Early Lupus Project: one-year follow-up of an Italian cohort of patients with systemic lupus erythematosus of recent onset.

Objective To describe the clinical and serological features of a prospectively followed cohort of early diagnosed systemic lupus erythematosus (SLE) patients during a one-year follow-up period. Methods SLE patients with disease duration less than 12 months were consecutively enrolled in a multicentre, prospective study. At study entry and then every 6 months, a large panel of data was recorded. Results Of 260 patients enrolled, 185 had at least 12 months of follow-up; of these, 84.3% were female, 92.4% were Caucasians. Mean diagnostic delay was about 20 months; higher values of European Consensus Lupus Activity Measurement (ECLAM) and of organs/systems involved were both associated with shorter diagnostic delay. Clinical and serological parameters improved after study entry. However, patients' quality of life deteriorated and cardiovascular risk factors significantly increased. About one-third of patients with active disease at study entry went into remission (ECLAM = 0). Negative predictors for remission were: oral ulcers, arthritis, low C4, anti-SSB (Ro) antibodies and therapy with mycophenolate. There was a widespread use of glucocorticoids both at baseline and during follow-up. Conclusion Clinical symptoms and serological parameters improve during the first period after diagnosis. However, patients' quality of life deteriorates. The widespread use of glucocorticoids is probably the reason for the early significant increase of some cardiovascular risk factors.

Morbidity and mortality in the antiphospholipid syndrome during a 10-year period: a multicentre prospective study of 1000 patients.

OBJECTIVES: To assess the prevalence of the main causes of morbi-mortality in the antiphospholipid syndrome (APS) during a 10-year-follow-up period and to compare the frequency of early manifestations with those that appeared later. METHODS: In 1999, we started an observational study of 1000 APS patients from 13 European countries. All had medical histories documented when entered into the study and were followed prospectively during the ensuing 10 years. RESULTS: 53.1% of the patients had primary APS, 36.2% had APS associated with systemic lupus erythematosus and 10.7% APS associated with other diseases. Thrombotic events appeared in 166 (16.6%) patients during the first 5-year period and in 115 (14.4%) during the second 5-year period. The most common events were strokes, transient ischaemic attacks, deep vein thromboses and pulmonary embolism. 127 (15.5%) women became pregnant (188 pregnancies) and 72.9% of pregnancies succeeded in having one or more live births. The most common obstetric complication was early pregnancy loss (16.5% of the pregnancies). Intrauterine growth restriction (26.3% of the total live births) and prematurity (48.2%) were the most frequent fetal morbidities. 93 (9.3%) patients died and the most frequent causes of death were severe thrombosis (36.5%) and infections (26.9%). Nine (0.9%) cases of catastrophic APS occurred and 5 (55.6%) of them died. The survival probability at 10 years was 90.7%. CONCLUSIONS: Patients with APS still develop significant morbidity and mortality despite current treatment. It is imperative to increase the efforts in determining optimal prognostic markers and therapeutic measures to prevent these complications.

Evaluation of the effect of Bosentan treatment on proinflammatory cytokine serum levels in patients affected by Systemic Sclerosis.

Systemic sclerosis (SSc) is a connective tissue disease characterized by vascular and fibrotic changes in the skin and in internal organs. Endothelin-1 (ET-1) is a peptide that has a role in promoting both vascular injury and the fibrotic process in SSc; indeed, patients with systemic sclerosis have higher levels of ET-1 compared with healthy subjects. Moreover, ET-1 enhances expression of pro-inflammatory cytokines in animal model. Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension and digital ulcers in scleroderma patients. In animal models and in vitro models, after treatment with Bosentan, a significant reduction of cytokine (TNF α, IFN γ,IL-8, IL-4) levels was observed. The aim of the study is to verify whether Bosentan treatment in SSc patients can reduce circulating cytokines levels. We enrolled 10 patients affected by SSc with digital ulcers and/or pulmonary hypertension, treated with Bosentan 125 mg twice daily. Patients were tested for cytokines and ET-1 level before treatment and after 12 months. The cytokines tested were IL-10, IL-2, IL-4, IL-5, IL-6, IL-8, GM-CSF, IFN-γ and TNF. Levels of ET-1, IL-10, IL-4, IL-5, GM-CSF and TNFalpha did not show consistent modification during treatment with Bosentan in respect to baseline, while IL-2, IL-6, IL-8 and IFN-γ were significantly decreased. Bosentan significantly reduced IL-2, IL-6, IL-8 and IFN- γ levels in SSc patients, probably slowing progression to fibrosis and vascular damage. This is the first report showing a decrease of profibrotic and proinflammatory cytokines levels in humans during treatment with Bosentan.

Validation of a diagnostic score for the diagnosis of autoinflammatory diseases in adults.

Most autoinflammatory disorders typically come out in the pediatric population, although a limited number of patients may experience disease onset during adulthood. To date, a late disease onset has been described only in familial Mediterranean fever, caused by mutations in the MEFV gene, and in tumor necrosis factor receptor-associated periodic syndrome, caused by mutations in the TNFRSF1A gene. The relative rarity and lack of information on adult-onset autoinflammatory diseases make it likely that mutations will be found in an even smaller percentage of cases. With the aim of improving the genetic diagnosis in adults with suspected autoinflammatory disorders, we recently identified a set of variables related to the probability of detecting gene mutations in MEFV and TNFRSF1A and, in addition, we have also proposed a diagnostic score for identifying those patients at high risk of carrying mutations in these genes. In the present study we evaluated the preliminary score sensitivity and specificity on a wider number of patients in order to validate the goodness of fit of the model. Two hundred and nineteen consecutive patients with a clinical history of periodic fever attacks were screened for mutations in MEFV and TNFRSF1A genes; detailed information about family/personal history and clinical manifestations were also collected. For the validation of the score we considered data both from the 110 patients used to build the preliminary diagnostic score and from the additional 219 patients enrolled in the present study, for a total number of 329 patients. Early age at disease onset, positive family history for recurrent fever episodes, thoracic pain, abdominal pain and skin rash, which are the variables that had previously been shown to be significantly associated with a positive genetic test result (12), were used for validation. On univariate analysis the associations with a positive genetic test were: age at onset (odds ratio [OR] 0.43, p=0.003), positive family history for recurrent fever episodes (OR 5.81, p<0.001), thoracic pain (OR 3.17, p<0.001), abdominal pain (OR 3.80, p<0.001) and skin rash (OR 1.58, p=0.103). The diagnostic score was calculated using the linear combination of the estimated coefficients of the logistic multivariate model (cut-off equals to 0.24) revealing good sensitivity (0.778) and good specificity (0.718). In conclusion, our score may serve in the diagnostic evaluation of adult patients presenting with recurrent fever episodes suspected of having an autoinflammatory disorder, helping identify the few subjects among them who may be carriers of mutations in MEFV and TNFRSF1A genes.

Morbidity and mortality in the antiphospholipid syndrome during a 5-year period: a multicentre prospective study of 1000 patients.

OBJECTIVES: To identify the main causes of morbidity and mortality in patients with antiphospholipid syndrome (APS) during a 5-year period and to determine clinical and immunological parameters with prognostic significance. METHODS: The clinical and immunological features of a cohort of 1000 patients with APS from 13 European countries who had been followed up from 1999 to 2004 were analysed. RESULTS: 200 (20%) patients developed APS-related manifestations during the 5-year study period. Recurrent thrombotic events appeared in 166 (16.6%) patients and the most common were strokes (2.4% of the total cohort), transient ischaemic attacks (2.3%), deep vein thromboses (2.1%) and pulmonary embolism (2.1%). When the thrombotic events occurred, 90 patients were receiving oral anticoagulants and 49 were using aspirin. 31/420 (7.4%) patients receiving oral anticoagulants presented with haemorrhage. 3/121 (2.5%) women with only obstetric APS manifestations at the start of the study developed a new thrombotic event. A total of 77 women (9.4% of the female patients) had one or more pregnancies and 63 (81.8% of pregnant patients) had one or more live births. The most common fetal complications were early pregnancy loss (17.1% of pregnancies) and premature birth (35% of live births). 53 (5.3% of the total cohort) patients died. The most common causes of death were bacterial infection (21% of deaths), myocardial infarction (19%) and stroke (13%). No clinical or immunological predictor of thrombotic events, pregnancy morbidity or mortality was detected. CONCLUSION: Patients with APS still develop significant morbidity and mortality despite current treatment (oral anticoagulants or antiaggregants, or both).

Safety and efficacy of etanercept in children with juvenile-onset Behcets disease.

Behcets disease (BD) is a chronic, relapsing, multi-system inflammatory disorder, clinically characterized by recurrent oral and genital ulcers, skin lesions, and uveitis. Other manifestations include arthritis, a positive pathergy test, thrombophlebitis, central nervous system disease and gastrointestinal ulcerations. The majority of affected individuals do not have life-threatening disease, although mortality can be associated with vascular-thrombotic and neurological manifestations. Currently, treatment of BD is symptomatic and empirical, and is tailored according to the severity of clinical features. In the past few years, isolated reports and case-series have been published on adult BD patients suggesting that inhibition of TNF-alpha is a promising therapeutic approach for severe ocular and various extra-ocular manifestations, including central nervous system involvement. In this study we present our promising experience with Etanercept therapy in juvenile-onset BD patients, characterized by refractory multiorgan involvement.

Efficacy and safety of anti-TNF-alpha therapy combined with cyclosporine A in patients with rheumatoid arthritis and concomitant hepatitis C virus infection.

This study further expands our previous observation demonstrating the usefulness of combination therapy of anti-TNF-alpha and cyclosporine A in the treatment of rheumatoid arthritis and concurrent hepatitis C virus infection, as well its efficacy and safety in controlling HCV viremia and liver toxicity. Seven patients were included in the study; transaminase levels remained unchanged, HCV RNA serum levels decreased significantly and DAS 28 significantly improved after twelve month follow-up. No side effects were registered.

Idiopathic recurrent pericarditis refractory to colchicine treatment can reveal tumor necrosis factor receptor-associated periodic syndrome.

Recurrences develop in up to 20-50% of patients with acute pericarditis. Although different causes of recurrent pericarditis have been identified, the etiology remains obscure in most cases which are therefore labelled as idiopathic. Autoinflammatory syndromes include familial Mediterranean fever (FMF), due to mutations in the MEFV gene, and tumor necrosis factor receptor-associated periodic syndrome (TRAPS), due to mutations in the TNFRSF1A gene. Recurrent pericarditis is a common feature of both conditions, but it rarely occurs alone. Colchicine is the standard treatment for FMF, while patients with TRAPS do not respond to colchicine therapy, but are responsive to corticosteroids. Based on the proven efficacy of colchicine in preventing polyserositis in FMF, colchicine has been proposed for the treatment of recurrent pericarditis and is able to decrease the recurrence rate. Our aim was to investigate the possible involvement of TNFRSF1A mutations in a group of patients with idiopathic recurrent pericarditis who were refractory to colchicine treatment. Thirty consecutive patients (17 males, 13 females) diagnosed with idiopathic recurrent pericarditis, who were characterized by a poor response to colchicine treatment, were enrolled in the study. Mutations of the TNFRSF1A gene were searched for by amplifying, using polymerase chain reaction (PCR), genomic DNA, and direct sequencing. TNFRSF1A mutations were found in 4 of the 30 patients. None of these 4 patients had a family history of recurrent inflammatory syndromes or history of pericarditis. One of the 4 patients had a novel heterozygous deletion (DeltaY103-R104) and three patients carried a heterozygous low-penetrance R92Q mutation. Our data suggest that TRAPS should be kept in mind in the differential diagnosis of recurrent pericarditis, and mutation analysis of the TNFRSF1A gene should be considered, in addition to MEFV analysis, in patients of Mediterranean origin. A poor response to colchicine treatment and/or a steroid-dependence may be the clue to investigate TNFRSF1A mutations in patients with idiopathic recurrent pericarditis.

[Viral genotype and HLA class II alleles influence on extra-hepatic manifestations of chronic HCV infection]. / Associazione tra genotipo virale, HLA e manifestazioni extra-epatiche dell'epatite C.

OBJECTIVE: To test whether an association between HCV genotype, HLA class II alleles distribution and extra-hepatic manifestations (EHM ) can be demonstrated in a group of Italian patients with chronic HCV infection . METHODS: Sixty patients affected by HCV infection with EHM were consecutively enrolled. 163 HCV patients without EHM were tested as controls for the prevalence of HCV genotypes, while we referred to literature as to the controls for HLA distribution. HCV-RNA was quantified by a RT-PCR. HLA class II alleles typing was performed using a standard microlymphocytotoxicity assay. We used chi-square or Fisher test (p<0.05 significant). Odds Ratio (OR) was performed by 2X2 contingency table. RESULTS: HCV 2c genotype was found in 63.46% of patients compared to 19.63% of controls (p<0.0001; OR=7.11). Furthermore, it correlated with carpal tunnel syndrome (p=0.03; OR=4.5) and autoimmune thyroiditis (p=0.02; OR=9.2). On the contrary, 1b genotype protected from EHM in toto (p=0.0004; OR=0.21) and particularly from carpal tunnel syndrome (p=0.0014; OR=0.07). Moreover, 3a genotype prevented HCV people from having cryoglobulinemia (p=0.05; OR=0.11). As to HLA, DR6 seemed to facilitate EHM in HCV patients (p=0.041; OR=1.61), while DQ2 (p=0.03; OR=0.5) and DQ3 (p=0.002; OR= 0.5) may play a protective role. In addition, HLA DR3 was associated with cryoglobulinemia (p=0.02; OR=9.5). CONCLUSIONS: According to our findings, 2c genotype can be considered as a major risk factor for developing HCVrelated EHM, while 1b genotype seems to prevent their onset; there are also evidences suggesting that HLA might play a role in chronic HCV infected patients.

Low serum level of COMP, a cartilage turnover marker, predicts rapid and high ACR70 response to adalimumab therapy in rheumatoid arthritis.

The aim of this study was to evaluate serum biomarkers, used in clinical routine, to predict the American College of Rheumatology (ACR) response to long-term anti-TNF alpha treatment (adalimumab). Sera from 29 consecutive rheumatoid arthritis patients were analysed for anti-cyclic citrullinated peptide (anti-CCP), cartilage oligomeric matrix protein (COMP) and IgM and IgA RFs (class-specific rheumatoid factors) at the start of treatment with adalimumab and after 3, 6 and 12 months. The response to the therapy was evaluated by ACR 20, 50, 70 and by DAS 28 scores. The mean serum COMP level of the population did not change after treatment. However, patients with low serum COMP levels (<10 U/l) at baseline showed a significant (p<0.02) higher ACR70 response (>50%) within 3 months, and also at 6 months, than patients with higher COMP values (ACR70<20%). This was also reflected by significantly higher decrease in DAS score at 3 (p<0.02) and 6 months (p<0.01) treatments. The IgM RF titre decreased significantly (p=0.02) after the therapy, but the percentage of serum positivity for anti-CCP and IgA/IgM RF did not change. No significant correlation was shown between serum COMP levels and C-reactive protein/erythrocyte sedimentation rate during the follow-up. Neither were any correlations shown between ACR/DAS 28 scores and anti-CCP, Ig M/IgA RFs. Our data indicate that low (<10 U/l) serum COMP before starting anti-TNF alpha treatment predicts a rapid (within 3 months) and high ACR70 response compared to RA patients with higher COMP values. This might reflect different mechanisms in the cartilage process in the RA disease at that time of treatment with different therapeutic sensitivity to anti-TNF alpha treatment.

Systemic lupus erythematosus in Europe at the change of the millennium: lessons from the "Euro-Lupus Project".

The "Euro-Lupus Cohort" is composed by 1000 patients with systemic lupus erythematosus (SLE) that have been followed prospectively since 1991. These patients have been gathered by a European consortium--the "Euro-Lupus Project Group". This consortium was originated as part of the network promoted by the "European Working Party on SLE", a working group created in 1990 in order to promote research in Europe on the different problems related to this disease. The "Euro-Lupus Cohort" provides an updated information on the SLE morbidity and mortality characteristics in the present decade as well as defines several clinical and immunological prognostic factors.

Association of extrahepatic manifestations with HLA class II alleles and with virus genotype in HCV infected patients.

It has been postulated that host factors, such as the human leucocyte antigen (HLA) system, may play a predominant role in the pathogenesis of HCV-related extra-hepatic manifestations. This study was performed to investigate the role of HLA- DR and DQ alleles in a group of Italian patients, with HCV infection and associated extrahepatic manifestations and to test whether an association between HCV genotype, HLA locus and clinical or serological manifestations can be demonstrated. Thirty unrelated patients affected by HCV infection with extra-hepatic manifestations were consecutively included in the study. One hundred and sixty-three HCV patients without extrahepatic manifestations were tested as controls for the prevalence of HCV genotypes, and 283 healthy donors were used as controls for HLA class II alleles distribution. HCV-RNA was quantified by an reverse transcription-PCR. HLA class II alleles typing was performed using a standard microlymphocytotoxicity assay on B lymphocyte purified. HCV 2c genotype was found in 53.3% compared to 18.4% of controls (p=0.00001; OR=5.1). Cryoglobulins were detected in 72.7% DR6+ patients and in 31.6% DR6- patients (p=0.05; OR=3.21). Rheumatoid factor was found in 90.9% of DR6+ patients and in 42.1% DR6- patients (p=0.018; OR 13.7). Only two DR5+ patients (20%) had cryoglobulinemia, while 6 patients (30%) in the DR5- group had cryoglobulinemia (p=0.02; OR=0.07). Associations were found between DR7 and ANA (OR=1.74) and between DQ2 and ANA (OR=1.97). According to our findings HLA-DR6 might play an important role in developing extra-hepatic manifestations and genotype 2c could be considered as a risk factor for their onset.

Dosage and characterization of circulating DNA: present usage and possible applications in systemic autoimmune disorders.

The discovery of extracellular nucleic acids in the circulation was firstly reported in 1948. In the last few years it has been demonstrated that the entire spectrum of genetic changes seen in primary tumors could also be detected in the serum of patients with solid tumors. This observation has also opened up exciting possibilities for tumor detection and monitoring. More recently investigators started looking for other forms of non-host DNA in the plasma/serum so that in 1997 the presence of fetal DNA in the plasma/serum of pregnant women was demonstrated. This finding suggested that maternal plasma fetal DNA would be a very valuable material for noninvasive prenatal diagnosis and monitoring. It has been also postulated that the presence of the two-way trafficking of nucleated cells and free DNA between the mother and fetus may have potential implications for the development of certain autoimmune diseases. Concerning autoimmune disorders, Tan was the first author to describe the presence of high levels of circulating DNA in patients with systemic lupus erythematosus (SLE) in 1986. Later on different authors demonstrated that elevated levels of serum DNA was also present in patients with other diseases including rheumatoid arthritis. We have analyzed both circulating free DNA and DNA extracted from nucleated blood cells in scleroderma and in lupus patients but, by using gel electrophoresis, we were able to define the pattern of the DNA, instead of simply dosing its amount in the circulation. We have found that SLE and SSc have anomalous patterns of DNA both in serum and in the Buffy-coat and that these patterns are typical for each disorder. It is possible that understanding the biological significance of the diversity in DNA pattern exhibition in white blood cells may give new insights into the pathophysiology of autoimmune disorders. It is also conceivable that circulating and immune-competent cellular DNA markers might offer the promise of precise quantitative analysis useful for diagnostic purposes, without the need to establish difficult cutoffs as is necessary for protein markers.

Comparison of different methods for the detection of anti-Ro/SSA antibodies in connective tissue diseases.

OBJECTIVE: To compare the performance characteristics of various tests commonly used to detect anti-SSA/Ro autoantibodies in the sera of patients affected by connective tissue diseases (CTD). METHODS: Indirect immunofluorescence (IIF) with HEp-2000 as substrate (ImmunoConcepts, USA), Ouchterlony's double immunodiffusion (ID) (home made), commercial Varelisa ReCombi anti-Ro kit (Pharmacia & Upjohn, Germany), research kits (60 kDa and 52 kDa) with human recombinant antigens (Pharmacia & Upjohn, Germany) and a commercial western blot (WB) kit (MarDx, USA) were evaluated in our study. Sixty-four sera from patients affected by CTD were tested: 15 had primary Sjögren's syndrome (SS), 34 only had sicca syndrome, and 15 had systemic lupus erythematosus (SLE). Thirty sera from healthy subjects were selected as controls. RESULTS: 54 sera were positive by at least one method. The specificity of all tests was good. The prevalence of anti-SSA antibodies on 54 positive sera was 76% (ID), 89% (IIF), 89% (Varelisa), 89% (ELISA Ro-60 kDa), 67% (ELISA Ro-52 kDa) and 85% (WB). Some differences were found between WB and ELISA in the detection of anti-60 kDa SSA and anti-52 kDa SSA; in 3 SS sera only anti-52 kDa protein was found by WB. CONCLUSION: Our data confirm that, although IIF HEp 2000 (Immuno Concepts) and Varelisa anti-Ro (Pharmacia & Upjohn) both performed well, a combination of 2 or more methods must still be recommended for anti-SSA antibody detection.

Association of 16/6 and SA1 anti-DNA idiotypes with anticardiolipin antibodies and clinical manifestations in a large cohort of SLE patients. European Concerted Action on the Immunogenetics of SLE.

OBJECTIVE: The SA1 and 16/6 idiotypes can be found in some patients with systemic lupus erythematosus (SLE) or anti-phospholipid syndrome (APS). The aim of this study was to ascertain the prevalence of these idiotypes in a large cohort of SLE patients, and to determine whether their presence is correlated with the anticardiolipin (aCL) and anti-dsDNA antibodies or with the clinical manifestations of SLE. METHODS: 492 SLE patients were evaluated for clinical manifestations of SLE and were assigned a disease severity score. ds-DNA autoantibodies, aCL autoantibodies of the IgM, IgG and IgA isotypes, and SA1 and 16/6 idiotypes were also determined in these patients. RESULTS: The prevalence of the SA1 and 16/6 idiotypes in the 492 SLE patients was found to be 11% and 5.1%, respectively, and these idiotypes were significantly more prevalent in the patients who had aCL antibodies of either the IgG, IgM or IgA isotypes. Moreover, while the 16/6 idiotype was not associated with the clinical manifestation of either SLE or APS, the SA1 idiotype was found significantly more frequently in patients who had vascular events Raynaud's phenomenon or hemolytic anemia (p = 0.016, 0.01, 0.01, respectively). CONCLUSION: SLE patients with the SA1 idiotype may run a higher risk of developing vascular events, Raynaud's phenomenon or hemolytic anemia. These clinical manifestations can be attributed to both SLE and secondary APS when aCL autoantibodies are also found. These results indicate that the possible pathogenicity of certain idiotypes in SLE cannot be excluded.

Anti-neutrophil cytoplasmic antibodies in 566 European patients with systemic lupus erythematosus: prevalence, clinical associations and correlation with other autoantibodies. European Concerted Action on the Immunogenetics of SLE.

OBJECTIVES: To evaluate, in a cohort of 566 patients with systemic lupus erythematosus (SLE) drawn from 11 European centres: (i) the prevalence of ANCAs and their subspecificities in a large series of European SLE patients; (ii) the possible associations of ANCA with the most common clinical manifestations of the disease; and (iii) whether ANCAs correlate with some of the autoantibodies commonly found in SLE. METHODS: ANCA detection was performed by indirect immunofluorescence (IIF), and by ELISA for lactoferrin (LF), myeloperoxydase (MPO), proteinase3 (PR3) and lysozyme (LZ) subspecificities. RESULTS: The prevalence of ANCA was 16.4% (IIF). The prevalence of LF was 14.3%, LZ 4.6%, MPO 9.3%, and PR3 1.7%. Our results show that ANCA is associated with certain clinical manifestations of SLE. In particular, positive correlations were found between IIF ANCA and serositis (p = 0.026), livedo reticularis (p = 0.01), venous thrombosis (p = 0.03) and arthritis (p = 0.04), while anti-LF antibodies were associated with serositis (p = 0.05) and livedo reticularis (p < 10(-3). Nevertheless, multivariate analysis demonstrated that other autoantibodies, such as aCL and SSA/Ro, are more closely correlated than ANCA with some of the aforementioned clinical features. CONCLUSION: Our results demonstrate that ANCA are detectable in SLE sera and that some of them are associated with particular clinical manifestations. Whether ANCA plays a direct pathogenetic role in the vascular damage of SLE or only represents an epiphenomenon or a marker of disease activity remains to be elucidated.

[Estrogens and cartilage: an ex vivo ultrastructural study]. / Estrogeni e cartilagine: studio ultrastrutturale ex vivo.

The ultrastructural change brought about by administration of oestrogens on articular cartilage were evaluated in the course of the present study. To this end 3 stabilized postmenopausal patients were selected who were to undergo bilateral hip prosthesis. After the first operation the patients were started on continuous estriol treatment by oral route for 6 months. For study purposes a cartilage fragment was obtained during the first (baseline time) operation and the second operation (final time, after 6 months of oestrogen therapy). The results of histologic examination of the semifine sections as well as the submicroscopic patterns of the various cell elements showed great similarity in all cases considered. Ultrastructure examination had evidenced signs of marked cell damage in the 3 initial samples. After treatment with oestrogens, chondrocyte metabolic activity was resumed, evidenced by the presence of extensive granular endoplasmic reticulum with hypertrophic cisternae, finely dispersed nuclear chromatin and more regular nuclear membrane. These findings would appear to support metabolic stimulation of articular cartilage by the oestrogens.

[Chromosome aberrations, valued as frequency of spontaneous micronuclei, in subjects with suspected presclerodermic Raynaud's phenomenon]. / Anomalie cromosomiche, valutate come frequenza di micronuclei spontanei, in soggetti con fenomeno di Raynaud sospetto presclerodermico.

OBJECTIVE: To evaluate the prevalence of spontaneous chromosome damage in cultured peripheral lymphocytes of subjects with suspected presclerodermic Raynaud's phenomenon (RP), by means of molecular cytogenetic analysis. METHODS: We studied 20 suspected presclerodermic RP, 20 idiopathic RP and 25 healthy subjects. As marker of chromosome alteration we used the micronucleus assay. All subjects were also classified as ANA-, ACA+ or Scl70+. To identify the mechanism of MN formation, a MN fluorescence in situ hybridisation (FISH) analysis using a pancentromeric DNA probe was also performed. RESULTS: Suspected presclerodermic RP subjects, showed significantly higher MN frequencies than idiopathic RP and controls (39+/-15.2 vs 10+/-2.1 and 9.8+/-3.5 respectively p<0.0001). Interestingly, subjects with idiopathic RP displayed MN frequency comparable to that of controls. Furthermore, ACA+ subjects showed the highest MN frequencies (44+/-8.1) as compared to subjects with different antibody pattern (26+/-7.1). CONCLUSIONS: Our results show the presence of higher levels of chromosomal damage in circulating lymphocytes of suspected presclerodermic RP. They also would suggest a key role of anti-centromere antibody in determining the observed cytogenetic anomalies. FISH analysis indicated that both aneuploidogenic and clastogenic events contribute to the formation of MN observed in suspected presclerodermic RP.

[Anti-proteinase 3 antibodies in diffuse systemic sclerosis (SSc) with normotensive renal impairment: is it suggestive for an overlapping between SSc and idiopathic vasculitis? ] / Anticorpi anti proteinasi 3 nella sclerosi sistemica (ScS) diffusa con impegno renale normotensivo: possono essere suggestivi di una sovrapposizione tra ScS e vasculite idiopatica?

OBJECTIVE: To test the prevalence of anti-neutrophil cytoplasmic antibodies (ANCA) in systemic sclerosis (SSc) and to verify a possible association of ANCA with normotensive renal involvement in SSc. PATIENTS AND METHODS: 51 patients affected by SSc, 35 with diffuse scleroderma (dSSc) and 16 with limited scleroderma (lSSc), were tested for ANCA by indirect immunofluorescence (IIF) on human ethanol and formalin-acetone-fixed granulocytes (before and after DNase treatment), by conventional enzyme linked immuno-sorbent assay (ELISA) and by capture-ELISA. RESULTS: Six out of 51 selected SSc patients had ANCA by IIF (11.7%) and five presented a perinuclear/nuclear atypical ANCA pattern. In all cases we only found anti-proteinase3 (aPR3) antibodies. All ANCA positive patients had diffuse form of SSc (17.1%), all were anti-Scl70 positive (aScl70), five patients had proteinuria, three had microscopic haematuria. All ANCA positive patients were normotensive with normal renin plasma levels, the mean erythrocyte sedimentation rate (ESR) was higher in this group compared to the other SSc patients. CONCLUSIONS: Our study shows that aPR3 is not rare in dSSc. According to the clinical and serological findings and to the recent literature, we can hypothesise that when ANCA are found in SSc, an overlapping of scleroderma with systemic necrotizing vasculitis should be suspected.