17-ß-estradiol affects BLyS serum levels and the nephritogenic autoantibody network accelerating glomerulonephritis in NZB/WF1 mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects fertile women, suggesting sex hormones are involved in disease pathogenesis. B lymphocyte stimulator (BLyS) has been found to be elevated in SLE patients and to drive a lupus-like syndrome in transgenic mice. Our aim was to evaluate the effects of estrogen administration on BLyS and nephritogenic anti-C1q and anti-dsDNA antibodies in lupus-prone NZB/WF1 mice. We implanted pellets releasing 17-ß-estradiol (18.8 µg/day) on the back side the ear of 10 NZB/WF1 mice (group 1), and compared them with 10 mice intraperitoneally injected with PBS 200 µl twice a week (group 2), as controls. We evaluated BLyS, anti-dsDNA and anti-C1q serum levels starting one week after pellet implantation. We also analyzed time to proteinuria onset, proteinuria-free survival and overall survival. Kidneys, spleen, liver and lungs were harvested for histological analysis. Mice were bred until natural death. BLyS serum levels were higher in group 1 than in group 2 mice at each evaluation. Group 1 mice developed nephritogenic antibodies and proteinuria significantly earlier and at higher levels than controls. Direct correlation between BLyS and anti-C1q (R (2 )= 0.6962, p < 0.0001) or anti-dsDNA (R (2 )= 0.5953, p < 0.0001), and between anti-C1q and anti-dsDNA autoantibodies (R (2 )= 0.5615, p < 0.0001) were found. Proteinuria-free and global survival rates were significantly lower in group 1 than in controls. Histological analyses showed more severe abnormalities in group 1 mice. Estrogen administration is associated with increased levels of BLyS as well as of anti-C1q and anti-dsDNA antibodies, leading to accelerated glomerulonephritis and disease progression in NZB/WF1 mice.

The interferon in idiopathic inflammatory myopathies: Different signatures and new therapeutic perspectives. A literature review.

Idiopathic inflammatory myopathies (IIM), even though sharing common clinical manifestations, are characterized by diversified molecular pathogenetic mechanisms which may account for the partial inefficacy of currently used immunomodulatory drugs. In the last decades, the role of interferon (IFN) in IIM has been extensively elucidated thanks to genomic and proteomic studies which have assessed the molecular signature at the level of affected tissues or in peripheral blood across distinct IIM subtypes. A predominant type I IFN response has been shown in dermatomyositis (DM), being especially enhanced in anti-melanoma differentiation-associated gene 5 (MDA5)+ DM, while a type 2 IFN profile characterizes anti-synthetase syndrome (ASyS) and inclusion body myositis (IBM); conversely, a less robust IFN footprint has been defined for immune-mediated necrotizing myopathy (IMNM). Intracellular IFN signaling is mediated by the janus kinase/signal transducer and activator of transcription (JAK/STAT) through dedicated transmembrane receptors and specific cytoplasmic molecular combinations. These results may have therapeutic implications and led to evaluating the efficacy of new targeted drugs such as the recently introduced janus kinase inhibitors (JAKi), currently approved for the treatment of rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. In this review we aim to summarize the most significant evidence of IFN role in IIM pathogenesis and to describe the current state of the art about the ongoing clinical trials on IFN-targeting drugs, with particular focus on JAKi.

Induction of the 'ASIA' syndrome in NZB/NZWF1 mice after injection of complete Freund's adjuvant (CFA).

Adjuvants, commonly used in vaccines, may be responsible for inducing autoimmunity and autoimmune diseases, both in humans and mice. The so-called 'ASIA' (Autoimmune/inflammatory Syndrome Induced by Adjuvants) syndrome has been recently described, which is caused by the exposure to a component reproducing the effect of adjuvants. The aim of our study was to evaluate the effect of injection of complete Freund's adjuvant (CFA) in NZB/NZWF1 mice, a lupus-prone murine model. We injected 10 NZB/NZWF1 mice with CFA/PBS and 10 with PBS, three times, 3 weeks apart, and followed-up until natural death. CFA-injected mice developed both anti-double-stranded DNA and proteinuria earlier and at higher levels than the control group. Proteinuria-free survival rate and survival rate were significantly lower in CFA-treated mice than in the control mice (p = 0.002 and p = 0.001, respectively). Histological analyses showed a more severe glomerulonephritis in CFA-injected mice compared with the control mice. In addition, lymphoid hyperplasia in spleen and lungs, myocarditis, and vasculitis were observed in the former, but not in the latter group. In conclusion, the injection of CFA in NZB/NZWF1 mice accelerated autoimmune manifestations resembling 'ASIA' syndrome in humans.

Vaccination of mice for research purpose: alum is as effective as and safer than complete Freund adjuvant.

Systemic lupus erythematosus (SLE) is an autoimmune disease involving many organ systems. Glomerulonephritis (GLN) is one of the major causes of morbidity and mortality in SLE. It has recently been demonstrated that adjuvants of vaccines could cause the so called ASIA syndrome. The study aimed to assess the effects of Complete Freund's Adjuvant (CFA) vs alum injections in NZB/NZWF1 mice. Mice (n=10 each group) were injected with a total volume of 200 µL of: CFA in PBS (group 1), alum in PBS (group 2), PBS (group 3) as controls, PTX3/CFA (group 4), PTX3/alum (group 5), 3 times, 3 weeks apart /given in each injection, three weeks apart from ten weeks of age. Urine samples were collected weekly to evaluate proteinuria. Blood samples were collected before every injection, at 21 weeks of age, and at death to evaluate levels of anti-PTX3 and anti-dsDNA. Proteinuria free survival and survival rates were analyzed by the Kaplan-Meier method using Mantel-Cox's test for comparisons. CFA-treated mice developed both anti-dsDNA antibodies and proteinuria earlier and at higher levels than alumtreated and PBS-injected mice, starting from 13 weeks of age. Proteinuria free survival rates (proteinuria ≥ 300 mg/dL) and survival rates were lower in CFA-treated mice than those treated with alum or injected with PBS (P<0.001 for all). No difference was observed between the alum-treated group and PBS-injected mice. Notably, groups 4 and 5, immunized with PTX3, developed anti-PTX3 antibodies and no significant difference was observed. Alum seems to be as effective as and safer than CFA as adjuvant, since it did not affect disease progression in immunized NZB/NZWF1 mice.

Polarization of TH2 response is decreased during pregnancy in systemic lupus erythematosus.

This study evaluated some cytokines involved in the Th1-Th2 shift during pregnancy in patients with systemic lupus erythematosus (SLE) and healthy women. Twenty-seven consecutive successful pregnancies in 26 SLE patients and 28 pregnancies in 28 matched healthy subjects, as controls, were enrolled and prospectively studied. Sera obtained at first and third trimesters of pregnancy were tested for IL-1α, IL-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, INF-γ, and TNF-α with a highly sensitive, multiplexed sandwich ELISA (SearchLight Human Inflammatory Cytokine Array). Statistics were performed by SPSS package. IL-8 serum levels were higher in the first (P<0.0001) and third (P=0.003) trimesters of pregnancy in SLE patients compared with controls, INF-γ serum levels in the third trimester (P=0.009), and IL-10 serum levels in the first and third trimesters (P=0.055 and P<0.0001, respectively). IL-2 (r=0.524 P=0.010), IL-12 (r=0.549 P=0.007), IFN-γ (r=0.492 P=0.017), and IL-6 (r=0.515 P=0.020) serum levels correlated with disease activity in SLE patients in the first trimester of pregnancy. Cytokine profile was similar in patients with and without lupus nephritis both in the first and in the third trimesters of pregnancy. IL-8 serum levels were lower in patients with a previous diagnosis of antiphospholipid antibody syndrome compared with those without, both in the first and in the third trimesters of pregnancy. In SLE patients, a lower than expected decrease in Th1 cytokine serum levels was observed in the third trimester of gestation which could contribute to a lower Th2 cytokine polarization during pregnancy.

IgG anti-pentraxin 3 antibodies in systemic lupus erythematosus.

OBJECTIVE: To evaluate the prevalence and correlates of anti-pentraxin 3 (PTX3) antibodies in systemic lupus erythematosus (SLE). METHODS: Serum samples from 130 patients with SLE, 130 age- and sex-matched healthy subjects and 130 patients with other autoimmune rheumatic diseases (oARD) were analysed by home-made ELISAs using as substrate human recombinant PTX3 and two peptides, PTX3_1 and PTX3_2, obtained from the complete protein, identified as potential antigenic sites using the Lasergene DNA program (DNA Star). Inhibition tests were performed to evaluate potential interferences between bovine serum albumin or C-reactive protein and anti-PTX3 or anti-PTX3 peptides, and between antigens and antibodies. Statistical analysis was performed using receiving operating characteristics curves, the Fisher exact test, two-tailed t test and Pearson correlations. RESULTS: Patients with SLE had higher levels and prevalence of anti-PTX3, anti-PTX3_1 and anti-PTX3_2 antibodies than patients with oARD or healthy controls (p<0.001 for all). No differences were observed between patients with oARD and healthy controls. A correlation was found between anti-PTX3 and anti-PTX3_2 antibodies (r=0.615, p<0.001). No association was observed between these antibodies and disease activity. Univariate and multivariate analyses showed that anti-PTX3 and anti-PTX3_2 antibody levels and prevalence were higher in patients without glomerulonephritis and in patients positive for antiphospholipid antibody. All inhibition tests were negative apart from PTX3 against anti-PTX3 antibody or, to a lesser extent, against anti-PTX3_2 antibody, and PTX3_2 against anti-PTX3_2 antibody, all in a dose-dependent manner. CONCLUSIONS: Anti-PTX3 antibodies are significantly prevalent in patients with SLE where they might provide protection from renal involvement. The antigenic properties of PTX3_2 peptide are similar to those of PTX3, suggesting its potential use in further analyses.

Neurolupus is associated with anti-ribosomal P protein antibodies: an inception cohort study.

OBJECTIVE: Serum IgG antibodies (Abs) to phosphorylated ribosomal (P ribosomal) proteins have been inconsistently associated with neuropsychiatric manifestations in systemic lupus erythematosus (SLE). Our aim was to assess whether serum IgG Abs to ribosomal P proteins are associated with neuropsychiatric SLE. PATIENTS AND METHODS: We examined an inception cohort of 219 SLE patients. Neuropsychiatric SLE manifestations were characterized using the American College of Rheumatology (ACR) definition. Serum Abs to P ribosomal proteins were searched for by immunoblotting. In a subgroup of patients, Abs were investigated also in cerebrospinal fluid (CSF). RESULTS: Abs to P ribosomal proteins were detected in 45 (21%) patients, 23 of whom (51%) with neuropsychiatric involvement. Abs to P ribosomal protein were present both in serum and CSF. Abs to P ribosomal proteins significantly correlated with psychosis (p=0.017), mononeuropathy multiplex (p=0.040), malar rash (p=0.004), serum anti-Sm Abs (p=0.042), and lupus anticoagulant (p=0.036). SLE onset age was significantly younger in patients with Abs to P ribosomal proteins. Logistic regression analysis confirmed the relationship between Abs to P ribosomal proteins and psychosis, malar rash, SLE onset age and lupus anticoagulant. CONCLUSIONS: Abs to ribosomal P proteins are associated with psychosis and might be associated with peripheral nervous system complications.

Recent advances in diagnostic technology: applications in autoimmune and infectious diseases.

Biomarkers are used ubiquitously as indicators of biological health. The development of genomic and proteomic multiplex technologies have enormously amplified biomarker discovery and application to diagnostic and therapeutic decisions in clinical practice. New technologies are now available that simultaneously identify a wide spectrum of biomarkers and save time and costs. Multiplexed assays can be coupled to other disease specific indicators (i.e., cytokines, single nucleotide polymorphisms) in order to get more powerful information. However, there is an urgent need for validation/standardization of the new assays before they are adopted into clinical diagnostics. It is worthy to note a new assay, T cell interferon gamma release (TIGRAs), which has recently been introduced in the diagnosis of latent tuberculosis infection. It seems to perform better than tuberculin skin test in patients with inflammatory rheumatic diseases. In this review, we focus on advantages and limits of novel approaches to the detection of autoantibody profiles in autoimmune diseases or pathogen signatures in microbiology.

Anti-prothrombin antibodies predict thrombosis in patients with systemic lupus erythematosus: a 15-year longitudinal study.

OBJECTIVE: To evaluate the role of anti-prothrombin (anti-PT) antibodies in predicting thrombosis in patients with systemic lupus erythematosus (SLE). METHODS: An inception cohort of 101 SLE patients (12 males, 89 females; mean age 30 +/- 8 years), was considered. Clinical and laboratory evaluations were regularly performed during a 15-year follow-up (median 108 months) with a special focus on thromboembolic events. Serum samples were collected at time of diagnosis and at least once a year thereafter. IgG and IgM anti-PT, anti-cardiolipin (aCL) and anti-beta(2)glycoprotein I (beta(2)GPI) antibodies were measured by enzyme-linked immunosorbent assay (ELISA); lupus anticoagulant (LAC) was assayed by the dilute Russell's viper venom time and activated partial thromboplastin time tests. The analytical specificity of anti-PT ELISA was investigated. The timing of thrombosis occurrence was calculated using the Kaplan-Meier method. RESULTS: In the 15-year follow-up, thrombosis occurred in 14 out of the 101 patients: venous thrombosis in nine cases and arterial thrombosis in five. IgG and/or IgM anti-PT, anti-beta(2)GPI and aCL antibodies, and LAC activity were detected in ten, nine, seven, and nine cases, with sensitivity for thrombosis of 71.4%, 64.3%, 50% and 64.3%, respectively. Thrombosis-free survival was 90% at 5 years and 85.8% at 10 and 15 years, respectively. Thrombosis was predicted by anti-PT (P = 0.001), anti-beta(2)GPI antibodies (P = 0.002) and LAC activity (P = 0.001). Moreover, the risk of thrombosis progressively increased with the number of positive antiphospholipid antibody tests. The presence of four positive antibody tests was associated with a risk of thrombosis thirtyfold higher than in their absence. CONCLUSIONS: This longitudinal study shows that IgG anti-PT antibodies are predictors of thrombosis in SLE patients.

OxLDL/beta2GPI-anti-oxLDL/beta2GPI complex and atherosclerosis in SLE patients.

It has been demonstrated that atherosclerosis (ATS) is enhanced in autoimmune rheumatic diseases, such as systemic lupus erythematosus (SLE). The reason for this accelerated process is still debatable and, although traditional risk factors are more prevalent in SLE patients than in general population, they do not seem to fully explain the enhanced risk. ATS has the characteristics of an autoimmune chronic disease, involving both the innate and the adaptive immunity. Moreover, it satisfies the four criteria defining an autoimmune disease, proposed by Witebsky and Rose. It has been shown that some autoantibodies, including anti-oxLDL, anti-beta(2)GPI, anti-HSP60/65, and more recently anti-oxLDL/beta(2)GPI, play a key role in the pathogenesis of ATS. However the role of these autoantibodies in accelerated ATS in SLE patients is still controversial. In fact, some of them seem to be proatherogenic and other protective; moreover, it has been demonstrated that induced oral tolerance has a protective role against ATS. We have recently observed that the levels of oxLDL/beta(2)GPI antigenic complexes and their antibodies were higher in patients with SLE than in healthy subjects, but we did not find a clear association between oxLDL/beta(2)GPI complexes and IgG or IgM anti-oxLDL/beta(2)GPI autoantibodies and subclinical ATS in SLE patients. Many other studies are required to explain the role of autoantibodies in the pathogenesis of ATS in SLE patients, because the characteristics of SLE seem to mask their effects for atherogenesis.

Anti-C1q antibodies in pregnant patients with systemic lupus erythematosus.

OBJECTIVE: To study anti-C1q antibodies in pregnant patients with systemic lupus erythematosus (SLE) and to evaluate their prognostic significance for the occurrence of disease flares or pregnancy complications. METHODS: Twenty-one pregnancies in 19 SLE patients prospectively followed were analyzed. Disease activity was evaluated on the basis of the physician's intention to treat and a modified version of the ECLAM index. Anti-C1q and anti-dsDNA antibodies were detected in the sera by an ELISA assay. Antinuclear antibodies, anti-ENA antibodies, anticardiolipin antibodies and lupus anticoagulant were also performed. RESULTS: In all the patients the disease was inactive at the beginning of the pregnancy. Four flares of disease activity were observed in 4 pregnancies (19%) and obstetric complications were encountered in 7 pregnancies (43%). Anti-C1q antibodies were positive in 4 (19%) pregnancies and anti-dsDNA antibodies in 8 (38%). The presence of anti-phospholipid antibodies at the first assessment was correlated with the occurrence of obstetric complications (p<0.05). The presence of anti-C1q and anti-dsDNA antibodies at the first assessment had no prognostic significance for the occurrence of flares or obstetric complications during the course of pregnancy. Although the small number of patients studied did not allow for statistically significant analysis, flares appeared to be more likely to occur in patients presenting with anti-dsDNA or anti-C1q antibodies during pregnancy compared to patients with no changes in these antibody titers (43% vs 8% respectively). CONCLUSIONS: The presence of anti-C1q and anti-dsDNA antibodies does not seem to be prognostic for the occurrence of flares during pregnancy. Further studies are warranted to explore this possibility.

Clinical implications of autoantibody screening in patients with autoimmune myositis.

OBJECTIVE: To evaluate the clinical usefulness of serum autoantibody profiling in patients with autoimmune myositis. METHODS: We retrospectively studied 74 consecutive patients: 68 had definite or probable myositis according to Bohan-Peter criteria, six suffered from antisynthetase syndrome with subclinical myopathy. Myositis specific antibodies (MSA) (anti-ARS, -SRP, -Mi-2) were determined by RNA immunoprecipitation or immunoblot, myositis associated antibodies (MAA) (anti-RoRNP, -U1RNP, -PM/Scl, -Ku) by immunoblot. RESULTS: Forty-three patients (58%) were positive for MSA: anti-Jo-1 in 15/27 polymyositis (PM) (55%), 4/33 dermatomyositis (DM) (12%), 1/8 overlap (12%) and 2/6 antisynthetase syndrome (33%); anti-ARS non-Jo-1 in 1/27 PM (4%), 2/33 DM (6%) and 4/6 antisynthetase syndrome (67%); anti-Mi-2 in 1/27 PM (4%) and 11/33 DM (33%); anti-SRP in 3/27 PM (11%) and 1/33 DM (3%). One patient was anti-Jo-1/Mi-2 positive, one anti-Jo-1/SRP positive. Moreover, 27 patients (36%) were positive for MAA: anti-Ro/SSA in 8/27 PM (30%), 7/33 DM (21%), 1/8 overlap (12%), and 3/6 antisynthetase syndrome (50%); anti-U1RNP in 1/27 PM (3.7%), 1/33 DM (3%), and 2/8 overlap (25%); anti-PM/Scl in 2/8 overlap (25%), anti-Ku in 2/8 overlap (25%). Anti-Jo-1 was predominantly associated with PM, anti-Mi-2 was almost exclusively found in DM patients. Anti-ARS antibodies were closely associated with interstitial lung disease and polyarthritis; notably, anti-ARS non-Jo-1 was more frequent in patients without overt muscle alterations. Anti-Ro/SSA antibody was not associated with any disease subset, but significantly more frequent in antisynthetase syndrome. CONCLUSIONS: Searching for MSA and MAA in patients with autoimmmune myositis is recommended because of its diagnostic and clinical value. Anti-ARS non-Jo-1 antibodies seem to preferentially target patients with pulmonary fibrosis without overt myopathy.

Increased titres of IgM anti-heparan sulfate antibody in Behçet's disease.

OBJECTIVE: Endothelial dysfunction is crucial in Behçet's disease (BD) pathogenesis, and measures of endothelial damage are potential markers of BD activity. Heparan sulfate (HS) is the most abundant proteoglycan in the endothelial cells, and anti-HS antibodies have been reported in subjects with vascular damage, due to vasculitis/vasculopathy. The aim of our study was to measure serum anti-HS antibodies in patients with BD and to determine whether their presence correlates with disease activity or clinical manifestations. METHODS: Thirty-two patients with BD (21 men, 11 women) (median age 36.81+/-12.0 years) were considered. Of these, 13 had clinically active disease at the time of study. The mean disease duration was 7.31+/- 8.2 years (median 6 years). Anti-HS antibodies were measured by ELISA. As controls, sera from 40 sex- and age-matched healthy subjects, and 78 age-matched patients with systemic lupus erythematosus (SLE) were studied. RESULTS: Anti-HS IgM antibody titres were significantly higher in BD patients compared to healthy subjects (p=0.016) and SLE controls (p=0.0008). No differences in anti-HS IgG antibody titres were observed among the 3 groups. Using categorical data, increased titres of IgM anti-HS antibodies were significantly more frequent in patients with BD vs patients with SLE (p=0.02). The presence of the antibodies, of either isotype, did not correlate with disease duration, disease activity or clinical manifestations. CONCLUSIONS: BD patients have increased IgM anti-HS antibody titres compared to healthy and SLE controls. These antibodies did not correlate with disease activity or discrete clinical features, but might be relevant for pathogenic mechanisms of the disease.

Autoinflammatory syndromes.

The autoinflammatory disorders are a new and expanding classification of inflammatory diseases characterized by recurrent episodes of systemic inflammation in the absence of pathogens, autoantibodies or antigen specific T cells. These disorders are caused by primary dysfunction of the innate immune system, without evidence of adaptive immune dysregulation. Innate immune abnormalities include aberrant responses to pathogen associated molecular patterns (PAMPs) like lipopolysaccharide and peptidoglycan, prominent neutrophilia in blood and tissues, and dysregulation of inflammatory cytokines (IL-1beta, TNF-alpha) or their receptors. The autoinflammatory diseases comprise both hereditary (Familial Mediterranean Fever, FMF; Mevalonate Kinase Deficiency, MKD; TNF Receptor Associated Periodic Syndrome, TRAPS; Cryopyrin Associated Periodic Syndrome, CAPS; Blau syndrome; Pyogenic sterile Arthritis, Pyoderma gangrenosum and Acne syndrome, PAPA; Chronic Recurrent Multifocal Osteomyelitis, CRMO) and multifactorial (Crohn's and Behçet's diseases) disorders. Mutations responsible for FMF, TRAPS, CAPS, PAPA are in proteins involved in modulation of inflammation and apoptosis.

Positive and negative effects of thalidomide on refractory cutaneous lupus erythematosus.

BACKGROUND: Thalidomide is used in cutaneous lupus erythematosus (CLE) refractory to conventional therapies. Peripheral neuropathy (PN) is the most severe side effect, but the incidence of PN and its relation to thalidomide dose are still unclear. OBJECTIVE: To prospectively evaluate the efficacy as well as the occurrence of PN in CLE patients treated with thalidomide, and to assess whether PN, when occurs, correlates with thalidomide dose and/or length of treatment. METHODS: Fourteen female patients with CLE in low-dose thalidomide therapy were followed for up to 24 months. Prior to, and regularly during treatment patients underwent rheumatological, dermatological, neurological and electrophysiological evaluations. A decline in sural SNAP of 50% or more from baseline value was considered as criterion of sensory axonal PN. RESULTS: All patients showed a dramatic improvement of skin manifestations. Ten patients (71.4%) developed a sensory axonal PN. The median time free from this complication was 14 months. No correlations were found between age of the patients nor thalidomide cumulative dose and occurrence of PN (Mann-Whitney U Test; p>0.16). Other adverse effects were: tremor, paresthesias, somnolence, amenhorrea, constipation and thoracic pain. CONCLUSIONS: Low does thalidomide is efficacious in treating CLE, but PN is a common complication whose occurrence does not seem to correlate with total thalidomide dose, whereas with the duration of therapy. A closer electrophysiological follow-up is therefore recommended in the long-term treatment.

[Myositis specific and myositis associated autoantibodies in idiopathic inflammatory myopathies: a serologic study of 46 patients]. / Gli anticorpi miosite specifici e miosite associati nelle miopatie infiammatorie idiopatiche: studio sierologico di 46 pazienti.

OBJECTIVE: To characterize serum autoantibody profiles of patients with idiopathic inflammatory myopathies (IIM) by searching for myositis-specific (MSA) and myositis-associated (MAA) antibodies with sensitive and specific laboratory tests. METHODS: We tested the sera from 46 Caucasian patients diagnosed as affected with IIM at the Division of Rheumatology of Padova University (21 polimyositis, PM; 22 dermatomyositis, DM; 3 myositis overlap syndrome). All patients had definite IIM according to the criteria of Bohan-Peter. MSA including anti-tRNA synthetase (anti-Jo-1 and others) and anti-Mi-2 were determined by RNA immunoprecipitation and a modified immunoblot test, respectively. MAA (-U1RNP, -U2RNP, RoRNP, PM/Scl, Ku) were detected by counterimmunoelectrophoresis and immunoblot. RESULTS: Serum MSA and/or MAA were found in 30/46 (65%) patients with IIM. Twenty-three patients (50%) were positive for at least one MSA: anti-Jo-1 in 15 (33%), anti-Mi-2 in 6 (13%), and other anti-tRNA synthetase in 3 (6%). One patient was anti-Jo-1/Mi-2 positive. Moreover, 18 patients (39%) were positive for at least one MAA: anti-Ro/SSA in 13 (28%), anti-U1RNP in 4 (9%), anti-PM/Scl in 1 (2%) and anti-Ku in 1 (2%). Coexisting MSA and MAA were observed in 8 patients (17%), anti-Jo-1/SSA positive in most cases. Anti-Jo-1 was predominantly associated with PM (57% in PM vs 14% in DM), whereas anti-Mi-2 was exclusively found in DM patients (27%). Anti-synthetase antibodies were closely associated with interstitial lung disease and polyarthritis; anti-Mi-2 positive DM patients did not have lung involvement. Notably, anti-Ro/SSA antibody was frequently observed and almost equally detected in either PM or DM (about 30%): in more than 50% of cases the antibody was associated with one MSA. CONCLUSIONS: By means of analytically reliable methods, MSA was detected in 50% of our IIM patients. Searching for MSA in patients with IIM is recommended because of its diagnostic and prognostic value.

The use of Tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases.

Autoantibodies directed against intracellular antigens can be detected by immunoblotting (IB). Due to its high sensitivity this technique has many advantages, but it can give misleading results when the specific bands are weak or blurred against the background staining. To decrease background staining, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are generally used as blocking agents. Moreover, these agents appear to have a renaturating action towards proteins and antigens. Tween 20 has a more pronounced renaturating effect on proteins than other detergents and thereby improves antigen-antibody binding. To evaluate the effect of Tween 20 on specific autoantibody detection by IB, we tested the sera of 162 patients with connective tissue diseases (CTDs) by adding this detergent at certain steps of the IB assay. We found that the use of Tween 20 in the IB procedure significantly improved the binding of autoantibodies to Jo-1, Scl70, (U1)RNP 68 kDa and C, Sm B/B' and D. Moreover, it increased the sensitivity for the detection of anti-Sm D peptide in systemic lupus erythematosus (SLE) sera with no decrease in specificity. In contrast, the addition of Tween 20 significantly decreased the binding of autoantibodies specific for ribosomal P proteins, La/SSB, Ro/SSA, but not the overall sensitivity and specificity of the method. We conclude that the addition of Tween 20 to standard IB is advantageous for anti-nuclear antigen antibody detection and improves the sensitivity of the method in revealing anti-Sm-positive sera in SLE. However, Tween 20 is not recommended for the detection of anti-cytoplasmic antibodies.

Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry.

This study was performed by the Italian Society of Laboratory Medicine (SIMeL) in order to establish the variability between the different analytical systems currently used in clinical laboratories for the detection of autoantibodies diagnostic of systemic autoimmune disease. Sixteen industrial, and two university laboratories participated in this study which entailed the determination of anti-nuclear (ANA), anti-dsDNA and anti-ENA antibodies in 11 sera from patients with clinically diagnosed systemic rheumatic disease, using reagents produced by these companies and different methodologies (indirect immunofluorescence, immunoenzymatic assay, counterimmunolectrophoresis, immuno and western blotting). We found 93.5% agreement between the methods used for the detection of ANA, 85.2% for anti-dsDNA antibodies, and 86.9% for anti-ENA antibodies. Among the anti-ENA antibodies, regardless of the method used, detection percentages were excellent for anti-RNP and anti-SSB/La (100%), good for anti-SSA/Ro (93%), but unacceptable for the anti-Jo-1 (67%), anti-Scl70 and anti-Sm (47%) antibodies. This further stresses the need for rigorous standardisation of commercial reagents and analytical procedures, as well as the introduction of external quality assessment (EQA) programs, and a complete definition of operative protocols adjusted to the sensitivity and specificity of the various methods.

Prevalence and characteristics of anti-single-stranded DNA antibodies in localized scleroderma. Comparison with systemic lupus erythematosus.

Prevalence, levels, and immunoglobulin classes of anti-single-stranded DNA antibodies were determined by an enzyme-linked immunosorbent assay in 52 patients with localized scleroderma (33 with morphea, four with generalized morphea, and 15 with linear scleroderma), in 60 healthy controls, and, for comparison, in 31 patients with systemic lupus erythematosus. Localized scleroderma revealed a significant prevalence of anti-single-stranded DNA antibodies, mainly characterized by high levels and IgM and IgA isotypes. Comparison of antibody characteristics in different clinical forms of localized scleroderma showed some significant differences (levels and immunoglobulin isotypes). Comparison with systemic lupus erythematosus showed that frequency, high levels, and IgG isotype of anti-single-stranded DNA antibodies significantly prevailed in systemic lupus erythematosus, while the IgM isotype significantly prevailed in localized scleroderma. However, generalized morphea and linear scleroderma did not significantly differ from systemic lupus erythematosus as regards antibody frequency and prevalence of high antibody levels.