocytes, which is mediated by kinase cascades involving the p42/44 MAP kinase and PI3-K/Akt signalling pathways.

Autoantibodies to peroxiredoxin I and IV in patients with systemic autoimmune diseases.

Anti-oxidative enzymes protect living bodies from various oxidative stresses. In the systemic autoimmune diseases, autoantibodies to oxidized molecules and to anti-oxidative enzymes have been reported. To promote understanding of the relationships between autoimmunity and oxidative stress, we here investigate whether autoimmunity to the anti-oxidative peroxiredoxin (Prxs) enzymes exists in patients with systemic autoimmune diseases. Specifically, we detected autoantibodies to recombinant Prx I and Prx IV respectively by ELISA and western blotting. Next, clinical parameters were compared between the anti-Prx I or IV-positive and-negative patients. We found that 33% of the 92 patients with autoimmune diseases tested possessed autoantibodies to Prx I (57% in systemic lupus erythematosus (SLE), 19% in rheumatoid arthritis (RA), 5% in Behçet disease, and 46% in primary vasculitis syndrome). In contrast, autoantibodies to Prx IV were detected in only 17% of the same patients. No significant correlation was found between occurrence of the two autoantibodies. Clinically, possession of anti-Prx I autoantibodies correlated with lower serum levels of CH50, C3, and C4. Taken together, our data demonstrate the existence of autoantibodies to Prxs for the first time. The autoantibodies to Prx I may be involved in the pathophysiology of systemic autoimmune diseases such as SLE and vasculitis.

Cushing's syndrome due to a large adrenocortical adenoma with histological features simulating ACTH-independent macronodular adrenocortical hyperplasia.

A 53-year-old woman presented with Cushing's syndrome resulting from an adrenocortical adenoma, 6.5 cm in diameter and 75 g in weight, which is larger than usual. Endocrinological data of this patient showed adrenocorticotropin (ACTH)-independent hypercortisolemia. A computed tomography scan of the adrenal glands revealed a single large and well-encapsulated tumor with an irregularly shaped area of calcification and loss of parenchyma on the left adrenal. The right adrenal gland was atrophic. Laparoscopic removal of the left adrenal tumor was performed. The tumor was lobulated and clearly encapsulated, and the non-neoplastic area of the left adrenal was atrophic without any nodularity. The histological analysis confirmed the diagnosis of adrenal adenoma. In addition, this adenoma displayed histopathological features in common with ACTH-independent macronodular adrenocortical hyperplasia (AIMAH), including clear cell predominance, a pattern of small compact cell nests in clear cell areas, and very long cord-like arrangement of small compact cells. In AIMAH, adrenals are extremely enlarged and are more massive than in any other subtype of Cushing's syndrome. The fact that the present adrenocortical adenoma was larger than those typical adenomas of Cushing's syndrome may reflect an AIMAH-type cellular composition of clear cell predominance and small compact cell nests.