Francesca Pregnolato

A non–complement-fixing antibody to β2 glycoprotein I as a novel therapy for antiphospholipid syndrome

A single-chain fragment variable (scFv) recognizing β2-glycoprotein 1 (β2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against β2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-β2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-β2GPI antibodies from APS patients and displaced β2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy.

Antinucleosome antibodies in primary antiphospholipid syndrome: A hint at systemic autoimmunity?

BACKGROUND Antinucleosome antibodies (anti-NCS) are reported to be highly sensitive and specific for systemic lupus erythematosus (SLE) and to correlate with disease activity. They may appear in early stages of the disease, in particular before anti-dsDNA antibodies, being a potential marker for identifying patients susceptible to SLE. Patients with primary antiphospholipid syndrome (PAPS) may develop full-blown SLE but there is no evidence for markers predictive for that. AIM To evaluate whether anti-NCS may be predictors for full-blown or lupus like disease (LL) in a cohort of PAPS patients. METHODS A multicentric cohort of 105 PAPS patients was tested for IgG/IgM anti-NCS by using a home made assay with H1-stripped chromatin as antigen. RESULTS Eighty-one out of 105 (77%) of the patients were positive for anti-NCS; medium-high titre results were present only in 49/105 (46%). Anti-NCS were more frequently detected in PAPS+LL, but no relationship with clinical/serological features was found, except for a weak correlation with anti-dsDNA antibodies. Two PAPS patients evolved into full-blown SLE during the follow-up and displayed high titre anti-NCS many years before. CONCLUSIONS Our findings suggest that anti-NCS might be added to the mosaic of autoimmune phenomena characterizing PAPS patients and in particular those with more chance to evolve to SLE.

Anti-phospholipid induced murine fetal loss: novel protective effect of a peptide targeting the β2 glycoprotein I phospholipid-binding site. Implications for human fetal loss

β2 glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) induce thrombosis and affect pregnancy. The CMV-derived synthetic peptide TIFI mimics the PL-binding site of β2GPI and inhibits β2GPI cell-binding in vitro and aPL-mediated thrombosis in vivo. Here we investigated the effect of TIFI on aPL-induced fetal loss in mice. TIFI inhibitory effect on in vitro aPL binding to human trophoblasts was evaluated by indirect immunofluorescence and ELISA. TIFI effect on aPL-induced fetal loss was investigated in pregnant C57BL/6 mice treated with aPL or normal IgG (NHS). Placenta/fetus weight and histology and RNA expression were analyzed. TIFI, but not the control peptide VITT, displayed a dose-dependent inhibition of aPL binding to trophoblasts in vitro. Injection of low doses of aPL at day 0 of pregnancy caused growth retardation and increased fetal loss rate, both significantly reduced by TIFI but not VITT. Consistent with observations in humans, histological analysis showed no evidence of inflammation in this model, as confirmed by the absence of an inflammatory signature in gene expression analysis, which in turn revealed a TIFI-dependent modulation of molecules involved in differentiation and development processes. These findings support the non-inflammatory pathogenic role of aPL and suggest innovative therapeutic approaches to aPL-dependent fetal loss.

Clinical Characterization of Antiphospholipid Syndrome by Detection of IgG Antibodies Against β2‐Glycoprotein I Domain 1 and Domain 4/5: Ratio of Anti–Domain 1 to Anti–Domain 4/5 As a Useful New Biomarker for Antiphospholipid Syndrome

It has been suggested that only antibodies against domain 1 (D1) of β2‐glycoprotein I (β2GPI) are pathogenic and diagnostic. The role of antibodies against other β2GPI domains is still debated. This study was undertaken to evaluate the clinical relevance of domain specificity profiling of anti‐β2GPI IgG antibodies in antiphospholipid syndrome (APS) patients and in control groups of patients with systemic autoimmune rheumatic diseases and in asymptomatic antiphospholipid antibody (aPL) carriers.

Measuring IgA Anti-β2-Glycoprotein I and IgG/IgA Anti-Domain I Antibodies Adds Value to Current Serological Assays for the Antiphospholipid Syndrome

Introduction Currently available clinical assays to detect antiphospholipid antibodies (aPL) test for IgG and IgM antibodies to cardiolipin (aCL) and β2-glycoprotein I (aβ2GPI). It has been suggested that testing for IgA aPL and for antibodies to Domain I (DI), which carries the key antigenic epitopes of β2GPI, could add value to these current tests. We performed an observational, multicenter cohort study to evaluate the utility of IgG, IgM and IgA assays to each of CL, β2GPI and DI in APS. Methods Serum from 230 patients with APS (n = 111), SLE but not APS (n = 119), and 200 healthy controls were tested for IgG, IgM and IgA aCL, aβ2GPI and aDI activity. Patients with APS were further classified into thrombotic or obstetric APS. Logistic regression and receiver operator characteristic analyses were employed to compare results from the nine different assays. Results All assays displayed good specificity for APS; IgG aCL and IgG aβ2GPI assays however, had the highest sensitivity. Testing positive for IgA aβ2GPI resulted in a higher hazard ratio for APS compared to IgM aβ2GPI. Positive IgG, IgM or IgA aDI were all associated with APS, and in subjects positive for aCL and/or aβ2GPI, the presence of aDI raised the hazard ratio for APS by 3–5 fold. IgG aCL, aβ2GPI, aDI and IgA aDI were associated with thrombotic but not obstetric complications in patients with APS. Conclusion Measuring IgG aDI and IgA aβ2GPI and aDI may be useful in the management of patients with APS, particularly thrombotic APS.

Clinical usefulness of autoantibodies to M-type phospholipase A2 receptor (PLA2R) for monitoring disease activity in idiopathic membranous nephropathy (IMN)

Autoantibodies to M-type phospholipase A2 receptor (PLA2R) are specific markers of idiopathic membranous nephropathy (IMN). They can differentiate IMN from other glomerular diseases and primary from secondary forms of MN. Preliminary data suggest that anti-PLA2R antibody titer correlates with disease activity but more solid evidence is needed. To evaluate the performance of anti-PLA2R antibody for monitoring nephropathy activity, 149 anti-PLA2R antibody measurements were performed during the follow-up of 42 biopsy proven IMN consecutive patients. Patients were enrolled either at time of diagnosis (33 cases, inception cohort) or after diagnosis (9 patients, non-inception cohort). Anti-PLA2R detection was performed using the highly sensitive transfected cell-based indirect immunofluorescence (IIFT). Over the follow-up there was a linear time-trend of decreasing proteinuria (P<0.001), increasing serum albumin (P<0.001) and decreasing PLA2R antibody levels (P=0.002). There was a statistically significant association between changes in PLA2R antibody levels and the clinical course of PLA2R-positive IMN. The positive PLA2R serum antibody status was linearly associated with increasing proteinuria and decreasing serum albumin over time, compared with negative antibody status. Moreover, the strong correlation between the clinical conditions and PLA2R antibody levels allowed the prediction of prevalence distribution of patients with active disease, partial and complete remission. Over the course of the follow-up, the probability of halving proteinuria increased 6.5 times after disappearance of PLA2R antibodies. Our data suggest that the serial evaluation of anti-PLA2R antibodies could help in optimal timing and duration of the immunosuppressive therapy, reducing over(under)-treatment and associated side-effects.

Preliminary evaluation of the first international reference preparation for anticitrullinated peptide antibodies

Background A lyophilised reference serum from one patient with rheumatoid arthritis (RA) diluted with serum samples from healthy subjects was evaluated as a possible first international standard for anticitrullinated peptide antibodies (ACPAs). Methods The authors used 12 commercial ELISAs for ACPA detection in the reference serum and for testing the linearity of the assays by studying twofold serial dilutions. To test the effectiveness of the standardisation, sera from 20 RA patients with variable antibody concentrations were analysed, and the relative concentrations were calculated using both the kits own curve and the six dilutions of the reference serum as a calibration curve. Fifty sera from normal healthy subjects were used to calculate cut-off values for the reference serum using each commercial kit. Results The calibration curve obtained for each of the 12 methods using the reference sample dilutions as calibrator allowed harmonisation of the ACPA concentration of the 20 RA serum samples, significantly reducing the dispersion of the values. The mean coefficient of variation (CV) was reduced from 76.4% to 27.9% (p=0.018) and from 85.9% to 33.5% (p=0.028) for the medium/high and negative samples, respectively. Low positive sera CV was also reduced, but to a smaller degree, from 82.5% to 55.5% (p=0.043). Conclusion This first evaluation of the behaviour of the ACPA reference serum demonstrated that it tested positive in all the assays and that it may be used as a reference standard for establishing calibration curves, reducing the dispersion of antibody values and better comparing results obtained from different methods/laboratories.

The role of biologic agents in damage progression in rheumatoid arthritis: indirect comparison of data coming from randomized clinical trials

Objectives: All biologic agents approved for the treatment of rheumatoid arthritis (RA) have been tested versus methotrexate (MTX) for efficacy on damage progression in several randomized clinical trials (RCTs), but direct head-to-head comparisons have never been conducted. The purpose of this investigation is to analyse data coming from main RA RCTs and to perform an indirect comparison. Methods: A systematic review of literature from 1988 to 2011 was conducted. Only randomized, double-blind, controlled, comparative trials, with evaluation of radiographic progression were included. The radiographic score was standardized and mean difference in the percentage of the annual radiographic progression rate was used as the effect measure. Heterogeneity between studies was estimated by I2 test. For each trial, the effect was plotted according to its standard error in a funnel plot. Results: Of 44 potentially relevant trials, 12 RCTs were included in the study. In order to optimize RCTs comparison, studies were stratified in early and late RA group. Main population characteristics were similar in both early and late RA groups, whereas the standardized baseline radiographic score value significantly differs among trials in both early (range 2.7–21.9) and late (range 23.46–75) RA groups. The standardized annual estimated progression is similar across the late RA group. Strong evidence of heterogeneity (I2 = 97%, p = 0.00001) but no asymmetry of the funnel plot was observed in the early RA group. Total mean difference was −16.28 (95% confidence interval [CI] −24.42 to −8.14). For the late RA group a random model was used (I2 = 99%, p = 0.00001) and a total mean difference of −39.25 (95% CI −53.77 to −24.73) was found. Conclusions: All biologic agents provide a favourable effect on disease progression both in early and late RA. The significant heterogeneity among various RCTs did not allow an effective comparison of the performance of biologic agents in each study.

Antiglutamate Receptor Antibodies and Cognitive Impairment in Primary Antiphospholipid Syndrome and Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) and antiphospholipid syndrome have an increased risk to develop cognitive impairment. A possible role for antiphospholipid antibodies (aPL) and antiglutamate receptor (anti-NMDA) antibodies in the pathogenesis of neurological manifestations of these two conditions, have been suggested. In particular, the role of anti-NMDA antibodies in the pathogenesis of neuropsychiatric SLE is supported by several experimental studies in animal models and by the finding of a correlation between anti-NMDA positivity in cerebrospinal fluid and neurological manifestations of SLE. However, data from the literature are controversial, as several studies have reported a correlation of these antibodies with mild cognitive impairment in SLE, but more recent studies have not confirmed this finding. The synergism between anti-NMDA and other concomitant autoantibodies, such as aPL, can be hypothesized to play a role in inducing the tissue damage and eventually the functional abnormalities. In line with this hypothesis, we have found a high incidence of at least one impaired cognitive domain in a small cohort of patients with primary APS (PAPS) and SLE. Interestingly, aPL were associated with low scoring for language ability and attention while anti-NMDA titers and mini-mental state examination scoring were inversely correlated. However, when patients were stratified according to the presence/absence of aPL, the correlation was confirmed in aPL positive patients only. Should those findings be confirmed, the etiology of the prevalent defects found in PAPS patients as well as the synergism between aPL and anti-NMDA antibodies would need to be explored.

The comparison of effects of biologic agents on rheumatoid arthritis damage progression is biased by period of enrolment: Data from a systematic review and meta-analysis

OBJECTIVES To indirectly compare the 12-month effects of available biologic agents in slowing RA radiographic progression. METHODS A systematic review of literature of randomised, double-blind, controlled trials (RCTs) evaluating RA radiographic progression as end point was conducted using a PubMed searching of MEDLINE from January 1995 to May 2012. For each trial, the mean change from baseline of the standardised annual radiographic progression score (weighted for estimated annual progression rate) was estimated, and the effect size was calculated as the difference between biologic and non-biologic-treated groups. In order to optimise data homogeneity and improve RCTs comparison, a mixed-effect model was applied including previous responsiveness to methotrexate (MTX-experienced or MTX-naïve populations) and period of study enrollment as moderators. RESULTS The PubMed search resulted in 183 references, and 14 were eligible for the meta-analysis. The analysis of study distribution in forest plots showed a high correlation between the study period of enrollment and the impact of biological therapy in both MTX-naïve and MTX-experienced subgroups. In particular, effect size was the highest for older trials and progressively decreased in the most recent ones, suggesting a highest propensity to radiographic progression in populations enrolled in older trials. Some statistically significant differences among RCTs were found in both subgroups but were significantly biased by the different propensity to radiographic progression due to period of enrollment. CONCLUSIONS Our meta-analysis demonstrated that period of enrollment deeply influence study population propensity to radiographic progression in each trial. This finding does not allow the indirect comparison of various biologic agents, despite our mixed-model significantly reducing heterogeneity among RCTs.