Anna Ghirardello

Diagnostic performance and validation of autoantibody testing in myositis by a commercial line blot assay

OBJECTIVEnSerological testing for myositis-specific or associated autoantibodies [myositis-specific antibody (MSA) and myositis-associated antibody (MAA)] is useful for the diagnosis of idiopathic inflammatory myopathies (IIMs). However, available assays are neither standardized nor validated. The objective is to evaluate the accuracy of a commercial line blot assay for myositis diagnosis.nnnMETHODSnIgG antibodies against Jo-1, PL-7, PL-12, PM/Scl, Ku, Mi-2 and Ro52 antigens were detected by a line blot and in-house RNA immunoprecipitation or immunoblot. We tested sera from 208 IIM patients, 50 healthy subjects and 180 control patients (11 non-autoimmune myopathy, 23 muscular dystrophy, 11 UCTD, 68 SLE, 36 SSc, 22 SS and 9 arthropathy).nnnRESULTSnMSAs or MAAs were detected in 98 (47%) out of the 208 IIM patients by line blot: anti-Jo-1 in 43 (21%), anti-PL-7 or anti-PL-12 in 8 (4%), anti-Mi-2 in 9 (4%), anti-PM/Scl in 9 (4%), anti-Ku in 10 (5%) and anti-Ro52 in 49 (24%). Overall specificity was: 100% for anti-Jo-1, anti-PL-7 or PL-12 and anti-PM/Scl; 96% for anti-Ku; 98% for anti-Mi-2; and 76% for anti-Ro52. In-house testing confirmed line blot results regarding anti-Jo-1, anti-PM/Scl and anti-Ku, while it was more accurate than line blot in detecting anti-Mi-2 (7 vs 4% sensitivity, 100 vs 98% specificity), and anti-aminoacyl-tRNA synthetase (anti-ARS) non-Jo-1 antibodies (11 vs 4% sensitivity, 97 vs 99% specificity).nnnCONCLUSIONSnLine blot could be a suitable serological test in the diagnostic workup for myositis, and it represents a reliable alternative to more time-consuming procedures. Continuous effort is recommended in order to improve its accuracy.

Thalidomide neurotoxicity Prospective study in patients with lupus erythematosus

The authors prospectively followed 14 patients treated with thalidomide for cutaneous lupus erythematosus (CLE), in order to evaluate the occurrence of peripheral neuropathy (PN) and to assess whether PN correlates with thalidomide dose. The patients were followed for up to 24 months with neurologic and electrophysiologic evaluations. Seven patients (50%) developed sensory axonal PN. The median time free from PN was 14 months. PN occurred after 10 months in the majority of patients. No correlations were found between thalidomide cumulative dose and occurrence of PN (Mann-Whitney U test; p > 0.16).

Pregnancy in rare autoimmune rheumatic diseases: UCTD, MCTD, myositis, systemic vasculitis and Beçhet disease

The physiological adaptation of the immune system to pregnancy can potentially affect the course of all autoimmune rheumatic diseases (ARD), conversely the autoimmune processes characteristic of these conditions may compromise the foetal outcome. Unfortunately, very few reports on pregnancy outcome in patients with rare ARD are available. In this paper, we briefly review the data published until now on these disorders. Some general guidelines which were elaborated for more prevalent ARD seem to be valid also for such rare conditions: 1) patients should be correctly informed on the risk of becoming pregnant; 2) pregnancies should be planned when the disease is in remission since it increases the probability of successful maternal and foetal outcome; 3) patients should be regularly monitored during gestation and postpartum by a multidisciplinary team including rheumatologist, obstetrician, and neonatologist; 4) in the case of disease relapse an adequate treatment, even aggressive if necessary, should be recommended since active disease can be more detrimental for foetus than drugs; 5) pregnancies complicated by the onset of rare ARD have a particularly severe prognosis; in these cases a prompt treatment and very close clinical surveillance are indicated.

Anti-ribosomal P protein antibodies and neuropsychiatric systemic lupus erythematosus: cross-sectional vs. prospective studies.

Sir, Whether anti-ribosomal P (anti-P) antibodies have a prognostic value for neuropsychiatric manifestations in systemic lupus erythematosus (NPSLE) remains a matter of debate. Anti-P antibodies are directed to the functional centre of ribosomal P (phospho) proteins. P proteins are post-translationally modified (dephosphorylated) during apoptosis, and a dysregulation in the normal clearance of apoptotic cells leads to aberrant exposure to the immune system of modified non self-antigens. This could be one of the triggering events for the development of anti-P autoimmune response in SLE. Haddouk et al. recently investigated the diagnostic value of anti-P in SLE patients and the relationship between anti-P antibodies, detected at disease onset, and SLE manifestations prospectively registered during a 5-year follow-up in a large cohort of Tunisian patients. Haddouk’s results confirmed our already published findings about anti-P antibody sensitivity and specificity for SLE and the relevant association with antiphospholipid antibodies. Indeed, the high specificity of anti-P antibody (99.3%) was confirmed in a recent international multi-centre study of 947 SLE patients and more than 1000 controls. Anti-P antibody positive/anti-dsDNA negative/ anti-Sm negative sera can occur in a significant proportion of SLE patients, further stressing the diagnostic relevance of this biomarker. Thus, we agree with the Authors on the fact that the American College of Rheumatology (ACR) criteria for SLE classification should be revised by including anti-P serology, due to its specificity and positive predictive value. Conversely, we suggest caution in interpreting the results of the second part of Haddouk’s study regarding the clinical correlates of anti-P antibodies. A number of limitations can be noted in their study, including the detection of anti-P antibodies at a single time point and the grouping of NPSLE manifestations in some sketchy clusters each one encompassing a broad spectrum of manifestations potentially induced by different pathogenic mechanisms. Since their first description in 1987, one of the most intriguing aspects of anti-P antibodies has been their possible pathogenic role in SLE psychosis and depression. It is worth noting that depressive features were induced in a lupus-prone mouse model by injecting affinity purified human anti-P antibodies into cerebral ventriculi. We have evaluated longitudinally both anti-P antibodies and NPSLE in an inception cohort of 219 SLE Italian patients, followed-up in a single centre for over 10 years. The method used for the anti-P assaying was as previously described and NPSLE involvement was carefully investigated according to ACR guidelines. In our study, anti-P antibodies were associated with psychosis both in univariate and multivariate analysis. We observed a significant variation of antibody levels in 27% of cases as well as a fluctuation between negative and positive results concomitantly with NPSLE events in some patients (Figure 1). In keeping with our results, a longitudinal study carried out on an international inception cohort of SLE patients applying a very stringent methodology showed a clear association between anti-P antibodies and NPSLE episodes. Thus, a prospective study longitudinally investigating both anti-P antibody and NPSLE occurrence should certainly be more appropriate and informative for NP attribution and outcome (Table 1).

oxLDL/beta2GPI complex and anti-oxLDL/beta2GPI in SLE: prevalence and correlates.

High levels of oxidized low-density liprotein/beta2 glycoprotein 1 (oxLDL/β2GPI) complexes and anti-complex IgG as well as IgM have been reported in SLE. We analysed this complex and Ab against the complex in SLE patients and evaluated their relationship with clinical and serological findings, traditional risk factors for atherosclerosis, and subclinical atherosclerosis. The prevalence and the levels of the complex and of anti-complex Ab were significantly higher in systemic lupus erythematosus (SLE) patients than in normal healthy donors (NHD). The titers of oxLDL/β2GPI were significantly higher in patients with renal involvement and previous thromboembolic episodes and were correlated with the number of risk factors for atherosclerosis, whereas they were significantly lower in patients with neurological involvement. Both IgG and IgM anti-complex Ab were associated with antiphospholipid (APL). In conclusion, the oxLDL/β2GPI complex as well as Ab against the complex are prevalent in SLE where they seem to be involved in organ damage.

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 Freunds 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-Coxs 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.

Seltene autoimmune rheumatische Erkrankungen in der Schwangerschaft@@@Rare autoimmune rheumatic illnesses during pregnancy: systemic sclerosis, polymyositis/dermatomyositis and vasculitis: Systemische Sklerose, Polymyositis/Dermatomyositis und Vaskulitiden

ZusammenfassungAutoimmune rheumatische Erkrankungen (ARE) betreffen auch junge Frauen im gebärfähigen Alter. In den letzten Jahrzehnten führte die verbesserte Überlebensrate und Lebensqualität der Patientinnen mit ARE dazu, dass mehr Frauen mit diesen Erkrankungen schwanger wurden. Der systemische Lupus erythematodes (SLE) ist die häufigste während der Schwangerschaft beobachtete ARE. Es konnte gezeigt werden, dass die immunoendokrinen Veränderungen in der Schwangerschaft den Erkrankungsverlauf beeinflussen können.Schwangerschaften sind auch möglich bei Patientinnen mit seltenen ARE, z.xa0B. systemischer Sklerose, Polymyositis/Dermatomyositis, systemischer Vaskulitis einschließlich Wegener-Granulomatose, Churg-Strauss-Syndrom, Polyarteriitis nodosa, mikroskopischer Polyangiitis, Takayasu-Arteriitis und Behçet-Krankheit.Diese Übersichtsarbeit konzentriert sich auf die Schwangerschaftskomplikationen dieser seltenen ARE und befasst sich kurz mit den zu diesen Erkrankungen veröffentlichten Daten. Außerdem werden Leitlinien für die Behandlung dieser Erkrankungen in der Schwangerschaft vorgestellt. Zu beachten ist, dass die Daten zu diesen Patientinnen sehr spärlich sind und dass in Ermangelung prospektiver Studien die meisten Erkenntnisse aus Fallberichten und retrospektiven Studien stammen.AbstractAutoimmune rheumatic diseases (ARD) affect young females durrng childbearing age. Over the last decades, improvements in survival as well as quality of life in patients affected with ARD have led to an increased number of pregnancies observed during the course of such diseases. Systemic lupus erythematosus (SLE) is the most frequently observed ARD during pregnancy, and the immunoendocrine changes occurring during pregnancy may influence the course of this disease.Pregnancy can also occur in patients with rare ARD, namely systemic sclerosis, polymyositis/dermatomyositis, systemic vasculitis including Wegener’s granulomatosis, Churg-Strauss syndrome, polyarteritis nodosa, microscopic polyangiitis, Takayasu arteritis and Behçet disease.This review focuses on the complications during pregnancy caused by these rare ARD, and we briefly discuss the data published on these disorders. Some guidelines for the management of these conditions during pregnancy will also be provided. However, it is important to note that data on pregnancy outcome are very limited and, in the absence of prospective studies, most of the information derives from case reports and retrospective studies.

Detection of autoantibodies to the p200-epitope of SSA/Ro52 antigen. A comparison of two laboratory assays

Abstract Background: Anti-p200 antibodies have been receiving growing interest in view of findings associating their presence to risk of fetal autoimmune congenital heart block (CHB). The study compares and evaluates the performance of two assays currently being used for their detection. Methods: One hundred and sixteen pregnant women positive for anti-SSA/Ro52 antibodies were considered as the study population. Fifty women negative for anti-SSA/Ro52 antibodies were considered as the control population. Anti-p200 antibodies were analyzed using two home-made ELISA assays: one with biotinylated antigen and the other with free antigen. Results: The specificity of the p200-free assay was significantly higher with respect to that of the p200-biotin assay (p=0.023). Both methods showed a high area under curve (AUC), thus, a good accuracy. There was a significant prevalence of anti-p200 antibodies when the p200-free assay was used to analyze the sera of the pregnant women with CHB fetuses (p=0.007). Cohen’s κ and Spearman’s ρ coefficients showed a good concordance (0.71) and a high correlation (0.93), respectively. Conclusions: The p200-free assay with respect to the biotin-based method was more specific in detecting p200 antibodies in women positive for anti-SSA/Ro52 antibodies. In addition, only the p200-free method significantly found p200 antibodies in patients with fetal CHB.

SERPINB3 Delays Glomerulonephritis and Attenuates the Lupus-Like Disease in Lupus Murine Models by Inducing a More Tolerogenic Immune Phenotype

Objective: To explore the effects of SERPINB3 administration in murine lupus models with a focus on lupus-like nephritis. Methods: 40 NZB/W F1 mice were subdivided into 4 groups and intraperitoneally injected with recombinant SERPINB3 (7.5 μg/0.1 mL or 15 μg/0.1 mL) or PBS (0.1 mL) before (group 1 and 2) or after (group 3 and 4) the development of proteinuria (≥100 mg/dl). Two additional mice groups were provided by including 20 MRL/lpr mice which were prophylactically injected with SERPINB3 (10 mice, group 5) or PBS (10 mice, group 6). Time of occurrence and levels of anti-dsDNA and anti-C1q antibodies, proteinuria and serum creatinine, overall- and proteinuria-free survival were assessed in mice followed up to natural death. Histological analysis was performed in kidneys of both lupus models. The Th17:Treg cell ratio was assessed by flow-cytometry in splenocytes of treated and untreated MRL/lpr mice. Statistical analysis was performed using non parametric tests and Kaplan-Meier curves, when indicated. Results: Autoantibody levels and proteinuria were significantly decreased and time of occurrence significantly delayed in SERPINB3-treated mice vs. controls. In agreement with these findings, proteinuria-free and overall survival were significantly improved in SERPINB3-treated groups vs. controls. Histological analysis demonstrated a lower prevalence of severe tubular lesions in kidneys of group 5 vs. group 6. SERPINB3-treated mice showed an overall trend toward a reduced prevalence of severe lesions in both strains. Th17:Treg ratio was significantly decreased in splenocytes of MRL/lpr mice treated with SERPINB3, compared to untreated control mice. Conclusions: SERPINB3 significantly improves disease course and delays the onset of severe glomerulonephritis in lupus-prone mice, possibly inducing a more tolerogenic immune phenotype.