Different contributions of angiostatin and endostatin in angiogenesis impairment in systemic sclerosis: a cohort study.

OBJECTIVES: To determine the concentrations of circulating endostatin and angiostatin in patients with systemic sclerosis (SSc) and to assess its relationship to disease subsets, evolution phase, organ involvement and nailfold capillaroscopic changes. METHODS: Endostatin and angiostatin serum levels were measured by ELISA in a cohort of 57 patients with SSc, and correlated with disease subsets, evolution phase, organ involvement and nailfold capillaroscopic changes. RESULTS: Endostatin and angiostatin serum levels were significantly higher in patients with SSc than in healthy controls. Also, angiostatin was elevated in diffuse cutaneous SSc (dcSSc) and limited cutaneous SSc (lcSSc), but not in pre-SSc, while endostatin was increased in all SSc subsets. Moreover, endostatin was augmented in lcSSc, with or without CREST syndrome, whereas angiostatin was increased exclusively in patients with CREST. Analysis according to disease evolution phase found that endostatin was elevated in all phases while angiostatin was only significantly higher in intermediate and late phases of disease. Analysis regarding organ involvement revealed that angiostatin was significantly higher in patients with osteoarticular involvement and with more serious lung affection; no significant differences were found for endostatin. Finally, endostatin was significantly increased in all nailfold capillaroscopy stages, while angiostatin was only elevated in active and late phases. CONCLUSIONS: In accordance with previous studies, we found that endostatin and angiostatin concentrations are elevated in SSc patients. Additionally, we recognised the important role that endostatin might play as an early disease marker and realized that angiostatin is a marker of late disease and relates to lung disease severity.

Detection of environmental clastogens and aneugens in human fibroblasts by cytokinesis-blocked micronucleus assay associated with immunofluorescent staining of CENP-A in micronuclei.

The cytokinesis-blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan-centromeric DNA probes, or with CREST antibodies that specifically stain kinetochore proteins, is widely used on several cell types. It distinguishes micronuclei containing one or several whole chromosomes, which are positively labeled (centromere positive micronucleus, C+MN, due to aneugenic effect), or acentric chromosome fragments, which are unlabeled due to the absence of centromere (centromere negative micronucleus, C-MN, due to clastogenic effect). However, the very slight level of the centromeric signals obtained with the FISH technique on primary human fibroblasts, a cell type commonly used in environmental genetic toxicology, leads to great difficulties in distinguishing C+MN and C-MN. Furthermore, the CREST technique may lead to inappropriate results particularly with regards to variations in antibody composition between patient sera. Our results show that the in vitro CBMN, in combination with immunofluorescence staining of CENP-A (centromere protein A), efficiently screens genotoxicants for their ability to induce clastogenic and/or aneugenic effects. We propose the in vitro CBMN assay in combination with immunofluorescence staining of CENP-A as a suitable tool in environmental genotoxicity testing of primary human fibroblasts.

Regulation of aurora B kinase by the lipid raft protein flotillin-1.

The lipid raft protein Flotillin-1 was previously shown to be required for cell proliferation. Here we show that it is critical for the maintenance of the levels of the mitotic regulator Aurora B. Knockdown of Flotillin-1 induced aberrant mitotic events similar to those produced by Aurora B depletion and led to a marked decline in Aurora B levels and activity. Transfection of wild-type full-length Flotillin-1 or forms directed to the nucleus increased Aurora B levels and activity. Flotillin-1 interacted with Aurora B directly through its SPFH domain in a complex distinct from the chromosomal passenger protein complex, and the two proteins co-purified in nuclear, non-raft fractions. These observations are the first evidence for a function of Flotillin-1 outside of lipid rafts and suggest its critical role in the maintenance of a pool of active Aurora B.

Increased prevalence of anti-third generation cyclic citrullinated peptide antibodies in patients with rheumatoid arthritis and CREST syndrome.

To investigate the prevalence of anti-third generation cyclic citrullinated peptide antibodies (anti-CCP3) in patients with systemic connective tissue diseases, we assembled a training set consisting of 115 patients with rheumatoid arthritis (RA), 52 with Calcinosis, Raynaud's phenomenon, oesophageal dysmotility, sclerodactyly, telangiectasia (CREST) syndrome, 21 with scleroderma, 20 with ankylosing spondylitis, 18 with reactive arthritis, 25 with juvenile rheumatoid arthritis (RA), 51 with osteoarthritis, 26 with mixed connective tissue disease, 23 with primary Sjogren's syndrome, 74 with systemic lupus erythematosus, 49 with Polymyalgia rheumatica, and 39 with polymyositis/dermatomyositis. The commercial enzyme-linked immunosorbent assay (ELISA) was used to detect anti-CCP antibodies, including anti-CCP2 (regular, second generation of CCP antigen) and anti-CCP3 (third generation of CCP antigen) in disease-related specimens and normal controls. These serum samples were also evaluated for anti-centomere antibodies by anti-centromere ELISA kit. The higher frequencies of anti-CCP3 and anti-CCP2 were detected in 75.6 and 70.4% patients with RA, respectively. At the same time, anti-CCP3 (not anti-CCP2) was significantly increased in samples isolated from patients with CREST syndrome. The clinical sensitivity of IgG anti-CCP3 for the patients with CREST syndrome was 29% (15 of 52) and the specificity was 96% (384 of 397), with the exception of the RA group. The anti-centromere antibodies were significantly higher in patients with CREST only. The results of our study suggest that compared to anti-CCP2 assay, the new anti-CCP3 assay can enhance the clinical sensitivity for diagnosis of RA and, as an associate marker combined with anticentromere, can distinguish CREST syndrome from other systemic connective tissue diseases, especially RA. The clinical specificity of anti-CCP3 was lower than anti-CCP2 assay in diagnosis of RA because of the crossreaction to the patients with CREST syndrome.

Intracellular Th1/Th2 balance of pulmonary CD4(+) T cells in patients with active interstitial pneumonia evaluated by serum KL-6.

The balance between CD4(+) T helper (Th1) lymphocytes producing interferon-gamma or Interleukin-4 (Th2) in the lungs may vary among diseases and during the progression of interstitial pneumonia (IP). Both idiopathic pulmonary fibrosis (IPF) and collagen vascular diseases (CVD) are associated with IP, but the clinical course and the response to treatment are different. Since Th1 or Th2 modulating drugs have been proven to alter the lymphocyte balance in vitro, it is important to elucidate the Th1/Th2 profile in patients with active IP. Bronchoalveolar lavage (BAL) was performed in patients who had IPF (n = 12) or CVD (n = 12) with IP, as well as in patients who had bronchoectasis and bronchopneumonia (n = 12). The CVD patients had rheumatoid arthritis (n = 6), Sjogren's syndrome (n = 2), dermatomyositis (n = 1), progressive systemic sclerosis (n = 2), and CREST syndrome (n = 1) as the underlying diseases. IP activity was evaluated by measuring serum KL-6, which is a clinically useful indicator for IP. The Th1/ Th2 balance and the CD4(+)/CD8(+) ratio were determined for lymphocytes obtained from BAL by flow cytometric analysis. In IPF patients, the CD4(+)/CD8(+) ratio was lower than in CVD patients. IPF patients showed Th2 dominance and CVD patients showed Th1 dominance when IP was active as evaluated by the serum KL-6 level. These data indicated that the Th1/Th2 balance of CD4(+) T cells in the BAL differs between active IPF and CVD, even though KL-6 is elevated in both diseases. Therefore, the Th1/Th2 profile should be investigated to determine the use of Th1/Th2 modulator therapy for active IP with elevation of KL-6.

Impact of external quality assessment on antinuclear antibody detection performance.

Our objective was to evaluate performance of the clinical laboratories for the detection of antinuclear antibodies (ANA) by using indirect immunofluorescence method (IIF), in France. A national external quality assessment (EQA) on ANA detection was organized by the French health products safety agency once a year since 1998. Between 606 to 687 laboratories together with six university reference laboratories experienced in performing tests in autoimmunity participated in the six-year consecutive survey. Each laboratory had to answer to methodological procedures and give coded responses. Variability in IIF methodological procedure was observed. Use of inappropriate microscope magnifications for reading slides or nonconventional cutoff dilution of serum were pointed out to concerned laboratories. Concerning ANA measurement, the rate of good responses ranged from 92.7% to 99.5% of the laboratories when the samples contained ANA. A wide dispersion of ANA titers obtained on a same sample was repeatly observed every year. Misinterpretation of particular fluorescence pattern was noticed. On ANA negative sample, the rate of good responses was 94.3%. In conclusion, ANA detection in routine practice is far from being standardized. However, EQA may have an impact on ANA detection performance when it is conducted on several consecutive year surveys, by providing advice for participating laboratories to limit inter laboratory variations related to methodological procedures.

[Analytic study of dot blotting for the detection of anti-Jo-1, anti-M2, anti-ribosomes and anti-LKM]. / Etude analytique d'un dot blot pour la détection des auto-anticorps anti-Jo-1, anti-M2, anti-ribosomes et anti-LKM1.

The Cyto-Dot 4 HM043 kit commercialised by BMD, has replaced the Cyto-Dot HM010 kit that allowed three auto-antibodies detection (anti-Jo-1, anti-M2 and anti-ribosomal protein). Detection of anti-LKM1 auto-antibody was added. These four auto-antibodies have in common only the intracytoplasmic localisation of their respective antigen. The aim of our study was to evaluate this new kit using 104 sera and to compare our results with reference techniques (indirect immunofluorescence IF for anti-M2, anti-ribosomal protein and anti-LKM1, double immunodiffusion ID for anti-Jo-1 and anti-LKM1, western blotting WB for anti-M2) and with Cyto-Dot HM010. The one hundred and four sera were divided into five groups: Group I (n = 12) with anti-Jo-1 detected by ID; Group II (n = 28) with 26 anti-M2 positive by IF and WB, 2 anti-M2 positive only by WB; Group III (n = 10) with anti-ribosomal protein detected by IF 5 of which precipitated by ID; Group IV (n = 32) with anti-LKM1 by IF and ID divided into 18 AIH2 and 14 HCV; Group V (n = 22) consisting of 14 healthy individuals and 8 patients with hypergammaglobulinemia. Results of this study are similar to those of Cyto-Dot HM010 for the three auto-antibodies already in use. Cyto-Dot 4 is a very good anti-LKM1 confirmation method as it is ID.

Human ninein is a centrosomal autoantigen recognized by CREST patient sera and plays a regulatory role in microtubule nucleation.

Centrosome is the major microtubule organizing center in mammalian cells that plays a critical role in a variety of cellular events by the microtubule arrays emanating from it. Despite its significance, the molecular mechanisms underlying the structure and function of the centrosome are still not clear. Herein we describe the identification of three isotypes of human ninein by expression library screening with autoimmune sera from CREST patients. All three ninein isotypes exhibit centrosomal localization throughout the cell cycle when GFP-tagged fusion proteins are expressed transiently in mammalian cells. Construction of serial deletions of GFP-tagged ninein reveals that a stretch of three leucine zippers with a flanking sequence is required and sufficient for centrosomal targeting. Overexpression of ninein results in mislocalization of gamma-tubulin, recruiting it to ectopic (noncentrosomal) ninein-containing sites which are not active in nucleating microtubules. In these cells, nucleation of microtubules from the centrosome is also inhibited. These results thus suggest a regulatory role for ninein in microtubule nucleation.

PTX3 in small-vessel vasculitides: an independent indicator of disease activity produced at sites of inflammation.

OBJECTIVE: To verify whether the prototypical long pentraxin PTX3 represents an indicator of the activity of small-vessel vasculitis. METHODS: Concentrations of PTX3, a pentraxin induced in endothelium by cytokines, were measured by enzyme-linked immunosorbent assay in the sera of 43 patients with Churg-Strauss syndrome, Wegener's granulomatosis, and microscopic polyangiitis. PTX3 was also measured in the sera of 28 patients with systemic lupus erythematosus (SLE), 22 with rheumatoid arthritis, and 16 with CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). Serum concentrations of C-reactive protein (CRP) were measured by immunoturbidimetry. The cells involved in PTX3 production in vivo were identified in skin biopsy samples. RESULTS: Patients with active vasculitis had significantly higher concentrations of PTX3 than did those with quiescent disease (P < 0.001). PTX3 levels in the latter group were similar to those in healthy controls. PTX3 levels were higher in patients with untreated vasculitis and lower in patients who underwent immunosuppressive treatments (P < 0.005). In contrast, patients with active SLE had negligible levels of the pentraxin. PTX3 levels did not correlate with CRP levels in vasculitis patients. Endothelial cells produced PTX3 in active skin lesions. CONCLUSION: PTX3 represents a novel acute-phase reactant produced at sites of active vasculitis.

Clinical, serological and genetic study in patients with CREST syndrome.

OBJECTIVE: To assess the clinical, serological and genetic features of Japanese patients with CREST syndrome. PATIENTS AND METHODS: Clinical features, autoantibodies and human histocompatibility leukocyte antigen (HLA) typing were studied in thirty patients with CREST syndrome, including 29 females and one male, with a mean age of 59.0 years (ranging from 40 to 76 years). RESULTS: Interstitial pneumonia on chest X-ray and renal involvement were rare. Mitral regurgitation and tricuspid regurgitation were present in 56.7% and 76.7%, respectively. Sjören's syndrome (SS) and primary biliary cirrhosis (PBC) were highly associated, however the positivity of the marker antibodies to those syndromes, such as anti-SSA, anti-SSB, anti-mitochondrial (AMA) and anti-smooth muscle autoantibodies were less frequent than that of primary SS and PBC without the other autoimmune diseases. The histological findings of PBC were all early stages in Scheuer's classification. HLA-Cw6 were associated with CREST-PBC overlap syndrome (p<0.05). However the HLA antigen was not correlated with CREST syndrome, and the frequency of HLA-DR2 between CREST syndrome with or without PBC was significantly different (p<0.01). CONCLUSION: It was suggested that there was a genetic difference between CREST syndrome alone and CREST-PBC overlap syndrome and there were differences (the positivity of AMA and the severity of bile duct lesion) between PBC and CREST-PBC overlap syndrome.

Kinetics of anti-fibrillin-1 autoantibodies in MCTD and CREST syndrome.

Using a highly sensitive Radioimmunoassay (RIA), the kinetics of synthesis of anti-fibrillin (Fbn-1) autoantibodies were studied in 17 patients with mixed connective tissue disease (MCTD) and two with CREST syndrome calcinosis, Raynaud's oesophageal dismotility, sclerodectyly and teleangiectasis who were found to be positive for this autoimmune response. IgG autoantibodies specific for recombinant Fbn-1 (rFbn-1) (aa 369-425) were found in all patients excepting one with MCTD, multiple sclerosis, and dermatomyositis. IgM were found in fewer cases. Several kinetics patterns of anti-Fbn-1 autoantibodies were observed: a) long lasting persistence of IgG and IgM autoantibodies up to 14 years; b) fluctuation of antibodies during various periods up to 16 years; c) disappearance of antibody response after several years, and d) patients producing IgG but not IgM autoantibodies. No differences in the synthesis of autoantibodies were observed between MCTD patients with a stable disease, and those developing during the course features of systemic sclerosis (SSc), Sjogren's syndrome, or rheumatoid-like arthritis. In one patient displaying a lupus-like syndrome for 3 years, the appearance of anti-Fbn-1 autoantibodies coincided with the occurrence of MCTD and scleroderma. While the detection of anti-Fbn-1 autoantibodies may be clinically useful in differential diagnosis or eventual prognosis of patients with connective tissue diseases, their role in the pathogenesis of scleroderma syndromes requires further investigation.

[CREST syndrome presenting as pulmonary hypertension]. / Hypertension artérielle pulmonaire révélatrice d'un CREST syndrome.

The authors report the case of a 62-year-old patient complaining of recent onset of disabling breathlessness on exertion, and presenting clinical signs of previously undiagnosed scleroderma. Echocardiography revealed a diagnosis of precapillary pulmonary hypertension (74/14 mmHg) (PHT), with no pulmonary cause revealed by pulmonary ventilation-perfusion scintigraphy or by thoracic fine section computed tomography. The diagnosis of PHT in the context of circumscribed scleroderma was confirmed by x-rays of the hands, capillaroscopy, oesophageal investigations and positive anticentromere antinuclear antibodies. The clinical course was marked by rapid deterioration of the symptoms, requiring treatment with prostacyclin by continuous intravenous infusion. The appearance of PHT in a context of circumscribed scleroderma, usually a relatively benign disease, is a rare, late event, exceptionally revealing the disease, as in this case, and indicating a very unfavourable prognosis.

A novel cell-cycle-dependent 350-kDa nuclear protein: C-terminal domain sufficient for nuclear localization.

We have screened human scleroderma patients for immunoreactivity with the components of the nucleus and the mitotic apparatus. We announce the identification of a novel cell-cycle-dependent nuclear protein using serum from a CREST patient AH. AH protein first appears at the nucleus of G2-phase and associates with the centrosome throughout the cell cycle. As chromosomes condense during the prophase, AH protein becomes enriched at the kinetochores. During mitosis, AH protein progressively disperses from the kinetochore and becomes diffusely localized in the cytoplasm and in telophase; it appears to be enriched within the intracellular bridge. Molecular cloning and transfection studies reveal that the 350-kDa AH protein contains a coiled-coil and a globular domain at the C-terminus that is sufficient for nuclear localization.

C-band-positive double minutes have kinetochore proteins.

Double minutes (DMs) have been shown generally to lack centromeres. In the present paper we report on C-band-positive DMs in Colcemid-resistant SEWA cells. These DMs were heavily labeled after in situ hybridization with mouse major satellite DNA and, furthermore, reacted positively with antikinetochore antibodies. Upon antikinetochore antibody labeling, the immunofluorescent signals of some DMs were strong, indicating that they had functional centromeres.

Increased mitogenic activity of scleroderma serum: inhibitory effect of human recombinant interferon-gamma.

OBJECTIVES: To investigate the role of platelet activation in the development of systemic sclerosis and the role of interferon-gamma (IFN gamma) in the inhibition of mitogenic activity induced by whole blood serum of patients with systemic sclerosis. METHODS: The mitogenic activity of whole blood serum in the absence or presence of different concentrations of IFN gamma (a potent inhibitor of induced collagen synthesis in dermal fibroblasts) and platelet-poor plasma derived serum were tested on human dermal fibroblasts by measuring incorporation of [3H]thymidine. Platelet activation was determined by quantification of plasma beta-thromboglobulin (beta-TG) using a beta-TG radioimmunoassay kit. RESULTS: The mitogenic activity was significantly increased in whole blood serum and in platelet-poor plasma derived serum of the patients compared with controls. In contrast, no significant increase in beta-TG concentration was observed in scleroderma platelet-poor plasma compared with control. Recombinant human IFN gamma had a greater inhibitory effect on the mitogenic activity induced by whole blood serum of patients than on that produced with control sera, at any concentration of IFN gamma tested. CONCLUSIONS: Our results suggest that mitogenic activity observed in the plasma of sclerodermic patients could originate from cells other than platelets and could be involved in the development of fibrosis. The potent inhibitory effect of IFN gamma on this proliferative activity may account for the beneficial effect of this cytokine in the treatment of progressive systemic sclerosis.

Analysis of centromere structure in the fly Megaselia scalaris (Phoridae, Diptera) using CREST sera, anti-histone antibodies, and a repetitive DNA probe.

We have used CREST anti-centromere sera, rabbit anti-histone antibodies, and repetitive DNA analysis to study centromere structure in the fly Megaselia scalaris (Phoridae). In a panel of eight CREST sera, four were positive in immunofluorescence experiments for prekinetochores, ie centromeres in interphase nuclei. The access of the antibodies to CREST antigens may be compromised in the condensed state, since centromeres of prometaphase and metaphase chromosomes remained unstained. When Western blots of embryonic nuclei were probed with these four CREST sera, three of them showed a 17 kDa band. Human CENP-A, likewise recognized by the CREST sera, is a 17 kDa protein. The remaining sera were negative for centromeres although some detected centrosomes and non-histone chromosomal proteins not confined to the centromeres. The use of antibodies generated against histone H4 acetylated at four different sites of the N-terminal domain revealed that heterochromatic regions of M scalaris mitotic chromosomes, ie pericentric and NOR-associated segments, are hyperacetylated. This is at variance with a variety of other systems, where transcriptionally active chromatin is hyperacetylated. Finally, a repetitive 165 base pair fragment was isolated from genomic DNA of the fly and sequenced. An oligonucleotide from this sequence mapped to the centromere region of interphase nuclei and the pericentric regions of condensed chromosomes.

[Anti-centromere antibodies. Study of 67 positive sera]. / Anticorps anti-centromères. Etude de 67 sérums positifs.

From a series of 67 sera containing anticentromere antibodies we endeavoured to determine the principal clinical or biological peculiarities of these antibodies. The titers of anticentromere antibodies were usually high, with few differences between patients. Humoral immunity was frequently perturbed, with antinuclear autoantibodies (without anti-Scl 70), anti-mitochondria antibodies, rheumatoid factors, circulating immune complexes, etc. The disease predominated in women (97%) whose age and duration of symptoms varied considerably. The most frequent clinical manifestation noted in the 47 reports analyzed was Raynaud's phenomenon (93%) which in most cases (90%) was part of a complete or incomplete CREST syndrome. Telangiectasias, calcinosis and acrosclerosis were the main witnesses to the duration of these sclerodermas. Our findings were concordant with those of previous studies. However, the frequency of sicca syndrome (76%) was unexpected, and must be related to 2 laboratory results: the quasi-absence of anti-SSA and anti-SSB antibodies in our patients and the presence of two monoclonal immunoglobulins (IgM kappa and IgG lambda). There may be some degree of independence between the sicca syndrome and the sclerodermal manifestations.