IL-6, IL-8, IP-10, MCP-1 and G-CSF are significantly increased in cerebrospinal fluid but not in sera of patients with central neuropsychiatric lupus erythematosus.

OBJECTIVE: To determine whether the intrathecal concentrations of cytokines/chemokines are associated with, or influenced by, serum concentrations in patients with central neuropsychiatric systemic lupus erythematosus (NPSLE), and to ascertain whether the increased production of cytokines/chemokines intrathecally relative to serum levels is associated with the presence of central NPSLE. METHODS: 52 SLE patients (30 with central NPSLE and 22 with non-NPSLE), for whom the CSF and serum samples were obtained at the same time, were enrolled. 27 kinds of cytokine/chemokine concentrations other than IFN-α in the cerebrospinal fluid (CSF) and serum samples were measured by Bio-Plex Pro Assays. IFN-α concentration and anti-ribosomal P protein antibody (anti-P) titres in CSF and serum samples were measured by ELISA. RESULTS: The mean concentrations of IL-6, IL-8, IP-10, MCP-1, G-CSF and GM-CSF were higher in the CSF than in the sera, respectively, while the mean concentrations of other 22 cytokines/chemokines, including RANTES and IFN-α, in the CSF were much lower than those in the sera, respectively. Furthermore, the concentrations of IL-6, IL-8, IP-10, MCP-1 and G-CSF in the CSF of the 30 patients with NPSLE were significantly higher than in the 22 patients with non-NPSLE (p = 6.82 × 10(-5), p = 0.00037, p = 0.0028, p = 0.00065, and p = 0.0001, respectively), while the concentration of GM-CSF in the CSF of the 30 patients with NPSLE was not significantly higher than in the 22 patients with non-NPSLE. Most importantly, the largest difference occurred in CSF IL-6 concentrations. A significant positive correlation between CSF anti-P titres and serum anti-P titres in 52 patients with SLE (r = 0.6316, p = 6.44 × 10(-6)) was found, while no significant positive correlation was observed between CSF levels and serum levels of each cytokine/chemokine in the 52 SLE patients. CONCLUSION: In central NPSLE the production of IL-6, IL-8, IP-10, MCP-1 and G-CSF might take place in the central nervous system (CNS). These increased CSF cytokines/chemokines along with anti-P might have a prerequisite role in the pathogenesis of central NPSLE.

Efficacy of serum angiopoietin-1 measurement in the diagnosis of early rheumatoid arthritis.

OBJECTIVES: Previous studies showed that angiopoietin-1(Ang-1) expression was increased in the synovium in early rheumatoid arthritis (RA) patients. The present study was therefore designed to examine whether determination of serum Ang-1 might be effective in diagnosis of early RA. METHODS: One hundred and five serum samples of RA (21 males, 84 females) were studied for serum Ang-1 level. Serum samples were also collected from other collagen diseases, including 35 cases of SLE, 29 cases of systemic sclerosis, 16 cases of polymyositis/dermatomyositis. Serum samples were additionally obtained from 34 patients who visited our clinic for evaluation of symmetrical polyarthritis with morning stiffness. After one year of follow-up, those patients who satisfied the ACR 1987 classification criteria for RA were defined as 'early RA'. Serum Ang-1 levels were measured by sandwich ELISA using anti-angiopoietin-1 antibodies (both monoclonal and polyclonal antibodies). Serum anti-CCP antibody and rheumatoid factor (RF) were measured by ELISA and by laser nepherometry, respectively. RESULTS: Serum Ang-1 in RA patients was significantly higher than those in other collagen diseases. Serum Ang-1 levels in 50 normal healthy individuals were 5.8 ± 0.31 pg/ml (mean ± SEM). There was no significant difference in CRP and serum RF at the first visit between early RA patients and non-RA patients, whereas serum Ang-1 levels at the first visit were significantly higher in early RA (58.7 ± 17.9 pg/ml [mean ± SEM]) than those in non-RA (8.2 ± 4.5 pg/ml). ROC analysis revealed that serum Ang-1 (cut-off 23.91 pg/ml) could diagnose early RA at sensitivity 57.1% and specificity 84.6%, providing comparable area under the curve (0.71, 95% CI: 0.54-0.88) to that of serum anti-CCP antibody (0.72, 95% CI: 0.53-0.92). There was no significant correlation between anti-CCP antibody and Ang-1. CONCLUSIONS: These results indicate that serum Ang-1 is as useful a marker for the diagnosis of early RA as serum anti-CCP antibody.

Regulation of mechanical stress-induced MMP-13 and ADAMTS-5 expression by RUNX-2 transcriptional factor in SW1353 chondrocyte-like cells.

OBJECTIVE: To investigate the mechanism of mechanical stress-induced expression and regulation of aggrecanases and examine the role of runt-related transcription factor 2 (RUNX-2) in chondrocyte-like cells. METHODS: SW1353 cells were seeded onto stretch chambers at a concentration of 5×104 cells/chamber, and a uni-axial cyclic tensile strain (CTS) (0.5 Hz, 10% stretch) was applied for 30 min. Total RNA was extracted, reverse transcribed, and analyzed by polymerase chain reaction (PCR) and real-time PCR. RUNX-2 overexpression and small interfering RNA (siRNA) targeting RUNX-2 were used to investigate the role of RUNX-2 in CTS-induced gene expression. The involvement of diverse mitogen-activated protein kinase (MAPK) pathways in the activation of RUNX-2, MMP-13 and ADAMTS-5 during CTS was examined by Western blotting. RESULTS: CTS induced expression of RUNX-2, MMP-13, ADAMTS-4, -5, and -9. Overexpression of RUNX-2 up-regulated expression of MMP-13 and ADAMTS-5, whereas RUNX-2 siRNA resulted in significant down-regulation of mechanically-induced MMP-13 and ADAMTS-5 expression. CTS induced activation of p38 MAPK, and CTS induction of RUNX-2, MMP-13 and ADAMTS-5 mRNA was down-regulated by the selective p38 MAPK inhibitor SB203580 but not by the p44/42 MAPK inhibitor U0126, or the JNK MAPK inhibitor JNK inhibitor II. CONCLUSIONS: RUNX-2 might have a role as a key downstream mediator of p38's ability to regulate mechanical stress-induced MMP-13 and ADAMTS-5 expression.

Enhanced expression of mRNA for FK506-binding protein 5 in bone marrow CD34 positive cells in patients with rheumatoid arthritis.

OBJECTIVE: Recent studies have disclosed that several genes are up-regulated in bone marrow (BM) mononuclear cells from rheumatoid arthritis (RA) patients. However, it remains unclear whether such abnormalities result from systemic inflammation or from abnormalities at stem cell level. The current study therefore examined the expression of several representative genes, including amphiregulin (AREG), chemokine receptor 4 (CXCR4), and FK506-binding protein 5 (FKBP5) in RA BM CD34+ cells. METHODS: BM samples were obtained from 52 patients with RA and 35 patients with osteroarthritis (OA) during joint operations. CD34+ cells were purified from the BM mononuclear cells by positive selection with magnetic beads. The mRNA expression for AREG, CXCR4, and FKBP5 was measured using quantitative real-time PCR. RESULTS: The expression of mRNA for FKBP5, but not that of AREG or CXCR4, was significantly higher in RA BM CD34+ cells than in OA BM CD34+ cells. The FKBP5 mRNA expression level was not correlated with serum CRP or treatment. In addition, tumour necrosis factor-alpha did not enhance the expression of FKBP5 mRNA in BM CD34+ cells from healthy donors. CONCLUSION: The results suggest that the enhanced expression of FKBP5 in BM CD34+ cells might be an intrinsic abnormality of RA BM CD34+ cells, whereas the enhanced expression of AREG and CXCR4 in BM mononuclear cells might be secondary to systemic inflammation.

Identification of autoantigens specific for systemic lupus erythematosus with central nervous system involvement.

Using proteomic analysis, we identified candidate autoantigens specific for central nervous system (CNS) involvement in systemic lupus erythematosus (SLE). Proteins, extracted from cultured human neuroblastoma cells, were separated both by SDS-PAGE (1-DE) and two-dimensional electrophoresis (2-DE), and transferred to membranes. Western blot analysis was performed using serum samples from 30 SLE patients with CNS involvement (CNS-Lupus) and from 30 SLE patients without CNS involvement (non-CNS-SLE). The detected autoantigens were identified using MALDI-TOF/TOF MS. On the 1-DE Western blot, we detected 32 antigenic bands in the serum samples from the CNS-Lupus patients. Among them, four bands were detected significantly more frequently in the CNS-Lupus patients than in the non-CNS-SLE patients. Three bands were detected in four or more of the CNS-Lupus patients but in only one or none of the non-CNS-SLE patients. We thus selected these seven bands for the next investigations. Next, we detected protein spots corresponding to the selected seven bands by 2-DE Western blot and identified four proteins. They are peroxiredoxin-4, ubiquitin carboxyl-terminal hydrolase isozyme L1, splicing factor arginine/serine-rich 3, and histone H2A type 1. These four candidate autoantigens for the anti-neuronal cell antibodies would be a useful marker for CNS-Lupus.

Trichostatin A, a histone deacetylase inhibitor, suppresses synovial inflammation and subsequent cartilage destruction in a collagen antibody-induced arthritis mouse model.

OBJECTIVE: To investigate the effect of the histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), on joint inflammation and cartilage degeneration in a collagen antibody-induced arthritis (CAIA) mouse model. METHODS: CAIA mice were given daily subcutaneous injections of various concentrations of TSA (0, 0.5, 1.0, and 2.0 mg/kg) and various parameters were monitored for 14 days. On Day 15, the hind paws were examined histologically. To investigate the effects of TSA on the expressions of matrix metalloproteinase (MMP)-3, MMP-13, tissue inhibitor of MMP-1 (TIMP-1), and acetyl-H4 by chondrocytes, another group of mice was sacrificed on Day 6. In vitro direct effect of TSA was examined by real-time PCR for mRNA of type II collagen, aggrecan, MMP-3, and MMP-13 in murine chondrogenic ATDC5 cells after pro-inflammatory cytokine stimulation. RESULTS: In the TSA-treated group, clinical arthritis was significantly ameliorated in a dose-dependent manner. The severity of synovial inflammation and the cartilage destruction score were significantly lower in the TSA 2.0 mg/kg group compared to the other TSA-treated groups. On immunohistochemistry, the number of MMP-3 and MMP-13-positive chondrocytes was significantly lower in the TSA 2.0 mg/kg group than in the control group. In contrast, the number of TIMP-1-positive cells and acetyl-histone H4-positive cells was significantly higher in the TSA 2.0mg/kg group than in the control group. TSA suppressed interleukin 1-beta and tumor necrosis factor-alpha-stimulated up-regulation of MMP-3, but not MMP-13 mRNA expression by ATDC5. CONCLUSION: The systemic administration of TSA ameliorated synovial inflammation in CAIA mice. Subsequently cartilage destruction was also suppressed by TSA, at least in part, by modulating chondrocyte gene expression.

Myocardial infarction caused by rheumatoid vasculitis: histological evidence of the involvement of T lymphocytes.

Rheumatoid arthritis (RA) is a chronic joint disease that can be complicated with extra-articular manifestations due to vasculitis. We describe a patient with RA who developed systemic vasculitis and died of myocardial infarction. Autopsy demonstrated vasculitis of the left anterior descendent and circumflex coronary arteries, which were narrowed or occluded with organizing thrombosis. Formation of granuloma with multinucleated giant cells was also observed in media of the circumflex artery. There was no microscopic evidence of atheroma formation in the coronary arteries. Of note, there was a follicle-like infiltration of CD45RO-positive T lymphocytes in interna of the left coronary arteries and the right renal artery. Although not frequently reported, coronary vasculitis as a cause of myocardial infarction should be considered in patients with RA. Moreover, our results suggest that infiltration of T lymphocytes might be involved in the development of rheumatoid vasculitis.

Autoimmune hepatitis in a patient with systemic lupus erythematosus.

We report a female patient with systemic lupus erythematous (SLE), hyperbilirubinemia and high serum value of ALT. International autoimmune hepatitis (AIH) score showed definite AIH before treatment, but autoantibodies could not make a differential diagnosis of AIH and SLE-associated hepatitis. Liver biopsy showed periportal hepatitis with lymphoplasmacytic infiltration, but neither parenchymal collapse nor rosette formation could be found. Pericarditis, pleuritis and nephritis were improved as well as liver injury after administration of prednisolone, and no repeated attack has been present these 4 years. Our case suggested invalidity of AIH score among patients of SLE, and liver histology should be inferred most important at present to make a differential diagnosis of lupus hepatitis or AIH in patients with SLE.

Differential regulation of Th1 responses and CD154 expression in human CD4+ T cells by IFN-alpha.

Like interleukin (IL)-12, interferon (IFN)-alpha has been shown to play an important role in inducing human Th1 responses. Recent studies have shown that human Th1 responses driven by IL-12 are associated with enhanced expression of CD154. The present study examined the effects of IFN-alpha on CD154 expression in human CD4+ T cells, with special attention to the relationship with Th1 responses. Highly purified CD4+ T cells from healthy donors were stimulated with immobilized anti-CD3 with or without IFN-alpha and IL-12 in the complete absence of accessory cells. IFN-alpha suppressed CD154 protein and mRNA expression in CD4+ T cells at the initial phase of activation with immobilized anti-CD3, but enhanced it in the subsequent maturation phase irrespective of the presence of IL-12. By contrast, IFN-alpha by itself did not enhance IFN-gamma production or mRNA expression in CD4+ T cells in the absence of IL-12 even in the presence of stimulation with anti-CD28, but enhanced it in the presence of IL-12. Accordingly, IFN-alpha enhanced IL-12Rbeta2 mRNA expression in anti-CD3-stimulated CD4+ T cells. Neither IFN-alpha nor IL-12 influenced the stability of CD154 mRNA in anti-CD3-activated CD4+ T cells. These results indicate that IFN-alpha by itself enhances CD154 expression in CD4+ T cells independently of the induction of IFN-gamma mRNA expression. The data also suggest that the optimal induction of human Th1 responses by IFN-alpha might require the presence of IL-12 and that the induction of Th1 responses and CD154 expression in human CD4+ T cells might be regulated through different mechanisms.

A novel treatment of human malignant gliomas in vitro and in vivo: FADD gene transfer under the control of the human telomerase reverse transcriptase gene promoter.

Telomerase activity has a close relationship with malignancies in many cell types and it is tightly regulated at the transcriptional level of human telomerase reverse transcriptase (hTERT). Utilizing the hTERT promoter, the authors developed a gene delivery system of Fas associated protein with death domain (FADD) (hTERT/FADD). FADD is a protein which plays an important role in the apoptotic pathway of Fas. Over-expression of FADD induces apoptosis in the cells regardless of Fas expression on the cell surface. We hypothesized that we might be able to restrict the expression of FADD in malignant glioma cells if we use the gene transfer system under the control of hTERT promoter. This study was designed to investigate whether the hTERT/FADD construct induces apoptosis effectively in malignant glioma cells while keeping normal cells intact. First, using the reverse transcription-polymerase chain reaction (RT-PCR) technique, we confirmed that hTERT mRNA was expressed in human malignant glioma cells (U373-MG, A172 and GB-1), but not in cultured astrocytes (TEN) or fibroblasts (MRC5). After transient transfection with the hTERT/FADD construct, a significant number of FADD-positive cells and apoptotic cells were detected in hTERT-positive malignant glioma cells. In contrast, hTERT-negative astrocytes and fibroblasts remained intact. Furthermore, subcutaneously implanted U373-MG tumors treated with the hTERT/FADD construct reduced in volume significantly compared to the conrol treatment (p=0.0001). Gene transfer of FADD under the control of the hTERT promoter may be a novel and promising therapy to kill hTERT-positive malignant glioma cells while sparing normal brain cells lacking hTERT.

Systemic sclerosis complicated with nephrotic syndrome: relevance with antiribosomal P antibody.

The nephrotic syndrome is an extremely rare occurrence in systemic sclerosis (SSc). Here we describe a 39-year-old woman with SSc who developed the nephrotic syndrome along with the expression of antiribosomal P antibody but not of anti-double-stranded DNA antibody in her serum. Although a renal biopsy specimen showed minimal changes on light microscopy, immunofluorescence studies showed granular deposition of C3 and IgM in the glomeruli. The patient recovered from the nephrotic syndrome with the decrease in serum antiribosomal P antibody after the daily administration of 60 mg of prednisolone. It is strongly suggested that antiribosomal P antibody might have been involved in the development of the nephrotic syndrome in our patient.

FADD gene therapy using the human telomerase catalytic subunit (hTERT) gene promoter to restrict induction of apoptosis to tumors in vitro and in vivo.

Gene transfer vectors will dramatically increase the safety and effectiveness of cancer gene therapy, if they could restrict expression of the therapeutic products to the target tumors. To realize such a tumor-targeting system, telomerase is one of the most promising candidates. It is because telomerase activity is detected in the vast majority of tumors, but not in most normal cells. Activation of telomerase is tightly regulated at the transcriptional level of the telomerase catalytic subunit (hTERT). Therefore, the use of the hTERT promoter-driven vector system could restrict the expression of therapeutic products to telomerase-positive tumors. In this study, we constructed the expression vector of FADD gene with death domain afforded by the hTERT promoter (hTERT/FADD) and investigated its effect on tumors in vitro and in vivo. Transient transfection with the hTERT/FADD construct induced apoptosis in telomerase-positive tumor cells of wide range. In contrast, normal fibroblast cells without telomerase did not undergo apoptosis following the hTERT/FADD transfer. Furthermore, the growth of subcutaneous tumors in nude mice was significantly suppressed by the intratumoral injection of the hTERT/FADD construct (every day for one week) compared to the control (P<0.0005). The findings described here indicate the high potentiality of a novel telomerase-specific gene therapy of tumors with telomerase.

Induction of fibroblast-like cells from CD34(+) progenitor cells of the bone marrow in rheumatoid arthritis.

To assess the role of bone marrow in the pathogenesis of rheumatoid arthritis (RA), we examined the capacity of CD34(+) cells from bone marrow to generate fibroblast-like type B synoviocytes. CD34(+) cells from the bone marrow of 22 RA patients differentiated into cells with fibroblast-like morphology, which expressed prolyl 4-hydroxylase, in the presence of stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha), much more effectively than CD34(+) cells from bone marrow of 15 control subjects (10 patients with osteoarthritis and 5 healthy individuals). The generation of fibroblast-like cells was not at all observed in cultures with SCF, GM-CSF, and interleukin 4 (IL-4) with or without TNF-alpha. Generation of fibroblast-like cells was correlated with matrix metalloproteinase (MMP)-1 levels in culture supernatants. Thus, MMP-1 levels were significantly higher in TNF-alpha-stimulated cultures of bone marrow CD34(+) cells from patients with RA than in those from the control group. These results indicate that bone marrow CD34(+) cells from patients with RA have abnormal capacities to respond to TNF-alpha and to differentiate into fibroblast-like cells producing MMP-1, suggesting that bone marrow CD34(+) progenitor cells might generate type B synoviocytes and thus could play an important role in the pathogenesis of RA.

Treatment of malignant glioma cells with the transfer of constitutively active caspase-6 using the human telomerase catalytic subunit (human telomerase reverse transcriptase) gene promoter.

Because the apoptotic pathway is often disrupted in tumor cells, its genetic restoration is a very attractive approach for the treatment of tumors. To treat malignant gliomas with this approach, it would be preferred to restrict induction of apoptosis to tumor cells by establishing a tumor-specific expression system. Telomerase is an attractive target because the vast majority of malignant gliomas have telomerase activity whereas normal brain cells do not. Activation of telomerase is tightly regulated at the transcriptional level of the telomerase catalytic subunit [human telomerase reverse transcriptase, (hTERT)]. Therefore, we hypothesized that using a hTERT promoter-driven vector system, an apoptosis-inducible gene may be preferentially restricted to telomerase- or hTERT-positive tumor cells. In this study, we constructed an expression vector consisting of the constitutively active caspase-6 (rev-caspase-6) under the hTERT promoter (hTERT/rev-caspase-6) and then investigated its antitumor effect on malignant glioma cells. The rationale for using the rev-caspase-6 gene is because it induces apoptosis independent of the initiator caspases. We demonstrated that the hTERT/rev-caspase-6 construct induced apoptosis in hTERT-positive malignant glioma cells, but not in hTERT-negative astrocytes, fibroblasts, and alternative lengthening of telomeres cells. In addition, the growth of s.c. tumors in nude mice was significantly suppressed by the treatment with hTERT/rev-caspase-6 construct. The present results strongly suggest that the telomerase-specific transfer of the rev-caspase-6 gene under the hTERT promoter is a novel targeting approach for the treatment of malignant gliomas.

Procollagen II amino propeptide processing by ADAMTS-3. Insights on dermatosparaxis.

The amino and carboxyl propeptides of procollagens I and II are removed by specific enzymes as a prerequisite for fibril assembly. Null mutations in procollagen I N-propeptidase (ADAMTS-2) cause dermatosparaxis in cattle and the Ehlers-Danlos syndrome (dermatosparactic type) in humans by preventing proteolytic excision of the N-propeptide of procollagen I. We have found that procollagen II is processed normally in dermatosparactic nasal cartilage, suggesting the existence of another N-propeptidase(s). We investigated such a role for ADAMTS-3 in Swarm rat chondrosarcoma RCS-LTC cells, which fail to process the procollagen II N-propeptide. Stable transfection of RCS-LTC cells with bovine ADAMTS-2 or human ADAMTS-3 partially rescued the processing defect, suggesting that ADAMTS-3 has procollagen II N-propeptidase activity. Human skin and skin fibroblasts showed 30-fold higher mRNA levels of ADAMTS-2 than ADAMTS-3, whereas ADAMTS-3 mRNA was 5-fold higher than ADAMTS-2 mRNA in human cartilage. We propose that both ADAMTS-2 and ADAMTS-3 process procollagen II, but ADAMTS-3 is physiologically more relevant, given its preferred expression in cartilage. The findings provide an explanation for the sparing of cartilage in dermatosparaxis and, perhaps, for the relative sparing of some procollagen I-containing tissues.

[Central nervous system involvement in Behçet's disease].

Central nervous system involvement in Behçet's disease, usually called neuro-Behçet's syndrome (NB), includes acute type and chronic progressive type. Acute NB is characterized by acute meningoencephalitis with focal lesions, presenting high intensity areas in T2-weightened images or FLAIR images on MRI scans. Cyclosporin A frequently causes acute NB. Acute NB responds to steroid therapy, and is usually self-limiting. By contrast, chronic progressive NB is characterized by intractable slowly progressive dementia, ataxia and dysarthria with persistent elevation of cerebrospinal fluid IL-6 activity (more than 20 pg/ml). In our series, most patients (approximately 90%) with chronic progressive NB were HLA-B51 positive, and had history of attacks of acute NB prior to the development of progressive neurological symptoms. Chronic progressive NB is resistant to conventional treatment with steroid, cyclophosphamide, or azathioprine. However, recent studies suggest the efficacy of low dose methotrexate in chronic progressive NB.

A novel telomerase-specific gene therapy: gene transfer of caspase-8 utilizing the human telomerase catalytic subunit gene promoter.

Apoptosis is a genetically encoded cell death process and is a pathway that may be disrupted in tumor cells. Therefore, therapies that restore the ability to undergo apoptosis are promising for the treatment of tumor cells. We have demonstrated that the transfer of apoptosis-inducible genes inhibits the growth of tumors in vitro and in vivo through induction of apoptosis. However, to restrict induction of apoptosis to tumor cells, we need to explore a tumor-specific expression system of these genes. In the present study, we developed the telomerase-specific transfer system of apoptosis-inducible genes, utilizing the promoter of the human telomerase catalytic subunit (hTERT) gene. Approximately 90% of tumors have telomerase activity whereas most normal cells do not express the activity. These observations indicate that telomerase is a particularly attractive target for the tumor-specific expression system of vectors. We demonstrate here that by using the hTERT promoter-driven caspase-8 expression vector (hTERT/caspase-8), apoptosis is restricted to telomerase-positive tumor cells of wide range, and is not seen in normal fibroblast cells without telomerase activity. Furthermore, treatment of subcutaneous tumors in nude mice with the hTERT/caspase-8 construct inhibited tumor growth significantly because of induction of apoptosis (p < 0.01). The telomerase-specific expression of apoptosis-inducible genes afforded by the hTERT promoter, therefore, may be a novel and promising targeting approach for the treatment of tumors with telomerase activity.