New clinical, laboratory and experimental insights, since the 1999 publication of the Sapporo preliminary classification criteria for antiphospholipid syndrome (APS), had been addressed at a workshop ...in Sydney, Australia, before the Eleventh International Congress on antiphospholipid antibodies. In this document, we appraise the existing evidence on clinical and laboratory features of APS addressed during the forum. Based on this, we propose amendments to the Sapporo criteria. We also provide definitions on features of APS that were not included in the updated criteria.
Thrombosis in the context of Cardiovascular disease (CVD) affects mainly the blood vessels supplying the heart, brain and peripheries and it is the leading cause of death worldwide. The ...pathophysiological thrombotic mechanisms are largely unknown. Heritability contributes to a 30% of the incidence of CVD. The remaining variation can be explained by life style factors such as smoking, dietary and exercise habits, environmental exposure to toxins, and drug usage and other comorbidities.
Epigenetic variation can be acquired or inherited and constitutes an interaction between genes and the environment. Epigenetics have been implicated in atherosclerosis, ischemia/reperfusion damage and the cardiovascular response to hypoxia. Epigenetic regulators of gene expression are mainly the methylation of CpG islands, histone post translational modifications (PTMs) and microRNAs (miRNAs). These epigenetic regulators control gene expression either through activation or silencing. Epigenetic control is mostly dynamic and can potentially be manipulated to prevent or reverse the uncontrolled expression of genes, a trait that renders them putative therapeutic targets.
In the current review, we systematically studied and present available data on epigenetic alterations implicated in thrombosis derived from human studies. Evidence of epigenetic alterations is observed in several thrombotic diseases such as Coronary Artery Disease and Cerebrovascular Disease, Preeclampsia and Antiphospholipid Syndrome. Differential CpG methylation and specific histone PTMs that control transcription of prothrombotic and proinflammatory genes have also been associated with predisposing factors of thrombosis and CVD, such us smoking, air pollution, hypertriglyceridemia, occupational exposure to particulate matter and comorbidities including cancer, Chronic Obstructive Pulmonary Disease and Chronic Kidney Disease. These clinical observations are further supported by in vitro experiments and indicate that epigenetic regulation affects the pathophysiology of thrombotic disorders with potential diagnostic or therapeutic utility.
•Thrombosis and anticoagulation related genes have differential methylation in CVDs.•In vitro models further support these findings.•Top ranked genes in several studies are the thrombin receptors.
Antiphospholipid syndrome (APS) is an acquired thrombophilia characterized by recurrent thrombosis and/or pregnancy morbidity, in the presence of antibodies to β2 glycoprotein-I (β2GPI), prothrombin ...or Lupus anticoagulant (LA). Anti-β2GPI antibodies recognize complexes of β2GPI dimers with CXCL4 chemokine and activate platelets. Thrombospondin 1 (TSP-1) is secreted by platelets and exhibits prothrombotic and proinflammatory properties. Therefore, we investigated its implication in APS.
Plasma from APS patients (n = 100), Systemic Lupus Erythematosus (SLE) (n = 27) and healthy donors (HD) (n = 50) was analyzed for TSP-1, IL-1β, IL-17A and free active TGF-β1 by ELISA. Human Umbilical Vein Endothelial Cells (HUVECs) and HD monocytes were treated with total HD-IgG or anti-β2GPI, β2GPI and CXCL4 and CD4+ T-cells were stimulated by monocyte supernatants. TSP-1, IL-1β, IL-17A TGF-β1 levels were quantified by ELISA and Real-Time PCR.
Higher plasma levels of TSP-1 and TGF-β1, which positively correlated each other, were observed in APS but not HDs or SLE patients. Patients with arterial thrombotic events or those undergoing a clinical event had the highest TSP-1 levels. These patients also had detectable IL-1β, IL-17A in their plasma. HD-derived monocytes and HUVECs stimulated with anti-β2GPI-IgG-β2GPI-CXCL4 secreted the highest TSP-1 and IL-1β levels. Supernatants from anti-β2GPI-β2GPI-CXCL4 treated monocytes induced IL-17A expression from CD4+ T-cells. Transcript levels followed a similar pattern.
TSP-1 is probably implicated in the pathogenesis of APS. In vitro cell treatments along with high TSP-1 levels in plasma of APS patients suggest that high TSP-1 levels could mark a prothrombotic state and an underlying inflammatory process.
•Thombospondin-1 (TSP-1) is elevated in patients with Antiphospholipid syndrome.•Patients with both arterial and venous thrombosis have the highest TSP-1 values.•anti-β2GPI-IgG, β2GPI and CXCL4 induce TSP-1 secretion from platelets and HUVECs.•TSP-1 silencing in HUVECs reduces SELE upon anti-β2GPI-IgG-β2GPI-CXCL4 activation.
Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thromboembolism and/or pregnancy morbidity in the presence of Antiphospholipid antibodies, mainly anti-β2 ...glycoprotein I (anti-β2GPI). The autoantibodies lead to monocyte and endothelial cell activation and subsequent secretion of tissue factor (F3) and proinflammatory cytokines, like interleukins 6 (IL6) and 8 (IL8). The etiology of the syndrome remains largely unknown, with the contribution of environmental, genetic and epigenetic factors considered significant.
We aimed to identify epigenetic changes and factors potentially implicated in the pathophysiology of APS. To this end, we compared DNA methylation levels of the IL8 and F3 genes between healthy donors (HDs) and APS patients, using whole blood as a source.
Methylation was significantly reduced in the IL8 promoter and significantly increased in the F3 gene body in APS patients compared to HDs and correlated with specific clinical parameters. In an ex vivo model partially mimicking APS, stimulation of monocytes with a mixture of β2GPI, anti-β2GPI and CXCL4 also induces DNA methylation changes in the above genes, along with increase of their expression. Stimulation of human umbilical vein endothelial cells (HUVECs) with the same mixture also results in transcriptional upregulation of epigenetic factors, including MΕCP2, DNMT3, TET1, HDAC9 and ARID5B.
The above data support that epigenetic alterations could be implicated in the pathophysiology of APS and prompt further investigation of their potential diagnostic or therapeutic utility.
•IL8 promoter methylation is decreased in APS.•In APS patients, IL8 promoter methylation associates with arterial clinical events and more recent events.•F3 first intron methylation is increased in APS.•Stimulation of monocytes and HUVECs causes dynamic changes in F3 and IL8 methylation levels and gene expression.
Abstract The 11th International Symposium for Sjogren’s syndrome was held in Athens, Greece in September 2011. This symposia is part of a long series of meetings that have attempted to meet the needs ...of both scientists and physicians in improving the healthcare of their patients with Sjogren’s syndrome. Sjogren’s syndrome affects almost 0.5% of the general population and is second only to rheumatoid arthritis amongst the systemic autoimmune diseases. More importantly, it has daily implications for the millions of sufferers around the world. The goal of this meeting, which included nearly 200 abstracts and invited lectures, was to address the critical needs in the clinical practice of Sjogren’s syndrome. This volume is a composite of select papers that were presented at this meeting and attempts to provide a critical overview of clinical and basic science. The volume includes a variety of themes and, importantly, raises issues that are still unresolved but which are important in our future diagnostic and therapeutic efforts.
Background:
The clinical significance of pANCA by indirect immunofluorescence is well-established. However, their clinical utility is sometimes hindered by the fact that pANCA are also detected in ...various autoimmune diseases. Myeloperoxidase (MPO) is considered as the major autoantigen recognized by pANCA in ANCA-associated vasculitides (AAV) and predominantly in microscopic polyangiitis (MPA). However, information regarding the targets of pANCA in other autoimmune diseases is still elusive.
Objectives:
To investigate the specific autoantigens recognized by pANCA in autoimmune diseases.
Methods:
Sera from all patients that were found positive for pANCA in the diagnostic laboratories of the Department of Pathophysiology, School of Medicine, National and Kapodistrian University of Athens and the Department of Immunology and Histocompatibility, Evangelismos General Hospital, Athens, Greece during the last two years were studied. The pANCA+ sera were evaluated for reactivity with the major antigens that are recognized by pANCA MPO, lactoferrin, neutrophil elastase, cathepsin G and bactericidal/permeability increasing protein (BPI) by a commercially available multiplex ELISA (ANCA profile ELISA, Euroimmun, Lubeck, Germany).
Results:
A total of 82 patients were included in the study. All patients had positive pANCA by indirect immunofluorescence with a title ranging from 1/160 to 1/640. According to respective classification criteria, 21 patients had systemic vasculitides (15 MPA, 1 granulomatosis with polyangiitis; GPA, 1 Behcet’s disease; BD, 1 aortitis, 2 Henoch-Schonlein purpura; HSP and 1 cryoglobulinemic vasculitis; CV), 29 had systemic lupus erythematosus (SLE), 6 antiphospholipid syndrome (APS), 8 Sjögren’s syndrome (SS), 2 rheumatoid arthritis (RA), 1 systemic scleroderma (SScl), 14 Hashimoto thyroiditis and 1 sarcoidosis. The specificities of pANCA in each entity are shown in the following table.
Autoimmune Diseases
Antigens recognized by pANCA+ sera
MPO
Elastase
Cathepsin G
BPI
Lactoferrin
Vasculitides
MPA
66.7 (10/15)
0 (0/15)
0 (0/15)
0 (0/15)
0 (0/15)
GPA
100 (1/1)
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/1)
BD
100 (1/1)
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/1)
Aortitis
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/1)
HSP
0 (0/2)
0 (0/2)
0 (0/2)
0 (0/2)
0 (0/2)
CV
0 (0/1)
0 (0/1)
0 (0/1)
0(0/1)
100 (1/1)
SLE
6.9 (2/29)
0 (0/29)
0 (0/29)
0 (0/29)
6.9 (2/29)
APS
16.6 (1/6)
16.6 (1/6)
0 (0/6)
0 (0/6)
0 (0/6)
SS
0 (0/8)
12.5 (1/8)
0 (0/8)
12.5 (1/8)
0 (0/8)
RA
50 (1/2)
0 (0/2)
0 (0/2)
0 (0/2)
0 (0/2)
SScl
100 (1/1)
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/1)
Hashimoto
0 (0/14)
0 (0/14)
0 (0/14)
0 (0/14)
0 (0/14)
Sarcoidosis
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/1)
0 (0/1)
Conclusion:
pANCA positive staining in AAVs is highly restricted to MPO specificity. On the contrary, pANCA staining pattern in other autoimmune diseases, involves unknown autoantigens that are under investigation in our laboratory.
Disclosure of Interests:
None declared
To assess the efficacy and safety of the IL-1b inhibitor canakinumab in all adults with refractory Still's disease identified from the National Organization For Medicines for off-label drug use.
: In ...a retrospective longitudinal multicenter cohort of 50 patients (median age 39 years) with active Still's disease despite treatment with corticosteroids (n = 11), conventional and synthetic (n = 34) and/or biologic disease modifying anti-rheumatic drugs (n = 30), we assessed the efficacy of canakinumab 150–300 mg administered every 4 (n = 47) or 8 weeks (n = 3) as combination therapy or monotherapy (n = 7) during a median follow-up of 27 (3–84) months.
Α complete response was initially observed in 78% of patients within 3 months (median), irrespective of age at disease onset. A partial response was evident in 20%. One patient had resistant disease. Treatment de-escalation was attempted in 15 of 39 complete responders and a complete drug discontinuation in 21 patients for 8 months (median). Eleven patients (22%) relapsed during treatment, one during de-escalation process, and 11 after treatment discontinuation. Overall, 9 of 11 relapses were successfully treated with canakinumab treatment intensification or re-introduction. At last visit, 18% of patients were off treatment due to remission and 26% due to disease activity. Canakinumab had a significant corticosteroid sparing effect allowing weaning in 21 of 41 cases. Infections (20%, severe 4%) and leucopenia (6%) led to treatment cessation in one patient.
High rates of sustained remission were observed in this, largest so far, real-life cohort of adult patients with refractory Still's disease treated with canakinumab.
Background:
We
1
and others
2
have previously shown that ICU admitted patients with COVID-19 developed high titers of anti-SARS-CoV-2 antibodies, but also autoantibodies, some of which are ...pathogenic. We re-evaluated 8 patients of those survived after admission to the ICU of Evangelismos Hospital of Athens -1
st
Department of Internal Medicine, Medical School, NKUA
3
6 months later. We did not know whether these autoantibodies still exist, are associated with COVID-19 or with ARDS as described after septic shock
4
.
Objectives:
To investigate the presence and titers of anti-SARS-CoV-2 antibodies and autoantibodies in patients survived after COVID-19 ICU stay, in the ICU and 6 months later.
Methods:
Case series to evaluate titers of anti-SARS-CoV-2 antibodies, specificities of autoantibodies as well as clinical features in ICU admitted COVID-19 patients, initially and 6 months after their discharge. Evaluation of current clinical status included evaluation of lung, heart, kidney, central and peripheral nervous system and mental status using standardized methods. Methods for detection of anti-SARS-CoV-2 antibodies and autoantibodies were described in our previous report
1
.
Results:
We had initially evaluated
1
29 ICU admitted COVID-19 patients’ files and sera, of which 4 had been already died during serum evaluation. Six more patients died thereafter. Out of 19 having been discharged, 8 were willing to be re-evaluated. On second evaluation 6 months later, serum anti-SARS-CoV-2 antibodies were highly positive, although at lower titers compared to the titers at disease onset (median range) 8.705 (range: 7.95-9.56) vs 6.640 (range: 6.29-6.76), p=0.0002, Mann-Whitney test. Initially 3 out of 8 patients expressed antinuclear antibodies (ANA) at titers 1/160, 1/320 and 1/320 with a fine speckled pattern with the second patient also expressing at a titer of 1/160, antimitochondrial (AMA) antibodies. Six months later the same patients and not anyone else expressed ANA of the same pattern at titers 1/640, 1/160 and 1/160 respectively. Two patients with 1/20 p-ANCA and 1/640 c-ANCA initially, lost their respective autoantibodies after 6 months. One patient initially negative for IgM anti-β2GPI became positive at low titer and an initially positive became negative. One patient initially positive for anti-Ro60 antibody continued to be positive 6 months later. One patient initially negative developed anti-Tg antibodies and 3 patients initially positive for anti-TPO antibodies remained positive 6 months later.
Conclusion:
Patients with COVID-19 survived after ICU admission still retain high titers of anti-SARS-CoV-2 antibodies but significantly lower that at disease onset, but they tend to lose autoantibodies with pathogenic potential.
References:
1Vlachoyiannopoulos P et al, Ann Rheum Dis 2020,
22. Wang EY et al, medRxiv preprint doi:
https://doi.org/10.1101/2020.12.10.20247205
3National and Kapodistrian University of Athens, Athens, Greece
4Burbelo et al. Journal of Translational Medicine 2010
Disclosure of Interests:
None declared
Β2-Glycoprotein I (β2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post ...translational modification of β2GPI was examined.The effects of nitrated (n)β2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups.
β2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. β2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated β2GPI loses this binding (p < 0.05) and had diminished activity in inhibiting platelet adhesion to vWF under high shear flow (p < 0.01). Levels of nβ2GPI were increased in patients with primary APS compared to patients with either secondary APS (p < 0.05), autoimmune disease without APS (p < 0.05) or non-autoimmune patients with arterial thrombosis (p < 0.01) and healthy individuals (p < 0.05).In conclusion tyrosine nitration of plasma β2GPI is demonstrated and has important implications with regards to the pathophysiology of platelet mediated thrombosis in APS. Elevated plasma levels of nβ2GPI in primary APS may be a risk factor for thrombosis warranting further investigation.
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•The major autoantigen, B2-GPI in antiphospholipid syndrome can be nitrated.•Nitration of B2-GPI inhibits binding to lipid peroxidation products.•Nitrated B2-GPI loses its anti-thrombotic activity.•Nitrated B2-GPI is increased in patients with primary APS compared to secondary APS and autoimmune patients without APS.
BackgroundNailfold video-capillaroscopy(NVC) is a non-invasive method to assess peripheral microangiopathy. Abnormal capillaroscopic patterns are almost universally found in patients with Systemic ...Sclerosis (SSc) and assist the diagnosis of SSc. However, little is known about the prognostic value of NVC in skin and lung involvement progression in these patients.ObjectivesTo test the hypothesis that baseline capillaroscopic indices, as well as possible changes in capillaroscopic indices over time, correlate with deterioration in skin thickening and lung function tests in a prospective SSc cohort.MethodsFifty-five consecutive SSc patients from a tertiary care university centre (49 women, 29 limited cutaneous SSc, mean age: 50.84±14.88 years, mean disease duration 6.74±6.25 years) were evaluated by NVC at baseline and after a median of 3.1 years. Qualitative assessment of NVC findings permitted categorization of patients to a predominantly normal, early, active or late capillaroscopic pattern. Capillary loss, capillary dilatation, giant or ramified capillaries and microhemorrhages were further assessed using a semi-quantitative rating scale (score 0–3), derived as the mean of three fields in each of the 2nd, 3rd, 4th and 5th finger of both hands. Scoring was performed by two different assessors. Skin thickening was measured using the modified Rodnan Skin Score (mRSS). FVC and DLCO were performed within 6 months from the NVC. Deterioration in FVC and DLCO was considered clinically significant when>10%. Between baseline and follow-up evaluation 36% of patients had been receiving both major antiproliferative and vasodilator therapy, while15% and 29% had been receiving only antiproliferative or vasodilator therapy, respectively.ResultsIntraclass correlation coefficient (ICC) for interrater reliability analyses was very good for all semi-quantitative capillaroscopy scores ICC: 0.97 (0.74–0.99) for capillary loss score, 0.94 (0.85–0.98) for dilatation score,0.98 (0.97–0.99) for giant score, 0.94 (0.84–0.97) for microhemorrhages score), except for the ramification score ICC: 0.52 (-0.2,–0.81) which was excluded from all analyses. Linear regression, adjusted for age and gender, showed no association between either baseline capillaroscopy scores or of their changes and changes in mRSS over time. FVC and DLCO deteriorated in 13 and 11 patients, respectively. Binary logistic regression analysis adjusted for age and gender showed no association either baseline capillaroscopy scores or of their changes with deterioration in FVC or DLCO, with the exception of the change in microhemorrhages score, which correlated inversely with deterioration in FVC p=0.013, Odds Ratio=0.051 (CI: 0.005–0.534). Regarding qualitative analysis of capillaroscopic findings, the baseline capillaroscopy pattern remained unchanged in 35, deteriorated in 15 and improved in 5 patients. No difference was found between these 3 groups regarding change in mRSS (ANOVA, p=0.634), deterioration in FVC (x2test, p=0.502) or deterioration in DLCO (x2 test, p=1.00).ConclusionsAlthough a possible confounding effect of treatment cannot be excluded, NVC seems to have poor prognostic value for the progression of skin thickening and interstitial lung disease in rigorously treated SSc patients.Disclosure of InterestNone declared
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