CCL13 is a promising diagnostic marker for systemic sclerosis.

BACKGROUND: Previous studies suggest that CCL13 may have some role in the pathogenesis of systemic sclerosis (SSc). OBJECTIVES: To determine serum levels of CCL13 and its clinical associations in patients with SSc. METHODS: Serum CCL13 levels were examined by enzyme-linked immunosorbent assay in 80 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), 20 patients with dermatomyositis (DM), 29 patients with atopic dermatitis (AD) and 50 healthy individuals. RESULTS: Mean + or - SD serum CCL13 levels were elevated in patients with SSc (81.3 + or - 55.8 pg mL(-1)) compared with healthy controls (15.0 + or - 9.9 pg mL(-1); P < 0.001) and patients with SLE (22.0 + or - 6.9 pg mL(-1); P < 0.001), DM (24.4 + or - 36.1 pg mL(-1); P < 0.001) and AD (18.0 + or - 6.4 pg mL(-1); P < 0.001). Among patients with SSc, there were no differences in serum CCL13 levels between limited cutaneous SSc and diffuse cutaneous SSc. In a longitudinal study, CCL13 levels were generally unchanged during the follow-up. CONCLUSIONS: Serum CCL13 was specifically increased in patients with SSc, but not in patients with SLE, DM or AD or in healthy controls. CCL13 could be a promising serological marker for SSc.

Autoantibody against activating transcription factor-2 in patients with systemic sclerosis.

OBJECTIVES: To determine the prevalence and clinical correlation of autoantibody to activating transcription factor (ATF)-2, a transcription factor of ATF/CREB family, in patients with systemic sclerosis (SSc). METHODS: Anti-ATF-2 Ab was examined by ELISA and immunoblotting using human recombinant ATF-2. ATF-2 activity to bind target DNA was evaluated by ELISA using a plate coated with oligonucleotide containing the consensus binding site for ATF-2. RESULTS: IgG anti-ATF-2 Ab levels in SSc patients (n=69) were significantly higher than those in normal controls (n=26). SSc patients positive for IgG anti-ATF-2 Ab had significantly longer disease duration, more frequent presence of decreased %VC and %DLco, and elevated levels of serum IgG, serum IgA, and erythrocyte sedimentation rates than those negative. More-over, IgG anti-ATF-2 Ab levels correlated inversely with %VC or %DLco. The presence of anti-ATF-2 Ab in SSc patients was confirmed by immunoblotting analysis. IgG isolated from serum samples of SSc patients positive for IgG anti-ATF-2 Ab by ELISA slightly but significantly inhibited ATF-2 activity compared with normal controls. CONCLUSIONS: These results suggest that anti-ATF-2 Ab is a new autoantibody in SSc and that it serves as a novel serological marker for inflammation and lung involvement in SSc.

Endoscopic sinus surgery for the odontogenic maxillary cysts.

OBJECTIVE: To evaluate the effectiveness and usefulness of transnasal endoscopic surgery for the treatment of odontogenic maxillary cysts. METHODS: Between February 2003 and February 2008, transnasal endoscopic surgery was performed under general anesthesia in 13 patients (male 6 and female 7, 19 to 75 years old) with odontogenic maxillary cysts that extended to the maxillary sinus. Ten patients had a radicular cyst and three patients had a dentigerous cyst. After the resection of anterior edge of the inferior turbinate, the lateral wall of the inferior nasal meatus was opened. Then, the cyst wall of the maxillary sinus was partially or completely removed under the endoscope. RESULTS: The cyst walls were completely removed in five often patients with a radicular cyst and in all three patients with a dentigerous cyst. Five patients with a radicular cyst received partial resection of the cyst wall. The affected teeth could be preserved in seven of ten patients with a radicular cyst and in one of three patients with a dentigerous cyst. There were no complications, and postoperative courses were uneventful. Follow-up period ranged from 11 to 72 months (mean 42 months), and no recurrence has been noted in any of the cases. CONCLUSIONS: Endoscopic transnasal surgery for the odontogenic maxillary cyst is less invasive than conventional dental approach, and most of the affected teeth can be preserved. This technique appears to be a simple and highly effective surgical treatment for the treatment of patients with odontogenic cysts that extend to the maxillary sinus.

Serum levels of heat shock protein 70, a biomarker of cellular stress, are elevated in patients with systemic sclerosis: association with fibrosis and vascular damage.

OBJECTIVE: To determine the clinical significance of heat shock protein (Hsp) 70, a sensitive biomarker for monitoring cellular stress, in systemic sclerosis (SSc), we investigated the prevalence and clinical correlation of serum Hsp70 levels in SSc patients. METHODS: Serum Hsp70 levels were examined in 48 patients with SSc by enzyme-linked immunosorbent assay. RESULT: Serum Hsp70 levels were significantly elevated in SSc patients compared to normal controls (n=30), and were similar between patients with diffuse cutaneous SSc (n=26) and those with limited cutaneous SSc (n=22). Serum Hsp70 levels were elevated in 27% of total SSc patients with 30% of diffuse cutaneous SSc patients and 23% of limited cutaneous SSc patients. Hsp70 levels were significantly increased in SSc patients with pulmonary fibrosis or contracture of phalanges compared with those without pulmonary fibrosis or contracture of phalanges. Serum Hsp70 levels correlated positively with modified Rodnan total skin thickness score, renal vascular resistance, serum levels of monocyte chemotactic protein-1, C-reacting protein, and serum levels of 8-isoprostane. CONCLUSION: Serum Hsp70 levels were increased in SSc patients and were associated with pulmonary fibrosis, skin sclerosis, renal vascular damage, oxidative stress, and inflammation. These results suggest that Hsp70 is a useful serological marker for evaluating cellular stresses and the disease severity in SSc.

Involvement of gaseous low molecular monoxides in the cutaneous reverse passive Arthus reaction: cytoprotective action of carbon monoxide.

The deposition of immune complexes (IC) induces an acute inflammatory response with tissue injury, for which the involvement of nitric oxide (NO) and carbon monoxide (CO) has been suggested. NO is induced by NO synthase (NOS) and CO is generated by haeme oxygenase (HO). Among HO isoenzymes, HO-1 is an induced type. To assess the role of NO and CO in the pathogenic process, the cutaneous reverse passive Arthus reaction was examined using NOS inhibitor, HO-1 stimulator and HO-1 inhibitor. To evaluate the reaction we considered oedema, tumour necrosis factor-alpha, interleukin-6, and neutrophil number. The values of these four parameters were significantly reduced in mice treated with HO-1 stimulator as compared with the positive control mice. Quite the reverse was observed in mice treated with HO-1 inhibitor. These results suggest that the HO-1/CO signalling pathway is a therapeutic target for human IC-mediated disease.

Elevation of IgG levels is a serological indicator for pulmonary fibrosis in systemic sclerosis with anti-topoisomerase I antibodies and those with anticentromere antibody.

It is unclear whether any clinical and laboratory features are associated with pulmonary fibrosis (PF) in systemic sclerosis (SSc). We assessed these features using a database of 29 patients with SSc and anti-topoisomerase I antibodies and 68 patients with SSc and anticentromere antibody (ACA). Clinical features were not associated with the incidence of PF in patients with SSc and anti-topoisomerase I antibodies, although severe skin sclerosis was correlated with the presence of PF in patients with ACA. Serum IgG levels were often raised in patients with SSc and PF. Serum IgG levels in patients with PF were significantly higher than those in patients without PF, and were negatively correlated with percentage vital capacity and percentage diffusing capacity of the lung for carbon monoxide. In addition, serum IgG levels were correlated with serum interleukin-6. Thus, serum IgG levels are associated with PF in patients with SSc and anti-topoisomerase I antibodies and in patients with SSc and ACA.

The clinical relevance of serum antinuclear antibodies in Japanese patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disorder with excessive fibrosis of the skin and various internal organs. Although SSc is a heterogeneous disease, it has been reported that the particular antinuclear antibodies (ANA) are often indicative of clinical features, disease course and overall severity. OBJECTIVE: To clarify the association of clinical and prognostic features with serum ANA in Japanese patients with SSc. METHODS: We studied 203 Japanese patients diagnosed with SSc, who visited our hospital during the period 1983-2005. Six SSc-related ANA were identified using indirect immunofluorescence, double immunodiffusion and immunoprecipitation assays. RESULTS: Patients with SSc were classified into six ANA-based subgroups and a group without ANA. As expected, antitopoisomerase I antibody (Ab, n=64), anti-RNA polymerases (RNAP) Ab (n=12) and anti-U3 RNP Ab (n=5) were associated with diffuse cutaneous SSc, whereas anticentromere Ab (ACA, n=75), anti-Th/To Ab (n=7) and anti-U1 RNP Ab (n=10) were frequently detected in patients with limited cutaneous SSc. Clinical features of the ANA-negative group (n=10) were heterogeneous. Consistent with previous findings in Caucasian and/or black African patients, antitopoisomerase I Ab was associated with the involvement of vascular and pulmonary fibrosis, leading to decreased survival rate. However, no patients with anti-RNAP Ab developed renal crisis and the frequency of isolated pulmonary hypertension in patients with ACA, anti-Th/To Ab or anti-U3 RNP Ab was similar to that in other ANA-based subgroups. CONCLUSION: These results indicate that the clinical relevance of SSc-related ANA in Japanese patients differs in some aspects from that in Caucasian and/or black African patients.

Autoantibody against a protease domain of caspase-8 in patients with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is characterized by autoantibodies against various cellular components. OBJECTIVE: To determine the presence or levels of antibodies (Abs) against a protease domain (PD) of caspase-8 and their clinical relevance in SSc. METHODS: Anti-caspase-8 PD Ab was examined by enzyme-linked immunosorbent assay and immunoblotting using human recombinant caspase-8 PD. Caspase-8 activity was evaluated by spectrophotometric detection of cleavage from p-nitroanilide-labeled IETD, a substrate of caspase-8. RESULTS: IgG anti-caspase-8 PD Ab levels in patients with SSc, systemic lupus erythematosus, or dermatomyositis were higher than in normal controls (CTL). Furthermore, anit-caspase-8 PD Ab levels in limited cutaneous SSc (ISSc) patients were elevated compared to diffuse cutameous SSc (dSSc) patients. To investigate the clinical correlation, laboratory findings were compared between SSc patients with high levels (>the mean+2SD of CTL) of anti-caspase-8 PD Ab and those with low levels. SSc patients with high level exhibited lower frequency of male and decreased C-reactive protein levels relative to those with low levels. Immunoblotting showed the anit-caspase-8 PD Ab was present in all SSc patients examined, while it was also detected in 75% of CTL. Caspase-8 activity was inhibited by IgG isolated from sera of SSc patients and CTL, although inhibitory effect was greater in SSc patients than CTL. CONCLUSION: These results suggest that immune response to caspase-8 occurs in healthy individuals, although it is greater in patients with systemic autoimmune diseases including SSc. Furthermore, high level of anti-caspase-8 PD Ab may be a serological indicator for a milder SSc subset.

Blockade of CD40/CD40 ligand interactions attenuates skin fibrosis and autoimmunity in the tight-skin mouse.

OBJECTIVE: To assess the association of CD40/CD40 ligand (CD40L) interactions with the development of skin fibrosis and autoimmunity in tight-skin (TSK/+) mouse, which is a mouse model for human systemic sclerosis. METHODS: Newly born TSK/+ mice were treated with murine anti-CD40L monoclonal antibody (100 microg intraperitoneally weekly). Hypodermal thickness of 8-week-old female mice (defined as the thickness of a subcutaneous loose connective tissue layer beneath the panniculus carnosus) was measured under a light microscope. All skin sections were taken from the para-midline, upper back region. Serum anti-topoisomerase I autoantibody levels, serum immunoglobulin levels and plasma soluble CD40L levels were determined by enzyme-linked immunosorbent assay. For analysis of lymphocyte surface molecules, single cell suspensions of lymphocytes were stained by monoclonal antibodies. Proliferation of TSK/+ B cells and fibroblasts to anti-CD40 antibodies was assessed by the uptake of [3H]-labelled thymidine and bromodeoxyuridine, respectively. RESULTS: The blockade of CD40/CD40L interactions by anti-CD40L monoclonal antibody significantly reduced cutaneous fibrosis (65%) and anti-topoisomerase I autoantibody in TSK/+ mice. Anti-CD40L monoclonal antibody also normalised B lymphocyte abnormal activation in TSK/+ mice, demonstrated by hyper-gamma-globulinaemia. Furthermore, augmented CD40/CD40L interactions in TSK/+ mice were suggested by upregulated expression of CD40L on CD4(+) T cells, elevated plasma soluble CD40L levels. The hyperresponsiveness to CD40 stimulation was also observed in TSK/+ B cells and fibroblasts. CONCLUSIONS: Cutaneous fibrosis and autoimmunity in TSK/+ mice are closely correlated with CD40/CD40L interactions.

Increased serum levels of nitrotyrosine, a marker for peroxynitrite production, in systemic sclerosis.

OBJECTIVE: To determine serum levels of nitrotyrosine (NT), an end product of peroxynitrite (ONOO-), and its clinical association in patients with systemic sclerosis (SSc). METHODS: Serum NT levels from 25 patients with limited cutaneous SSc (lSSc) and 34 patients with diffuse cutaneous SSc (dSSc) were examined by enzyme-linked immunosorbent assay. RESULTS: Serum NT levels were elevated in SSc patients compared with normal controls (n = 27), the levels being similar between lSSc and dSSc patients (P < 0.001). SSc patients with elevated NT had higher serum levels of anti- agalactosyl IgG Ab, IgG and IgA than those with normal NT levels (P < 0.05). NT levels correlated inversely with the percentage diffusion capacity for carbon monoxide (DLco) (P < 0.02, r = -0.414, n = 47). CONCLUSION: These results suggest that ONOO- may play an important role in the clinical manifestations of SSc, especially vascular damage to the lungs, and that ONOO- may be related to immunological abnormalities in SSc.

Autoantibody against peroxiredoxin I, an antioxidant enzyme, in patients with systemic sclerosis: possible association with oxidative stress.

OBJECTIVES: To determine the prevalence and clinical correlation of autoantibody to peroxiredoxin (Prx) I, an antioxidant enzyme, in patients with systemic sclerosis (SSc). METHODS: Serum samples from SSc patients (n = 70) and healthy controls (n = 23) were examined by ELISA using human recombinant Prx I. The presence of anti-Prx I antibody was further evaluated by immunoblotting analysis. To determine the functional relevance of anti-Prx I antibody in vivo, we assessed whether anti-Prx I antibody was able to inhibit Prx I enzymatic activity using yeast thioredoxin reductase system. RESULTS: IgG anti-Prx I antibody levels in SSc patients were significantly higher than healthy controls and this autoantibody was detected in 33% of SSc patients. The presence of IgG anti-Prx I antibody was associated with longer disease duration, more frequent presence of pulmonary fibrosis, heart involvement, and anti-topoisomerase I antibody and increased levels of serum immunoglobulin and erythrocyte sedimentation rates. IgG anti-Prx I antibody levels also correlated positively with renal vascular damage and negatively with pulmonary function tests. Furthermore, anti-Prx I antibody levels correlated positively with serum levels of 8-isoprostane, a marker of oxidative stress. Immunoblotting analysis confirmed the presence of anti-Prx I antibody. Remarkably, Prx I enzymatic activity was inhibited by IgG isolated from SSc sera containing IgG anti-Prx I antibody. CONCLUSIONS: These results suggest that elevated IgG anti-Prx I autoantibody is associated with the disease severity of SSc and that anti-PrxI antibody may enhance the oxidative stress by inhibiting Prx I enzymatic activity.

Identification of a novel autoantibody reactive with 155 and 140 kDa nuclear proteins in patients with dermatomyositis: an association with malignancy.

OBJECTIVE: Myositis-specific autoantibodies (MSAs) are a useful tool in diagnosis, defining clinical subsets and predicting prognosis of dermatomyositis (DM) and polymyositis (PM). In this study, we identified a novel MSA reactive with 155 and 140 kDa nuclear proteins [anti-155/140 antibody (Ab)] and determined the clinical feature of DM patients positive for this autoantibody (autoAb). METHODS: Sera from 52 Japanese patients with DM, 9 with PM, 48 with systemic lupus erythematosus (SLE), 126 with systemic sclerosis and 18 with idiopathic interstitial pneumonia were examined by immunoprecipitation assays. Positive sera were further characterized by immunodepletion and immunofluorescence staining. RESULTS: Seven of the 52 (13%) Japanese patients with DM immunoprecipitated 155 and 140 kDa proteins from 35S-labelled K562 leukaemia cell extract. No patients with SLE, systemic sclerosis or idiopathic interstitial pneumonia as well as healthy controls were positive for this autoAb. Patients with anti-155/140 Ab developed heliotrope rash, Gottron's papules or sign and flagellate erythema significantly more frequently than those negative. Notably, internal malignancy was found at significantly higher frequency in those positive than those negative (71 vs 11%; P < 0.005). In contrast, none of these patients positive for this autoAb had interstitial lung disease. CONCLUSIONS: This novel MSA is associated with cancer-associated DM and may serve as a diagnostic serological marker for this specific subset.

Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases.

The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Mutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

Serum levels of 8-isoprostane, a marker of oxidative stress, are elevated in patients with systemic sclerosis.

OBJECTIVE: To determine serum levels and clinical correlation of 8-isoprostane, which is produced in vivo through free radical-catalysed peroxidation of arachidonic acid and reflects oxidative stress, in patients with systemic sclerosis (SSc). METHODS: Serum 8-isoprostane levels from 32 patients with diffuse cutaneous SSc (dSSc) and 25 patients with limited cutaneous SSc (lSSc) were examined by enzyme-linked immunosorbent assay. RESULTS: Serum 8-isoprostane levels were elevated in dSSc and lSSc patients by 75-fold compared with normal controls (n=32). Serum 8-isoprostane levels correlated negatively with pulmonary function, such as percentage vital capacity and diffusion capacity for carbon monoxide, and correlated positively with renal vascular damage determined by colour flow Doppler ultrasonography. Serum 8-isoprostane levels also correlated positively with serum levels of IgG and anti-agalactosyl IgG autoantibody. CONCLUSION: Increased 8-isoprostane levels correlated with the severity of pulmonary fibrosis, the extent of renal vascular damage and immunological abnormalities in SSc, suggesting that enhanced oxidative stress is related to the development of SSc.

Dendritic cell vaccine therapy by immunization with fusion cells of interleukin-2 gene-transduced, spleen-derived dendritic cells and tumour cells.

We examined the preventive and therapeutic effects of fusion cells prepared from spleen-derived dendritic cells (DCs) transduced with the interleukin-2 (IL-2) gene and QRsP fibrosarcoma cells in a mouse lung metastasis model. The IL-2 or LacZ gene was introduced into spleen-derived DCs using an adenoviral vector. Irradiated QRsP tumour cells were fused with IL-2 gene-transduced DCs (fusion/IL-2) or LacZ gene-transduced DCs (fusion/LacZ) by polyethylene glycol. These fusion cells expressed major histocompatibility complexes (MHC) class I and II, CD86, CD11c and CD8alpha. Splenocytes from mice vaccinated with fusion cells showed increased production of interferon-gamma (IFN-gamma) and cytotoxic T-lymphocyte (CTL) activity as compared with those vaccinated with DCs or tumour cells alone, and CTL levels were higher in fusion/IL-2-vaccinated mice than in fusion/LacZ-vaccinated mice. In our experiments on the protective and therapeutic effects on lung metastasis, mice vaccinated with fusion/IL-2 fusion/LacZ or fusion showed a significant reduction in pulmonary metastasis compared with those given DCs, tumour or phosphate-buffered saline. The introduction of the IL-2 gene into fusion cells produced more potent preventive and therapeutic effects. These results suggest that immunization with fusion cells prepared from spleen-derived DCs and tumour cells is capable of inducing preventive and therapeutic anti-tumour immunity against lung metastasis, and modification by the IL-2 gene may increase anti-tumour efficacy.

Silicone gel sheets relieve pain and pruritus with clinical improvement of keloid: possible target of mast cells.

Silicone gel sheet treatment is widely used to treat hypertrophic scars and keloids since it is easily applied and prevents scar pain and itching. We used Cica-Care silicone gel sheets in the conservative treatment of six patients for 24 weeks and recorded pain, itching, redness, and scar elevation every 4 weeks. We also investigated the number of mast cells and Fas antigen expression in the lesional skin (one patient) before and after treatment. The pain and itching clearly decreased after 4 weeks of the silicone gel sheeting and disappeared after 12 weeks. Twelve weeks were required for a reduction in scar redness and elevation. After 24 weeks, a decrease in the number of mast cells and the enhanced expression of Fas antigen by lesional fibroblasts were observed. Thus, silicone gel sheeting is effective and safe, especially with more severe symptoms of pain and itching possibly induced by mediators derived from increased mast cells.

[Examination of serum nitric oxide in Yusho patients].

In order to evaluate the influence of oxidative stress by PCB, we measured serum nitric oxide (NO) in Yusho patients by the Griess method, which detected nitrite (NO2-) formed by the oxidization of NO. Serum NO2- concentrations were significantly increased in Yusho patients as compared to healthy controls, but no correlation was detected between serum NO2- concentration and PCB or PCQ level in Yusho patients. The relationship was analyzed between serum NO2- concentration and certain parameters such as clinical symptoms, habits and clinical data. Serum NO2- level had a positive correlation with CPK in male patients, but not in female and all Yusho patients. Furthermore, no mutual relation was detected between serum NO2- concentration and the other parameters such as blood pressure, smoking and each laboratory data. Therefore, the possibility is considered that Yusho is one of the diseases which show the increase of serum NO.

Recurrent leg ulcers and arterial thrombosis in a 33-year-old homozygous variant of antithrombin.

We report here a homozygous variant case of antithrombin (AT) associated with arterial thrombosis and recurrent leg ulcers. The deep vein thrombosis was recognized by the venogram of his pelvic veins. His leg ulcers were scattered around his left ankle and accompanied by lipodermatosclerosis, which was evident in venous insufficiency. The propositus had developed cerebral infarction 12 years prior to his leg ulcers. Coagulation study showed low heparin cofactor activity of his AT with a normal level of immunoreactive AT. Nucleotide sequence analysis of the exon 2 of his AT gene showed Arg47-Cys mutation, leading to the lack of affinity of AT for heparin. The propositus is a homozygote for this abnormality.

Chorioangioma: antenatal diagnosis with fast MR imaging.

We report a case of chorioangioma of the placenta, in which fast magnetic resonance imaging (MRI) was useful adjunct to ultrasonography for the antenatal diagnosis. MRI allowed clear demonstration of 6.8 x 6.0 cm solid placental mass along with hydramnios and anatomically normal fetus. On T(1)-weighted breath-hold spoiled gradient-echo (fast low-angle shot [FLASH]) images, chorioangioma was mostly isointense to the placenta, but had an area of high signal intensity near the base and at the periphery, suggestive of hemorrhage. On T(2)-weighted half-Fourier single-shot fast spin echo (HASTE) images, the mass showed heterogeneous high signal intensity, but had an area of low signal intensity near the surface.