Prediction of organ involvement in systemic sclerosis by serum biomarkers and peripheral endothelial function.

OBJECTIVES: To identify prognostic factors among serum biomarkers and endothelial vasodilator function findings in patients with systemic sclerosis (SSc). METHODS: This is a clinical observational study. We assessed 60 consecutive SSc patients (44 limited cutaneous-type, 16 diffuse cutaneous-type). Circulating growth differentiation factor-15 (GDF-15), placenta growth factor (PlGF), endostatin, vascular endothelial growth factor (VEGF), and pentraxin 3 (PTX3) were measured by ELISA. Peripheral endothelial function was measured by forearm blood dilatation response to brachial artery occlusion using noninvasive plethysmography (EndoPAT2000), which is associated with nitric-oxide-dependent vasodilatation and yields a reactive hyperemia index (RHI). We evaluated whether abnormalities in these values were associated with type of SSc - namely, diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc) - or organ involvement including interstitial lung disease (ILD), digital ulcer (DU) and estimated right ventricular systolic pressure (RVSP) by echocardiography >30 mmHg. RESULTS: SSc patients showed significantly elevated serum GDF-15, PlGF, endostatin and VEGF but not PTX3 compared with controls. GDF-15 and PlGF were high in dcSSc patients. EndoPAT-RHI was low, and incidence of RVSP >30 mmHg was high in dcSSc. Multivariate analysis revealed that elevated GDF-15 was highly predictive of dcSSc, ILD or RVSP >30 mmHg. PlGF for DU was also found. Conversely, a low EndoPAT-RHI value was predictive of the presence of dcSSc, ILD or DU. CONCLUSIONS: This is the first study to inclusively investigate the relationships among biomarkers, EndoPAT-RHI and organ involvement in patients with SSc. Our data suggest a complex pathological progression of SSc through fibrotic impairment and microvascular damage.

The wound/burn guidelines - 4: Guidelines for the management of skin ulcers associated with connective tissue disease/vasculitis.

The Japanese Dermatological Association prepared guidelines focused on the treatment of skin ulcers associated with connective tissue disease/vasculitis practical in clinical settings of dermatological care. Skin ulcers associated with connective tissue diseases or vasculitis occur on the background of a wide variety of diseases including, typically, systemic sclerosis but also systemic lupus erythematosus (SLE), dermatomyositis, rheumatoid arthritis (RA), various vasculitides and antiphospholipid antibody syndrome (APS). Therefore, in preparing the present guidelines, we considered diagnostic/therapeutic approaches appropriate for each of these disorders to be necessary and developed algorithms and clinical questions for systemic sclerosis, SLE, dermatomyositis, RA, vasculitis and APS.

The wound/burn guidelines - 5: Guidelines for the management of lower leg ulcers/varicose veins.

Varicose veins are treated at multiple clinical departments, but as patients often visit the dermatology clinic first due to leg ulcers, the present Guidelines for the Management of Lower Leg Ulcers/Varicose Veins were prepared in consideration of the importance of the dermatologist's role. Also, the disease concept of chronic venous insufficiency or chronic venous disorders and the CEAP classification of these disorders are presented. The objective of the present guidelines is to properly guide the diagnosis and treatment of lower leg ulcers/varicose veins by systematically presenting evidence-based recommendations that support clinical decisions.

The wound/burn guidelines - 6: Guidelines for the management of burns.

Burns are a common type of skin injury encountered at all levels of medical facilities from private clinics to core hospitals. Minor burns heal by topical treatment alone, but moderate to severe burns require systemic management, and skin grafting is often necessary also for topical treatment. Inappropriate initial treatment or delay of initial treatment may exert adverse effects on the subsequent treatment and course. Therefore, accurate evaluation of the severity and initiation of appropriate treatment are necessary. The Guidelines for the Management of Burn Injuries were issued in March 2009 from the Japanese Society for Burn Injuries as guidelines concerning burns, but they were focused on the treatment for extensive and severe burns in the acute period. Therefore, we prepared guidelines intended to support the appropriate diagnosis and initial treatment for patients with burns that are commonly encountered including minor as well as moderate and severe cases. Because of this intention of the present guidelines, there is no recommendation of individual surgical procedures.

Yusho patients show increased serum IL-17, IL-23, IL-1ß, and TNFα levels more than 40 years after accidental polychlorinated biphenyl poisoning.

The Yusho poisoning incident, caused by rice oil contaminated with polychlorinated biphenyls (PCBs), polychlorinated quarterphenyls (PCQs), and polychlorinated dibenzofurans (PCDFs) generated by heat-denatured PCBs, occurred in 1968 in western Japan. Although severe symptoms are rarely observed today, the levels of PCBs and PCDFs in the sera of Yusho patients remain high. The aryl hydrocarbon receptor (AhR), which also acts as a dioxin receptor, is a transcriptional regulator that mediates dioxin toxicity. Recent studies show that dioxin mediates its immune toxic effects via AhR and that AhR activation induces dysregulation of interleukin (IL)-17- producing T (TH17) cells. This study therefore hypothesized that Yusho patients would show dysregulated TH17 cell-mediated immune responses. To validate the hypothesis, levels of IL-17 and IL-22, each secreted by TH17 cells, along with IL-1ß and IL-23 were measured in serum samples from 40 Yusho patients and 40 age-matched controls. Levels of tumor necrosis factor (TNF)-α potentially secreted by TH17 cell-stimulated neutrophils and macrophages were also measured. The results indicated that serum IL-17 levels, as well as those of IL-1ß, IL-23, and TNFα, were significantly higher in Yusho patients than in controls. In contrast, serum IL-22 levels were significantly lower in the Yusho patients. These results suggest that Yusho patients have dysregulated TH17 cell-mediated immune responses that may be linked to inflammation.

Exogenous application of hydrogen sulfide donor attenuates inflammatory reactions through the L-selectin-involved pathway in the cutaneous reverse passive Arthus reaction.

H2S has been highlighted recently as an endogenous, gaseous signaling molecule, especially in inflammations. The deposition of IC induces an acute inflammatory response with tissue injury. To assess the roles of H2S in the IC-induced diseases, the cutaneous, reverse passive Arthus reaction was conducted using NaHS as a H2S donor. Furthermore, we conducted similar experiments using selectin(-/-) mice to determine the involvement of selectin molecules in the H2S-mediated pathway. Exogenous application of NaHS dramatically attenuated inflammatory reactions in WT mice associated with Arthus reaction. Namely, mRNA expressions of TNF-α, IFN-γ, and neutrophil numbers were reduced significantly in the lesional skins of NaHS-treated WT mice relative to untreated ones. NaHS treatment significantly reduced these three parameters in the lesional skins of E- and P-selectin(-/-) mice but not in those of L-selectin(-/-) mice. Quite similar results were obtained in the blocking study using WT mice injected with mAb to E-, P-, and L-selectin. Our results indicated that the exogenous application of NaHS attenuates inflammatory responses in reverse passive Arthus reaction through a L-selectin-involved pathway but not through E- or P-selectin pathways.

Attenuation of an adult T-cell leukemia skin lesion after treatment of a concomitant herpes simplex infection: a case study.

We report the development and treatment of eczema herpeticum in a 51-year-old male suffering from adult T-cell leukemia (ATL). Lesions of eczema herpeticum coexisted with the skin lesions of ATL. Treatment of eczema herpeticum resulted in a concomitant improvement in the symptoms of ATL, including a reduction in the size of the ATL plaques, for over 2 months before relapse.

Increased serum levels of soluble CD163 in patients with scleroderma.

CD163 is a 130-kDa, type I transmembrane protein belonging to group B of the cysteine-rich scavenger receptor family. Expression of CD163 is constitutive and/or induced by some stimuli on circulating monocytes and most tissue macrophages. An approximately 130-kDa soluble form of human CD163 is released from the cell surface by proteolysis after oxidative stress or inflammatory stimuli. Thus, an elevated level of circulating soluble CD163 (sCD163) has been reported in diabetes mellitus, which is one of the oxidative conditions. We have already acknowledged that scleroderma (SSc) is one of the oxidative conditions. Therefore, we conducted the measurement of serum sCD163 in SSc patients. After receiving the informed consents, 56 SSc patients were examined; 20 dermatomyositis patients were used as disease controls and 40 persons were used as healthy controls. Blood samples were collected, and the concentration of serum sCD163 was measured by ELISA (human CD163, R&D Systems). Other parameters in the blood of SSc patients were also examined. Statistical analyses were performed using Mann-Whitney U test, and the relationship between parameters was statistically examined by Spearman's rank test. Serum sCD163 levels were elevated in SSc patients compared with normal controls (p < 0.01), with similar levels between limited SSc and diffuse SSc patients. SSc patients with pulmonary fibrosis had increased serum levels of sCD163 than those without pulmonary fibrosis (p < 0.05). SSc patients with elevated sCD163 levels had increased serum levels of IgG than those with normal sCD163 levels (p < 0.05). Serum sCD163 levels correlated positively with pulsatility index in SSc patients (p = 0.0009, r = 0.534). These results suggest that oxidative stress may play an important role in immunological abnormalities, renal circulation, and pulmonary fibrosis of SSc.

Myositis-specific anti-155/140 autoantibodies target transcription intermediary factor 1 family proteins.

OBJECTIVE: To identify the 140-kd autoantigen recognized by anti-155/140 autoantibodies that are associated with adult cancer-associated dermatomyositis (DM) and juvenile DM and to determine the clinical relevance of anti-155/140 antibodies in a large cohort. METHODS: Sera from 456 DM patients were assessed for the presence of anti-155/140 antibodies by immunoprecipitation using K562 cell extracts as substrate. Using immunoprecipitation and Western blotting, we then examined whether anti-155/140-positive sera recognized transcription intermediary factor 1α (TIF-1α), TIF-1ß, and TIF-1γ. The clinical associations of antigen reactivity were also evaluated. RESULTS: Anti-155/140-positive sera reacted with 140-kd TIF-1α in addition to 155-kd TIF-1γ. Among sera from 456 DM patients, 52 were reactive with both TIF-1α and TIF-1γ, while another 25 were reactive with TIF-1γ alone. Additionally, 7 were reactive with TIF-1ß. Malignancy was more frequently found in adult patients with both anti-TIF-1α and anti-TIF-1γ antibodies than in those with anti-TIF-1γ antibodies alone (73% versus 50%; P < 0.05). In addition to juvenile DM patients and middle-aged and older DM patients with high percentages of malignancy, 8 "young adult" DM patients without malignancy had these autoantibodies. CONCLUSION: Anti-155/140 antibodies target TIF-1 family proteins, TIF-1α and TIF-1ß, in addition to TIF-1γ. Since TIF-1 proteins have significant roles in oncogenesis, these antibodies may be produced during misdirected antitumor immunity.

The roles of P- and E-selectins and P-selectin glycoprotein ligand-1 in primary and metastatic mouse melanomas.

BACKGROUND: Malignant melanoma is often accompanied by a host response of inflammatory cell infiltration that is highly regulated by multiple adhesion molecules. OBJECTIVE: To evaluate the role of adhesion molecules, including P-selectin glycoprotein ligand-1 (PSGL-1), P-selectin, and E-selectin. METHODS: Subcutaneous primary growth and metastasis to the lung of B16 melanoma cells were examined in mice lacking PSGL-1, P-selectin, or E-selectin. RESULTS: Primary subcutaneous growth of B16 melanoma was augmented by loss of PSGL-1, P-selectin, or E-selectin, while pulmonary metastasis was reduced by the loss of E-selectin. The enhancement of subcutaneous tumor growth was associated with a reduced accumulation of natural killer cells, CD4(+) T cells and CD8(+) T cells, while the attenuation of pulmonary metastasis was related to the numbers of CD8(+) T cells. The expressions of transforming growth factor (TGF)-ß and interleukin (IL)-6 were correlated with primary subcutaneous growth; TGF-ß, IL-6, and interferon-γ were related to number of metastatic lung nodules. Cytotoxicity against melanoma cells in splenocytes and in tumor-draining lymph node cells were not defective by the absence of adhesion molecules, suggesting that the enhancement of tumor growth and metastasis caused by the loss of selectins results from an impaired migration of effector cells into the tissue. CONCLUSIONS: The results indicate the complexity of anti-tumor responses mediated by adhesion molecules in primary subcutaneous tumors and pulmonary metastasis of murine experimental melanoma.

Autoantibody against survivin in patients with systemic sclerosis.

OBJECTIVE: In patients with systemic sclerosis (SSc), to determine concentrations of antibodies against survivin and their clinical association with SSc, and to evaluate serum survivin concentrations. METHODS: Anti-survivin antibody was examined by ELISA and immunoblotting using human recombinant survivin. Serum survivin levels were assessed by ELISA. RESULTS: IgG but not IgM anti-survivin antibody levels in patients with SSc were significantly higher than those in healthy controls and patients with systemic lupus erythematosus (SLE). When cutoff values were set as mean + 2 SD of control, IgG anti-survivin antibodies were positive in 41% (25/61) of patients with SSc, while they were detected in only 1 healthy individual (3%, 1/29) and 1 patient with SLE (5%, 1/20). Regarding the clinical correlation, patients with SSc who were positive for IgG anti-survivin antibody exhibited significantly longer disease duration than those who were negative. Immunoblotting analysis confirmed the presence of anti-survivin antibody in sera from patients with SSc. Serum survivin levels in patients with SSc were also significantly higher than in controls and patients with SLE. CONCLUSION: Our results suggest that autoantibody against survivin is generated in patients with SSc, especially those with long disease duration.

The proteasome inhibitor bortezomib inhibits T cell-dependent inflammatory responses.

CHS is a cutaneous, T cell-dependent, inflammatory reaction mediated mainly by antigen-specific effector T cells. Bortezomib is a proteasome inhibitor that has shown impressive efficacy for the treatment of multiple myeloma. In the current study, we have assessed the effect of bortezomib treatment of CHS in mice and found that bortezomib potently inhibited CHS responses. The attenuation of CHS responses was associated with decreased inflammatory cell infiltration in the challenged skin. Specifically, bortezomib-treated mice showed significantly decreased numbers of CD4(+) and CD8(+) T cells in the challenged skin and draining lymph nodes. Cytoplasmic IFN-gamma production by CD4(+) and CD8(+) T cells in the draining lymph nodes was decreased substantially by bortezomib treatment. Notably, bortezomib enhanced T cell apoptosis by inhibiting NF-kappaB activation during CHS responses. Thus, bortezomib treatment is likely to induce T cell death, thereby suppressing CHS responses by reducing IFN-gamma production. These findings suggest that bortezomib treatment could be a promising strategy for treating autoimmune and inflammatory disease.

Case of localized scleroderma associated with osteomyelitis.

We report a 4-year-old girl presenting with progressive linear scleroderma affecting the right leg. Biopsy specimen disclosed typical histopathological findings of localized scleroderma. Right leg magnetic resonance imaging (MRI) showed high signal areas on T(2)-weighted images on the subcutaneous fatty tissue, muscles and bone marrow, suggesting that skin inflammation extended to the bone marrow. Oral corticosteroid therapy was instituted with improvement of both skin sclerosis and MRI findings. Our observations suggest that MRI examination should be considered in patients with localized scleroderma to evaluate the extension of the inflammation.

Involvement of L-selectin in contact hypersensitivity responses augmented by auditory stress.

Stress affects the pathophysiology of cutaneous immune reactions, including contact hypersensitivity (CH) in individuals sensitized with sensitizing hapten, where local endothelial cell activation plays a critical role. To clarify the effects of stress in cutaneous immune reactions, we selected a CH model using annoying sound as a stress. Furthermore, we conducted the stress experiments by using selectin-deficient mice to determine the involvement of selectin molecules regarding local endothelial activation. Auditory stress augmented CH responses in the present study. Namely, ear thickness and mast cell numbers were significantly increased in stressed CH mice. mRNA expression of preprotachykinin-A, a precursor of substance-P; interferon-gamma; interleukin (IL)-4; IL-6; and tumor necrosis factor-alpha significantly increased in stressed CH mice. Furthermore, stressed L-selectin-deficient mice showed significant decreases in all parameters mentioned above relative to stressed wild-type mice in CH response. Meanwhile, treatment with anti-L-selectin Ab resulted in a significant decrease in ear thickness and mRNA levels of interferon-gamma, IL-4, IL-6, and tumor necrosis factor-alpha, but failed to significantly reduce preprotachykinin-A mRNA levels and mast cell numbers. Our results indicated that auditory stress enhances CH response and that the augmentation of this CH response might be mediated through L-selectin, but not through P- or E-selectin pathways.

Platelets control leukocyte recruitment in a murine model of cutaneous arthus reaction.

Platelets have been shown to be important in inflammation, but their role in the cutaneous Arthus reaction remains unclear. To assess the role of platelets in this pathogenetic process, the cutaneous Arthus reaction was examined in wild-type mice and mice lacking E-selectin, P-selectin, or P-selectin glycoprotein ligand-1 (PSGL-1) with or without platelet depletion by busulfan, a bone marrow precursor cell-specific toxin. Edema and hemorrhage induced by immune complex challenge significantly decreased in busulfan-treated wild-type mice compared with untreated mice. Busulfan treatment did not affect edema and hemorrhage in P-selectin- or PSGL-1-deficient mice, suggesting that the effect by busulfan is dependent on P-selectin and PSGL-1 expression. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells and reduced levels of circulating platelets. Increased cutaneous production of interleukin-6, tumor necrosis factor-alpha, and platelet-derived chemokines during Arthus reaction was inhibited in busulfan-treated wild-type mice relative to untreated mice, which paralleled the reduction in cutaneous inflammation. Flow cytometric analysis showed that immune complex challenge generated blood platelet-leukocyte aggregates that decreased by busulfan treatment. In thrombocytopenic mice, the cutaneous inflammation after immune complex challenge was restored by platelet infusion. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates and secreting chemokines at inflamed sites, mainly through the interaction of P-selectin on platelets with PSGL-1 on leukocytes.

Autoantibody against one of the antioxidant repair enzymes, methionine sulfoxide reductase A, in systemic sclerosis: association with pulmonary fibrosis and vascular damage.

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis and vascular changes in the skin and internal organs with autoimmune background. It has been suggested that oxidative stress plays an important role in the development of SSc. To determine the prevalence and clinical correlation of autoantibody to methionine sulfoxide reductase A (MSRA), one of the antioxidant repair enzymes, in SSc, serum anti-MSRA autoantibody levels were examined in patients with SSc by enzyme-linked immunosorbent assay using recombinant MSRA. The presence of anti-MSRA antibody was evaluated by immunoblotting. To determine the functional relevance of anti-MSRA antibody in vivo, we assessed whether anti-MSRA antibody was able to inhibit MSRA enzymatic activity. Serum anti-MSRA antibody levels in SSc patients were significantly higher compared to controls and this autoantibody was detected in 33% of SSc patients. Serum anti-MSRA levels were significantly elevated in SSc patients with pulmonary fibrosis, cardiac involvement, or decreased total antioxidant power compared with those without them. Anti-MSRA antibodies also correlated positively with renal vascular damage determined as pulsatility index by color-flow Doppler ultrasonography of the renal interlobar arteries and negatively with pulmonary function tests. Furthermore, anti-MSRA antibody levels correlated positively with serum levels of 8-isoprostane and heat shock protein 70 that are markers of oxidative and cellular stresses. Remarkably, MSRA activity was inhibited by IgG isolated from SSc sera containing IgG anti-MSRA antibody. These results suggest that elevated anti-MSRA autoantibody is associated with the disease severity of SSc and may enhance the oxidative stress by inhibiting MSRA enzymatic activity.

Elevated circulating TWEAK levels in systemic sclerosis: association with lower frequency of pulmonary fibrosis.

OBJECTIVE: To determine serum levels of tumor necrosis factor-related weak inducer of apoptosis (TWEAK) and its clinical associations in patients with systemic sclerosis (SSc). METHODS: Serum TWEAK levels from 70 patients with SSc were examined by ELISA. In a retrospective longitudinal study, sera from 23 patients with SSc were analyzed (followup 0.8-7.2 yrs). RESULTS: Serum TWEAK levels were elevated in patients with SSc (n = 70) compared with healthy controls (n = 31) and patients with systemic lupus erythematosus (n = 22). Among patients with SSc, there were no differences in serum TWEAK levels between limited cutaneous SSc and diffuse cutaneous SSc. Patients with SSc who had elevated TWEAK levels less often had pulmonary fibrosis and decreased vital capacity than those with normal TWEAK levels. In the longitudinal study, SSc patients with inactive pulmonary fibrosis or without pulmonary fibrosis consistently exhibited increased TWEAK levels, while those with active pulmonary fibrosis showed decreased TWEAK levels during the followup period. CONCLUSION: TWEAK levels were increased in patients with SSc, and associated with a lower frequency of pulmonary fibrosis in patients with SSc. TWEAK could be a protective factor against the development of pulmonary fibrosis in this disease and as such would be a possible therapeutic target.

Establishment of experimental eosinophilic vasculitis by IgE-mediated cutaneous reverse passive arthus reaction.

Prominent eosinophil infiltration is a characteristic of some forms of vasculitis, such as Churg-Strauss syndrome, also known as allergic granulomatous vasculitis. In the current study, we established a mouse model of cutaneous eosinophilic vasculitis by the cutaneous reverse passive Arthus reaction using IgE injection instead of IgG. Wild-type C57BL/6 mice were injected with IgE anti-trinitrophenyl antibodies, followed immediately by intravenous administration of trinitrophenyl bovine serum albumin. IgE-mediated immune complex challenge induced substantial hemorrhage with marked infiltration of eosinophils in which neutrophils, mast cells, and macrophages were also mixed. This finding contrasted remarkably with the neutrophil-dominant infiltration pattern in IgG-mediated immune complex challenge. In the lesion, the expression level of monocyte chemotactic protein-3 was increased, and anti-monocyte chemotactic protein-3 treatment resulted in a significant but incomplete blockade of eosinophil recruitment. Furthermore, mice lacking E-selectin, P-selectin, L-selectin, or intercellular adhesion molecule-1, as well as wild-type mice that received anti-vascular cell adhesion molecule-1-blocking antibodies were assessed for the IgE-mediated Arthus reaction. After 24 hours, the loss of P-selectin resulted in a significant reduction in eosinophil accumulation compared with both wild-type mice and other mouse mutants. Collectively, the Fc class of immunoglobulins, which forms these immune complexes, critically determines the disease manifestation of vasculitis. The IgE-mediated cutaneous reverse passive Arthus reaction may serve as an experimental model for cutaneous eosinophilic infiltration in vasculitis as well as in other diseases.

P-selectin glycoprotein ligand-1 contributes to wound healing predominantly as a p-selectin ligand and partly as an e-selectin ligand.

Cell adhesion molecules are critical to wound healing through leukocyte recruitment. Although P-selectin glycoprotein ligand-1 (PSGL-1) regulates leukocyte rolling by binding P-selectin, but also binding E- and L-selectins with lower affinity, little is known about a role of PSGL-1 in wound healing. To clarify a role of PSGL-1 and its interaction with E- and P-selectins in wound healing, we investigated cutaneous wound healing in PSGL-1-deficient (PSGL-1(-/-)) mice in comparison with E-selectin(-/-), P-selectin(-/-), and P-selectin(-/-) mice treated with an anti-E-selectin antibody. PSGL-1 deficiency inhibited early wound healing, which was accompanied by decreased inflammatory cell infiltration and growth factor expression. By contrast, E-selectin deficiency did not affect wound healing. In general, the inhibitory effect of PSGL-1 deficiency on wound healing was similar to that of P-selectin deficiency either alone or with E-selectin blockade. However, early granulation tissue formation, late angiogenesis, and early infiltration of neutrophils and macrophages in PSGL-1(-/-) mice were inhibited beyond the inhibition in P-selectin(-/-) mice, but to a similar level of inhibition in P-selectin(-/-) mice with E-selectin blockade. These results suggest that PSGL-1 contributes to wound healing predominantly as a P-selectin ligand and partly as an E-selectin ligand by mediating infiltration of inflammatory cells.