Publications by authors named "Maria Orietta Borghi"

62 Publications

An elevated polyclonal free light chain level reflects a strong interferon signature in patients with systemic autoimmune diseases.

J Transl Autoimmun 2021 2;4:100090. Epub 2021 Mar 2.

Laboratory of Immunology and Immunotherapy, CHRU Morvan, Brest, France.

High amount of polyclonal free light chains (FLC) are reported in systemic autoimmune diseases (SAD) and we took advantage of the PRECISESADS study to better characterize them. Serum FLC levels were explored in 1979 patients with SAD (RA, SLE, SjS, Scl, APS, UCTD, MCTD) and 614 healthy controls. Information regarding clinical parameters, disease activity, medications, autoantibodies (Ab) and the interferon α and/or γ scores were recorded. Among SAD patients, 28.4% had raised total FLC (from 12% in RA to 30% in SLE and APS) with a normal kappa/lambda ratio. Total FLC levels were significantly higher in SAD with inflammation, active disease in SLE and SjS, and an impaired pulmonary functional capacity in SSc, while independent from kidney impairment, infection, cancer and treatment. Total FLC concentrations were positively correlated among the 10/17 (58.8%) autoantibodies (Ab) tested with anti-RNA binding protein Ab (SSB, SSA-52/60 kDa, Sm, U1-RNP), anti-dsDNA/nucleosome Ab, rheumatoid factor and negatively correlated with complement fractions C3/C4. Finally, examination of interferon (IFN) expression as a potential driver of FLC overexpression was tested showing an elevated level of total FLC among patients with a high IFNα and IFNγ Kirou's score, a strong IFN modular score, and the detection in the sera of B-cell IFN dependent factors, such as TNF-R1/TNFRSF1A and CXCL10/IP10. In conclusion, an elevated level of FLC, in association with a strong IFN signature, defines a subgroup of SAD patients, including those without renal affectation, characterized by increased disease activity, autoreactivity, and complement reduction.
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http://dx.doi.org/10.1016/j.jtauto.2021.100090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010703PMC
March 2021

Vandetanib versus Cabozantinib in Medullary Thyroid Carcinoma: A Focus on Anti-Angiogenic Effects in Zebrafish Model.

Int J Mol Sci 2021 Mar 16;22(6). Epub 2021 Mar 16.

Laboratory of Geriatric and Oncologic Neuroendocrinology Research, Istituto Auxologico Italiano, IRCCS, 20095 Cusano Milanino, MI, Italy.

Medullary thyroid carcinoma (MTC) is a tumor deriving from the thyroid C cells. Vandetanib (VAN) and cabozantinib (CAB) are two tyrosine kinase inhibitors targeting REarranged during Transfection (RET) and other kinase receptors and are approved for the treatment of advanced MTC. We aim to compare the in vitro and in vivo anti-tumor activity of VAN and CAB in MTC. The effects of VAN and CAB on viability, cell cycle, and apoptosis of TT and MZ-CRC-1 cells are evaluated in vitro using an MTT assay, DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, respectively. In vivo, the anti-angiogenic potential of VAN and CAB is evaluated in transgenic fluorescent zebrafish embryos by analyzing the effects on the physiological development of the sub-intestinal vein plexus and the tumor-induced angiogenesis after TT and MZ-CRC-1 xenotransplantation. VAN and CAB exert comparable effects on TT and MZ-CRC-1 viability inhibition and cell cycle perturbation, and stimulated apoptosis with a prominent effect by VAN in MZ-CRC-1 and CAB in TT cells. Regarding zebrafish, both drugs inhibit angiogenesis in a dose-dependent manner, in particular CAB shows a more potent anti-angiogenic activity than VAN. To conclude, although VAN and CAB show comparable antiproliferative effects in MTC, the anti-angiogenic activity of CAB appears to be more relevant.
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http://dx.doi.org/10.3390/ijms22063031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8002338PMC
March 2021

A cytokine network profile delineates a common Th1/Be1 pro-inflammatory group of patients in four systemic autoimmune diseases.

Arthritis Rheumatol 2021 Feb 18. Epub 2021 Feb 18.

UMR1227, Lymphocytes B et Autoimmunité, Université de Brest, INSERM, CHU de Brest, Brest, France.

Objective: The effector T-cell and B-cell cytokine networks have been implicated in the pathogenesis of systemic autoimmune diseases (SAD), but an exploration of this association with the heterogeneity of clinical manifestations and immune profiles has not been carefully examined. The objective of this study was to examine whether cytokine profiles could delineate distinct groups of patients in four SAD (systemic lupus erythematosus, Sjögren syndrome, rheumatoid arthritis, and systemic sclerosis).

Methods: 179 patients and 48 healthy volunteers were enrolled in the multi-centric cross-sectional PRECISESADS. Multi-low-dimensional omics data (cytokines, autoantibodies, immune circulating cells) were examined. Co-culture experiments were used to test the impact of the cytokine microenvironment on the T- and B-cell cross-talk.

Results: A pro-inflammatory cytokine profile defined by high levels of CXCL10, IL-6, IL-2, and TNF-α characterized a distinct group of patients in the four SAD. In each disease, this pro-inflammatory cluster was associated with a specific immune circulating cell signature, a more severe disease, and higher levels of autoantibodies, suggesting an uncontrolled pro-inflammatory Th1 immune response. We observed in vitro that B cells reinforce Th1 differentiation and naïve T-cell proliferation leading to the induction of B-cell effector type-1 (Be1) cells and IgG production. This process is associated with an increase in CXCL10, IL-6, IL-2, and IFN-γproduction.

Conclusions: This composite analysis brings new insights into human B cell functional heterogeneity based on the T/B-cell crosstalk and, on the other hand, proposes a better stratification of SAD patients suggesting that combined biomarkers would be a great value to design personalized treatments.
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http://dx.doi.org/10.1002/art.41697DOI Listing
February 2021

Integrative Analysis Reveals a Molecular Stratification of Systemic Autoimmune Diseases.

Arthritis Rheumatol 2020 Dec 8. Epub 2020 Dec 8.

Andalusian Public Health System Biobank, Granada, Spain.

Objective: Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis.

Methods: Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time.

Results: Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient.

Conclusion: Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases.
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http://dx.doi.org/10.1002/art.41610DOI Listing
December 2020

β2 glycoprotein I participates in phagocytosis of apoptotic neurons and in vascular injury in experimental brain stroke.

J Cereb Blood Flow Metab 2021 Jan 14:271678X20984551. Epub 2021 Jan 14.

Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Department of Neuroscience, Milan, Italy.

Beta-2 Glycoprotein I (β2-GPI) is the main target of anti-phospholipid antibodies (aPL) in the autoimmune anti-phospholipid syndrome, characterized by increased risk of stroke. We here investigated the antibody independent role of β2-GPI after ischemia/reperfusion, modeled by transient middle cerebral artery occlusion (tMCAo) in male C57Bl/6J mice; by subjecting immortalized human brain microvascular endothelial cells (ihBMEC) to 16 h hypoxia and 4 h re-oxygenation. (coding for β2-GPI) was upregulated selectively in the liver at 48 h after tMCAo. At the same time β2-GPI circulating levels increased. β2-GPI was detectable in brain parenchyma and endothelium at all time points after tMCAo. Parenchymal β2-GPI recognized apoptotic neurons (positive for annexin V, C3 and TUNEL) cleared by CD68+ brain macrophages. Hypoxic ihBMEC showed increased release of IL-6, over-expression of and after re-oxygenation with β2-GPI alone. β2-GPI interacted with mannose-binding lectin in mouse plasma and ihBMEC medium, potentially involved in formation of thrombi. We show for the first time that brain ischemia triggers the hepatic production of β2-GPI. β2-GPI is present in the ischemic endothelium, enhancing vascular inflammation, and extravasates binding stressed neurons before their clearance by phagocytosis. Thus β2-GPI may be a new mediator of brain injury following ischemic stroke.
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http://dx.doi.org/10.1177/0271678X20984551DOI Listing
January 2021

Anti-Phospholipid Antibodies in COVID-19 Are Different From Those Detectable in the Anti-Phospholipid Syndrome.

Front Immunol 2020 15;11:584241. Epub 2020 Oct 15.

Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, Istituto di Ricovero Cura a Carattere Scientifico (IRCCS), Milan, Italy.

Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPLs) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-βGPI), and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-βGPI antibodies was not reported.

Objective: To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-βGPI antibodies.

Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events.

Results: Anti-βGPI IgG/IgA/IgM was the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM was detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of βGPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-βGPI nor with thrombosis.

Conclusions: aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against βGPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.
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http://dx.doi.org/10.3389/fimmu.2020.584241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593765PMC
December 2020

Scleroderma-specific autoantibodies embedded in immune complexes mediate endothelial damage: an early event in the pathogenesis of systemic sclerosis.

Arthritis Res Ther 2020 11 9;22(1):265. Epub 2020 Nov 9.

Experimental Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Via Zucchi 18, Cusano Milanino, 20095, Milan, Italy.

Background: Consistently with their diagnostic and prognostic value, autoantibodies specific for systemic sclerosis (SSc) embedded in immune complexes (ICs) elicited a pro-inflammatory and pro-fibrotic cascade in healthy skin fibroblasts, engaging Toll-like receptors (TLRs) via their nucleic acid components. The objective of this study was to investigate the pathogenicity of SSc-ICs in endothelial cells.

Methods: ICs were purified from the sera of SSc patients bearing different autoantibody specificities (antibodies against DNA topoisomerase I, centromeric proteins, RNA polymerase, and Th/To), patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS), or healthy controls (NHS) using polyethylene glycol precipitation. Human umbilical vein endothelial cells (HUVECs) were incubated with ICs, positive and negative controls. mRNA levels of endothelin-1 (et-1), collagenIα1 (colIα1), interferon (IFN)-α, and IFN-β were investigated by real-time PCR; et-1 and il-6 mRNA levels were assessed after pre-treatment with bafilomycin. ICAM-1 expression was evaluated by cell ELISA; secretion of IL-6, IL-8, and transforming growth factor (TGF)-β1 in culture supernatants was measured by ELISA. The expression of Fcγ receptors (CD64, CD32, and CD16) was assessed in endothelial cells at FACS analysis. Intracellular signaling pathways culminating with NFκB, p38MAPK, SAPK-JNK, and Akt were assessed by Western blotting. Healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with ICs, and TGF-β1 secretion and mRNA levels of colIα1 and matrix metalloproteinase (mmp)-1, protein expression of α smooth muscle actin (α-SMA), and IL-6 were evaluated by Western blotting; et-1 mRNA levels were assessed in fibroblasts pre-treated with IL-6 and TGF-β inhibitors and stimulated with ATA-ICs.

Results: All SSc stimulated IL-6 secretion; ACA-ICs and anti-Th/To-ICs increased ICAM-1 expression; all SSc-ICs but anti-Th/To-ICs augmented IL-8 levels; all SSc-ICs but ACA-ICs and ARA-ICs upregulated et-1, and all SSc-ICs but ARA-ICs affected TGF-β1 secretion. colIα1, IFN-α, and IFN-β mRNA levels were not affected by any SSc-IC. FcγRII (CD32) and FcγRIII (CD16) were not detectable on HUVECs, while FcγRI (CD64) was minimally expressed. A differential modulation of tlr expression was observed: tlr2, tlr3, and tlr4 were upregulated by ATA-ICs and ACA-ICs, while anti-Th/To-ICs resulted in tlr9 upregulation. Pre-treatment with bafilomycin did not affect the upregulation of et-1 and il-6 induced by ATA-ICs, ACA-ICs, and anti-Th/To-ICs; a 23% reduction in both genes was reported for ARA-ICs. All SSc-ICs activated p38MAPK and Akt, and all SSc-ICs but ARA-ICs yielded the activation of NFκB; ATA-ICs and ACA-ICs increased the activation rate of both subunits of SAPK-JNK. When healthy skin fibroblasts were stimulated with supernatants from HUVECs incubated with SSc-ICs, TGF-β1 secretion, colIα1, α-SMA, and IL-6 expression levels were significantly modulated. Pre-treatment with IL-6 and TGF-β inhibitors prevented et-1 upregulation induced by ATA-ICs by 85% and 77%, respectively.

Conclusions: These data provide the first demonstration of the pathogenicity of ICs from scleroderma patients with different autoantibodies on the endothelium. Endothelial activation induced by SSc-ICs ultimately led to a pro-fibrotic phenotype in healthy skin fibroblasts.
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http://dx.doi.org/10.1186/s13075-020-02360-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7654597PMC
November 2020

Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases.

Nat Rev Rheumatol 2020 12 5;16(12):715-726. Epub 2020 Nov 5.

Istituto Auxologico Italiano, IRCCS; Immunorheumatology Research Laboratory, Milan, Italy.

Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).
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http://dx.doi.org/10.1038/s41584-020-00522-wDOI Listing
December 2020

Complement activation and endothelial perturbation parallel COVID-19 severity and activity.

J Autoimmun 2021 01 29;116:102560. Epub 2020 Oct 29.

Università degli Studi di Milano, Department of Pathophysiology and Transplantation, Milan, Italy; Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Internal Medicine and Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Milan, Italy.

Background: Animal models and few clinical reports suggest the involvement of the complement system in the onset of severe manifestations of coronavirus disease-2019 (COVID-19). However, complement contribution to endotheliopathy and hypercoagulability has not been elucidated yet.

Objective: To evaluate the association among complement activation, endothelial damage and disease severity or activity in COVID-19 patients.

Methods: In this single-centre cohort study, 148 patients with COVID-19 of different severity were evaluated upon hospital admission and 30 days later. Markers of complement activation (SC5b-9 and C5a) and endothelial perturbation (von Willebrand factor [vWF], tissue-type plasminogen activator [t-PA], plasminogen activator inhibitor-1 [PAI-1], soluble thrombomodulin [sTM], and soluble endothelial selectin [sE-selectin]) were measured in plasma.

Results: The patients had high plasma levels of SC5b-9 and C5a (p = 0.0001 for both) and vWF, t-PA and PAI-1 (p = 0.0001 for all). Their SC5b-9 levels correlated with those of vWF (r = 0.517, p = 0.0001) and paralleled disease severity (severe vs mild p = 0.0001, severe vs moderate p = 0.026 and moderate vs mild p = 0.001). The levels of sE-selectin were significantly increased only in the patients with severe disease. After 30 days, plasma SC5b-9, C5a and vWF levels had significantly decreased (p = 0.0001 for all), and 43% of the evaluated patients had normal levels.

Conclusions: Complement activation is boosted during the progression of COVID-19 and dampened during remission, thus indicating its role in the pathophysiology of the disease. The association between complement activation and the biomarkers of endothelial damage suggests that complement may contribute to tissue injury and could be the target of specific therapy.
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http://dx.doi.org/10.1016/j.jaut.2020.102560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598768PMC
January 2021

Efficacy of a novel second-generation somatostatin-dopamine chimera (TBR-065) in human medullary thyroid cancer: a preclinical study.

Neuroendocrinology 2020 Oct 19. Epub 2020 Oct 19.

Introduction: Somatostatin and dopamine receptors have a pivotal role in control of hormone secretion and cell proliferation in different neuroendocrine neoplasms, including medullary thyroid cancer (MTC). In the present preclinical study, we evaluated the antitumor activity of TBR-065 (formerly BIM-23B065), a second-generation somatostatin-dopamine chimera, in two human MTC cell lines.

Methods: the effects of lanreotide (LAN) and TBR-065 on the cell growth proliferation, calcitonin secretion, cell cycle, apoptosis, cell migration and tumor-induced angiogenesis have been evaluated through MTT assay, DNA flow cytometry with propidium iodide, and Annexin V-FITC/propidium iodide staining, ECLIA assay, wound-healing assay and zebrafish platform, respectively.

Results: TBR-065 exerted a more prominent antitumor activity compared to LAN in both MTC cell lines, as shown by inhibition of cell proliferation (maximal inhibition in TT: -50.3% and -37.6%, respectively; in MZ-CRC-1: -58.8% and -27%, respectively) and migration (in TT: -42.7% and -22.9%, respectively; in MZ-CRC-1: -75.5% and -58.2%, respectively). Only the new chimera decreased significantly the fraction of cells in S phase (TT: -33.8%, MZ-CRC-1: -18.8%), and increased cells in G2/M phase (TT: +13%, MZ-CRC-1: +30.5%). In addition, TBR-065 exerted a more prominent pro-apoptotic effect compared to LAN in TT cells. A concomitant decrease of calcitonin secretion was observed after 2 days of incubation with both drugs, with a more relevant effect of TBR-065. However, neither LAN nor TBR-065 showed any effect on tumor-induced angiogenesis, as evaluated using a zebrafish/tumor xenograft model.

Discussion/conclusion: In MTC cell lines a second generation somatostatin-dopamine analogue, TBR-065, exerts a more relevant anti-tumor activity, as compared with LAN, through modulation of cell cycle, induction of apoptosis and reduction in migration. Further studies are required to establish whether TBR-065 has comparable potent inhibitory effects on tumor growth in vivo.
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http://dx.doi.org/10.1159/000512366DOI Listing
October 2020

Prevalence, specificity, and clinical association of anti-phospholipid antibodies in COVID-19 patients: are the antibodies really guilty?

medRxiv 2020 Jun 19. Epub 2020 Jun 19.

Background: Critically ill patients with coronavirus disease 2019 (COVID-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. Because of a prolonged activated partial-thromboplastin time (aPTT), a relationship with anti-phospholipid antibodies (aPL) has been proposed, but results are controversial. Functional assays for aPL (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of C reactive protein. The presence of anti-cardiolipin (aCL), anti-beta2-glycoprotein I (anti-β GPI and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies was not investigated systematically. Epitope specificity of anti-β GPI antibodies was not reported.

Aim: To evaluate the prevalence and the clinical association of aPL in a large cohort of COVID-19 patients, and to characterize the epitope specificity of anti-β GPI antibodies.

Methods: ELISA and chemiluminescence assays were used to test 122 sera of patients suffering from severe COVID-19. Of them, 16 displayed major thrombotic events.

Results: Anti-β GPI IgG/IgA/IgM were the most frequent in 15.6/6.6/9.0% of patients, while aCL IgG/IgM were detected in 5.7/6.6% by ELISA. Comparable values were found by chemiluminescence. aPS/PT IgG/IgM were detectable in 2.5 and 9.8% by ELISA. No association between thrombosis and aPL was found. Reactivity against domain 1 and 4-5 of β GPI was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with aCL/anti-β GPI nor with thrombosis.

Conclusion: aPL show a low prevalence in COVID-19 patients and are not associated with major thrombotic events. aPL in COVID-19 patients are mainly directed against β GPI but display an epitope specificity different from antibodies in antiphospholipid syndrome.
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http://dx.doi.org/10.1101/2020.06.17.20134114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310661PMC
June 2020

EUREKA algorithm predicts obstetric risk and response to treatment in women with different subsets of anti-phospholipid antibodies.

Rheumatology (Oxford) 2021 Mar;60(3):1114-1124

Experimental Laboratory of Immunological and Rheumatologic Researches, Istituto Auxologico Italiano, IRCCS, Cusano Milanino, Milan, Italy.

Objectives: aPL, the serum biomarkers of APS, are the most common acquired causes of pregnancy morbidity (PM). This study investigates the impact of aPL positivity fulfilling classification criteria ('criteria aPL') and at titres lower than thresholds considered by classification criteria ('low-titre aPL') on PM and assesses the effectiveness of low-dose aspirin (LDASA), low molecular weight heparin (LMWH) and HCQ in reducing the probability of PM (PPM).

Methods: Longitudinal data on 847 pregnancies in 155 women with persistent aPL at any titre and 226 women with autoimmune diseases and negative aPL were retrospectively collected. A generalized estimating equations model for repeated measures was applied to quantify PPM under different clinical situations.

Results: EUREKA is a novel algorithm that accurately predicts the risk of aPL-associated PM by considering aPL titres and profiles. aPL significantly impact PPM when at low titres and when fulfilling classification criteria. PPM was further stratified upon the aPL tests: aCL IgG/IgM and anti-β2-glycoprotein I (β2GPI) IgM, alone or combined, do not affect the basal risks of PPM, an increase occurs in case of positive LA or anti-β2GPI IgG. LDASA significantly affects PPM exclusively in women with low-titre aPL without anti-β2GPI IgG. The LDASA + LMWH combination significantly reduces PPM in all women with low-titre aPL and women with criteria aPL, except those carrying LA and anti-β2GPI IgG. In this group, the addition of HCQ further reduces PPM, although not significantly.

Conclusion: EUREKA allows a tailored therapeutic approach, impacting everyday clinical management of aPL-positive pregnant women.
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http://dx.doi.org/10.1093/rheumatology/keaa203DOI Listing
March 2021

In utero exposure to Azathioprine in autoimmune disease. Where do we stand?

Autoimmun Rev 2020 Sep 30;19(9):102525. Epub 2020 Mar 30.

Synapse Research Institute, Maastricht, the Netherlands; Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands.

Azathioprine (AZA), an oral immunosuppressant, is safe during pregnancy. Some reports suggested different impairments in the offspring of mothers with autoimmune diseases (AI) exposed in utero to AZA. These observations are available from retrospective studies or case reports. However, data with respect to the long-term safety in the antenatally exposed child are still lacking. The aim of this study is to summarize the current knowledge in this field and to focus on the need for a prospective study on this population. We performed a PubMed search using several search terms. The actual data show that although the risk of congenital anomalies in offspring, as well as the infertility risk, are similar to those found in general population, there is a higher incidence of prematurity, of lower weight at birth and an intra-uterine delay of development. There is also an increased risk of materno- fetal infections, especially cytomegalovirus infection. Some authors raise the interrogations about neurocognitive impairment. Even though the adverse outcomes might well be a consequence of maternal illness and disease activity, interest has been raised about a contribution of this drug. However, the interferences between the external agent (in utero exposure to AZA), with the host (child genetic susceptibility, immune system anomalies, emotional status), environment (public health, social context, availability of health care), economic, social, and behavioral conditions, cultural patterns, are complex and represent confounding factors. In conclusion, it is necessary to perform studies on the medium and long-term outcome of children born by mothers with autoimmune diseases, treated with AZA, in order to show the safety of AZA exposure. Only large-scale population studies with long-term follow-up will allow to formally conclude in this field. TAKE HOME MESSAGES.
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http://dx.doi.org/10.1016/j.autrev.2020.102525DOI Listing
September 2020

Personalized medicine in rheumatoid arthritis: How immunogenicity impacts use of TNF inhibitors.

Autoimmun Rev 2020 May 12;19(5):102509. Epub 2020 Mar 12.

Istituto Auxologico Italiano, IRCCS, Experimental Laboratory of Immuno-rheumatology, Milan, Italy; Department of Clinical Sciences and Community Health, University of Milan, Italy.

Up to 40% of patients treated with tumor necrosis factor alpha inhibitors (TNFi) do not respond to therapy. Testing drug bioavailability and/or anti-drug antibody (ADAb) levels may justify dosage adjustment or switch to different drugs, enabling a personalized medicine approach. We report a multicenter cross-sectional study on different methods [ELISA and a cell based functional assay (reporter gene assay - RGA)] for drug/ADAb detection, and on the relationship between drug bioavailability and ADAb. 163 patients with rheumatoid arthritis (RA) treated with infliximab (IFX; n = 67), adalimumab (ADL; n = 49) or etanercept (ETA; n = 47) were tested for drug and ADAb levels. Furthermore, we report prospective data from additional 70 patients (59 RA and 11 juvenile idiopathic arthritis - JIA) tested for drug and ADAb levels at baseline (T0) and after 3 (T3) and 6 months (T6) of treatment with ADL or ETA only. IFX-treated patients were not included because of the increasing use of IFX biosimilars. Stringent inclusion criteria were used in order to avoid unwanted variables in both studies; none of the patients used TNFi before the study, and TNFi was used only in combination with methotrexate. Clinical response was defined according to EULAR response criteria. The two assays performed comparably in the comparison study. Accordingly, ELISA was selected for the prospective study because of its feasibility in the clinical setting. The cross-sectional study found ADAb in IFX and ADL treated groups only, that were associated with a decrease in pharmacological drug availability in the blood. Comparable results were found for the ADL-treated group in the prospective study which also showed a relationship between drug/ADAb levels and the loss of clinical response. Altogether our findings support drug and anti-drug Ab monitoring in the real-world clinical setting thus enabling individualized treatment and reducing disability in chronic inflammatory arthritis.
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http://dx.doi.org/10.1016/j.autrev.2020.102509DOI Listing
May 2020

Two Novel Technologies for the Detection of Anti-cardiolipin and Anti β2-Glycoprotein Antibodies in the Real Life: Chemiluminescent in Comparison to the Addressable Laser Bead Immunoassays.

Immunol Invest 2020 Feb 12;49(1-2):58-68. Epub 2019 Aug 12.

Immunology and Allergology Laboratory Unit, S.Giovanni di Dio Hospital, Azienda USL Toscana Centro, Florence, Italy.

In the present study, we evaluated two novel technologies, the chemiluminescent immunoassay (CIA) QUANTA Flash on BIO-FLASH (Inova Diagnostics, San Diego, CA, USA) and the addressable laser bead immunoassay (ALBIA) on BioPlex™ 2200 (Bio-Rad, Hercules, CA, USA) for the detection of anti-cardiolipin IgG/IgM (aCL) and anti-β2-glycoprotein IgG/IgM (aβ2GPI) antibodies. The study was performed on 134 samples from consecutive patients (59 males and 75 females, mean age 54 ± 10 years) who consulted a rheumatologist because thrombosis and/or pregnancy complications were present or another immunological disease (Sjogren's syndrome, inflammatory arthritis). Fourteen patients of the total fulfilled 25the Sydney criteria for APS and for these patients previous results of aPLs were available. Sera were tested for aCL and aβ2GPI of IgG and IgM isotypes using CIA (BIO-FLASH) and ALBIA (BioPlex™ 2200). Overall agreement between CIA and ALBIA ranged from 88.1% (aCL IgG) to 97.8% (aβ2GPI IgG). Cohen's kappa coefficient ranged from 0.53 to 0.91, implying moderate to almost perfect agreement. Almost perfect agreement was found between BioPlex™ 2200 and BIO-FLASH aβ2GPI IgG and aCL IgM with Cohen's kappa of 0.91 and 0.88, respectively. On the other hand, moderate agreement was found between BioPlex™ 2200 and BIO-FLASH aCL IgG and β2GPI IgM assays with Cohen's kappa of 0.57 and 0.53, respectively. The two novel technologies look promising and comparable but further studies with larger cohorts are needed to contribute to the better understanding of the new aPLs antibodies assays performance.
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http://dx.doi.org/10.1080/08820139.2019.1647233DOI Listing
February 2020

Only monospecific anti-DFS70 antibodies aid in the exclusion of antinuclear antibody associated rheumatic diseases: an Italian experience.

Clin Chem Lab Med 2019 Oct;57(11):1764-1769

SOS Laboratorio Immunologia e Allegologia Ospedale S. Giovanni di Dio Firenze, Florence, Italy.

Background The dense fine speckled (DFS) is one of the most common patterns that can be observed as a result of the anti-nuclear antibodies (ANA) test on HEp-2 cells and is mostly caused by antibodies to DFS70 as the main antigenic target. As was recently demonstrated, isolated anti-DFS70 positivity can be used as an aid in the exclusion of ANA associated rheumatic diseases (AARD) due to the opportunity to better interpret unexplained positive IIF ANA results. Methods Our study included 333 subjects with AARD, 51 undifferentiated connective tissue disease (UCTD) patients, 235 disease controls and 149 healthy blood donors from an Italian cohort. All samples were tested for anti-DFS70 and anti-ENA antibodies using QUANTA Flash assays (Inova Diagnostics, San Diego, CA, USA). Results No differences in the prevalence of anti-DFS70 antibodies were seen among AARD, non-AARD and UCTD (2.1% [7/333] vs. 2.3% [9/384] vs. 5.9% [3/51], respectively; p-value = 0.188). AARD patients positive for anti-DFS70 antibodies showed in all cases an accompanying anti-ENA specificity. In contrast, monospecific anti-DFS70 antibodies showed a significantly different distribution with a clear trend across the main groups (AARD vs. non-AARD vs. UCTD: 0% [0/7] vs. 22% [2/9] vs. 100% [3/3], p = 0.007). Anti-DFS70 antibody levels among AARD, non-AARD and UCTD patients were not significantly different (p = 0.094). Within the anti-DFS70 antibody positive cases, AARD cohort showed a higher variability (median [min-max]: 3.2 [3.2-450.8] CU) compared to non-AARD (median [min-max]: 3.2 [3.2-75.7] CU) and UCTD patients (median [min-max]: 3.2 [3.2-59.0] CU). Conclusions Our preliminary data showed a similar frequency of anti-DFS70 antibodies in AARD, UCTD and non-AARD cohorts. Monospecificity of anti-DFS70 antibodies but not their mere presence is the key element in the diagnostic algorithm. Mono-specific anti-DFS70 antibodies might be a helpful biomarker to discriminate individuals with AARD from non-AARD presenting with a positive ANA.
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http://dx.doi.org/10.1515/cclm-2019-0454DOI Listing
October 2019

Blood Cell-Bound C4d as a Marker of Complement Activation in Patients With the Antiphospholipid Syndrome.

Front Immunol 2019 12;10:773. Epub 2019 Apr 12.

Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, IRCCS, Milan, Italy.

Antiphospholipid syndrome (APS) is a chronic and disabling condition characterized by recurrent thrombosis and miscarriages mediated by antibodies against phospholipid-binding proteins (aPL), such as betaglycoprotein I (βGPI). Complement is involved in APS animal models and complement deposits have been documented in placenta and thrombotic vessels despite normal serum levels. Analysis of circulating blood cells coated with C4d displays higher sensitivity than the conventional assays that measure soluble native complement components and their unstable activation products in systemic lupus erythematosus (SLE). As C4d-coated blood cell count has been reported to be more sensitive than serum levels of complement components and their activation products in systemic lupus erythematosus (SLE) patients, we decided to evaluate the percentage of C4d positive B lymphocytes (BC4d), erythrocytes (EC4d), and platelets (PC4d) in primary APS patients and asymptomatic aPL positive carriers as marker of complement activation in APS. We assessed by flow cytometry the percentages of BC4d, EC4d, and PC4d in primary APS (PAPS; n. 23), 8 asymptomatic aPL positive carriers, 11 APS-associated SLE (SAPS), 17 aPL positive SLE, 16 aPL negative SLE, 8 aPL negative patients with previous thrombosis, 11 immune thrombocytopenia (ITP) patients, and 26 healthy subjects. In addition, we used an model to evaluate the ability of a monoclonal anti-βGPI antibody (MBB2) to bind to normal resting or activated platelets and fix complement. EC4d and PC4d percentages were significantly higher in PAPS and aPL carriers as well as aPL positive SLE and SAPS than in aPL negative controls. The highest values were found in PAPS and in SAPS. The EC4d and PC4d percentages were significantly correlated with serum C3/C4 and anti-βGPI/anti-cardiolipin IgG. studies showed that MBB2 bound to activated platelets only and induced C4d deposition. The detection of the activation product C4d on circulating erythrocytes and platelets supports the role of complement activation in APS. Complement may represent a new therapeutic target for better treatment and prevention of disability of APS patients.
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http://dx.doi.org/10.3389/fimmu.2019.00773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6474283PMC
August 2020

Interleukin-17/Interleukin-21 and Interferon-γ producing T cells specific for β2 Glycoprotein I in atherosclerosis inflammation of systemic lupus erythematosus patients with antiphospholipid syndrome.

Haematologica 2019 12 14;104(12):2519-2527. Epub 2019 Mar 14.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

Systemic lupus erythematosus is frequently associated with antiphospholipid syndrome. Patients with lupus-antiphospholipid syndrome are characterized by recurrent arterial/venous thrombosis, miscarriages, and persistent presence of autoantibodies against phospholipid-binding proteins, such as β2-Glycoprotein I. We investigated the cytokine production induced by β2-Glycoprotein I in activated T cells that infiltrate atherosclerotic lesions of lupus-antiphospholipid syndrome patients. We examined the helper function of β2-Glycoprotein I-specific T cells for tissue factor production, as well as their cytolytic potential and their helper function for antibody production. Lupus-antiphospholipid syndrome patients harbor activated CD4 T cells that recognize β2-Glycoprotein I in atherosclerotic lesions. β2-Glycoprotein I induces T-cell proliferation and expression of both Interleukin-17/Interleukin-21 and Interferon-γ in plaque-derived T-cell clones. β2-Glycoprotein I-specific T cells display strong help for monocyte tissue factor production, and promote antibody production in autologous B cells. Moreover, plaque-derived β2-Glycoprotein I-specific CD4 T lymphocytes express both perforin-mediated and Fas/FasLigand-mediated-cytotoxicity. Altogether, our results indicate that β2-Glycoprotein I is able to elicit a local Interleukin-17/Interleukin-21 and Interferon-γ inflammation in lupus-antiphospholipid syndrome patients that might lead, if unabated, to plaque instability and subsequent arterial thrombosis, suggesting that the T helper 17/T helper 1 pathway may represent a novel target for the prevention and treatment of the disease.
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http://dx.doi.org/10.3324/haematol.2018.209536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6959190PMC
December 2019

Detection of anti-adalimumab antibodies in a RA responsive cohort of patients using three different techniques.

Anal Biochem 2019 02 22;566:133-138. Epub 2018 Nov 22.

Laboratory of Peptide and Protein Chemistry and Biology, PeptLab, Italy; Department of Neurosciences, Psychology, Drug Research and Child Health, Section of Pharmaceutical Sciences and Nutraceutics, University of Florence, Via Ugo Schiff 6, I-50019, Sesto Fiorentino, Italy. Electronic address:

Reliable monitoring of clinical relevant anti-drug antibodies is fundamental in the follow-up of patients under adalimumab treatment. The aim of this study is to compare anti-adalimumab antibodies by using three methods based on different technologies. A cross-sectional study was performed in 50 patients with rheumatoid arthritis (RA) treated with adalimumab. Anti-adalimumab antibodies were detected in patients' sera by different techniques: bridging ELISA, reporter gene assay (RGA), and surface plasmon resonance (SPR). Results showed that all methods recognized anti-adalimumab antibodies and the percentage of positives fluctuated among the assays. Five (10%) of the 50 patients were positive in ELISA, 4 (8%) in RGA, and 6 (12%) in SPR. Among positive patients, 4 were positive in the three assays, one patient uniquely in ELISA, and two in SPR. Spearman correlation between ELISA and RGA showed good agreement (Spearman r = 0.800). No correlation between RGA and SPR was observed (Spearman r = 0.108). Similar results were obtained between ELISA and SPR (Spearman r = - 0.241). Summarizing, ELISA, RGA and SPR recognized anti-adalimumab antibodies in few RA patients, showing good agreement among the methodology employed. On the other hand, differences observed between SPR and ELISA or RGA highlight the relevance of the employed technologies in anti-drug antibody identification.
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http://dx.doi.org/10.1016/j.ab.2018.11.018DOI Listing
February 2019

New insight into antiphospholipid syndrome: antibodies to β2glycoprotein I-domain 5 fail to induce thrombi in rats.

Haematologica 2019 04 15;104(4):819-826. Epub 2018 Nov 15.

Istituto Auxologico Italiano, IRCCS, Laboratory of Immuno-Rheumatology, Milan, Italy

Clinical studies have reported different diagnostic/predictive values of antibodies to domain 1 or 4/5 of βglycoproteinI in terms of risk of thrombosis and pregnancy complications in patients with antiphospholipid syndrome. To obtain direct evidence for the pathogenic role of anti-domain 1 or anti-domain 4/5 antibodies, we analyzed the pro-coagulant effect of two groups of 5 sera IgG each reacting selectively with domain 1 or domain 5 in lipopolysaccharide (LPS)-treated rats. Antibody-induced thrombus formation in mesenteric vessels was followed by intravital microscopy, and vascular deposition of βglycoproteinI, human IgG and C3 was analyzed by immunofluorescence. Five serum IgG with undetectable anti-βglycoproteinI antibodies served as controls. All the anti-domain 1-positive IgG exhibited potent pro-coagulant activity while the anti-domain 5-positive and the negative control IgG failed to promote blood clot and vessel occlusion. A stronger granular deposit of IgG/C3 was found on the mesenteric endothelium of rats treated with anti-domain 1 antibodies, as opposed to a mild linear IgG staining and absence of C3 observed in rats receiving anti-domain 5 antibodies. Purified anti-domain 5 IgG, unlike anti-domain 1 IgG, did not recognize cardiolipin-bound βglycoproteinI while being able to interact with fluid-phase βglycoproteinI. These findings may explain the failure of anti-domain 5 antibodies to exhibit a thrombogenic effect , and the interaction of these antibodies with circulating βglycoproteinI suggests their potential competitive role with the pro-coagulant activity of anti-domain 1 antibodies. These data aim at better defining "really at risk" patients for more appropriate treatments to avoid recurrences and disability.
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http://dx.doi.org/10.3324/haematol.2018.198119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442945PMC
April 2019

HIBISCUS: Hydroxychloroquine for the secondary prevention of thrombotic and obstetrical events in primary antiphospholipid syndrome.

Autoimmun Rev 2018 Dec 12;17(12):1153-1168. Epub 2018 Oct 12.

MITOVASC institute and CARFI facility, UMR CNRS 6015, INSERM U1083, University of Angers, Angers, France.

The relapse rate in antiphospholipid syndrome (APS) remains high, i.e. around 20%-21% at 5 years in thrombotic APS and 20-28% in obstetrical APS [2, 3]. Hydroxychloroquine (HCQ) appears as an additional therapy, as it possesses immunomodulatory and anti-thrombotic various effects [4-16]. Our group recently obtained the orphan designation of HCQ in antiphospholipid syndrome by the European Medicine Agency. Furthermore, the leaders of the project made the proposal of an international project, HIBISCUS, about the use of Hydroxychloroquine in secondary prevention of obstetrical and thrombotic events in primary APS. This study has been launched in several countries and at now, 53 centers from 16 countries participate to this international trial. This trial consists in two parts: a retrospective and a prospective study. The French part of the trial in thrombosis has been granted by the French Minister of Health in December 2015 (the academic trial independent of the pharmaceutical industry PHRC N PAPIRUS) and is coordinated by one of the members of the leading consortium of HIBISCUS.
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http://dx.doi.org/10.1016/j.autrev.2018.05.012DOI Listing
December 2018

Effects of human recombinant type I IFNs (IFN-α2b and IFN-β1a) on growth and migration of primary endometrial stromal cells from women with deeply infiltrating endometriosis: A preliminary study.

Eur J Obstet Gynecol Reprod Biol 2018 Nov 3;230:192-198. Epub 2018 Oct 3.

Istituto Auxologico Italiano IRCCS, Laboratorio Sperimentale di Ricerche di Neuroendocrinologia Geriatrica ed Oncologica, Milan, Italy; Department of Clinical Sciences and Community Health (DISCCO), University of Milan, Milan, Italy. Electronic address:

Objective(s): Endometriosis is a major cause of infertility and disability for women, caused by the presence of inflammatory endometrial implants in extrauterine locations. Among the constituents involved in the immune response during the development of endometriosis, several chemokines, including interferons (IFNs) may have a role in the pathogenesis of this disease. The aim of this preliminary study was to investigate the anti-proliferative and anti-migratory activities of type I IFNs (IFN-α2b and IFN-β1a) in primary endometrial stromal cells (ESCs) isolated from women with deeply infiltrating endometriosis (DIE).

Study Design: The study subjects included 7 women ranged in the age from 27 to 37 years with diagnosis of DIE (Stage III and IV). Collected primary ESC monolayers, isolated from endometriotic nodules, were incubated with various concentrations (from 1 to 1000 IU/ml) of IFN-α2b or IFN-β1a.

Result(s): IFN-β1a had a significantly higher activity in hampering the proliferation of cells compared to IFN-α2b. This effect could be related to the induction of apoptosis and cell cycle arrest in S phase, observed in ESCs during incubation with IFN-β1a. Moreover, IFN-β1a was more potent than IFN-α2b in inhibiting migration and EGF-induced ERK activity of primary ESCs.

Conclusion(s): The inhibitory in vitro effect on ESC proliferation and migration of IFN-β1a was much more potent than IFN-α2b. These preliminary data offer the rationale for future preclinical and clinical trials using IFN-β1a as a new tool for the therapy and tertiary prevention in patients with DIE.
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http://dx.doi.org/10.1016/j.ejogrb.2018.10.004DOI Listing
November 2018

Immune complexes containing scleroderma-specific autoantibodies induce a profibrotic and proinflammatory phenotype in skin fibroblasts.

Arthritis Res Ther 2018 08 29;20(1):187. Epub 2018 Aug 29.

Experimental Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Via Zucchi 18, 20095 Cusano Milanino, Milan, Italy.

Background: In systemic sclerosis (SSc), autoantibodies provide the most accurate tool to predict the disease subset and pattern of organ involvement. Scleroderma autoantibodies target nucleic acids or DNA/RNA-binding proteins, thus SSc immune complexes (ICs) can embed nucleic acids. Our working hypothesis envisaged that ICs containing scleroderma-specific autoantibodies might elicit proinflammatory and profibrotic effects in skin fibroblasts.

Methods: Fibroblasts were isolated from skin biopsies obtained from healthy subjects and patients with diffuse cutaneous SSc (dcSSc). ICs were purified by polyethylene-glycol precipitation from sera of SSc patients bearing different autoantibodies. ICs from patients with systemic lupus erythematosus (SLE) and primary anti-phospholipid syndrome (PAPS) and from normal healthy subjects (NHS) were used as controls. After incubation with ICs, fibroblasts were evaluated for ICAM-1 expression, interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, matrix metalloproteinase (MMP)-2, tumor growth factor (TGF)-β1 and Pro-CollagenIα1 secretion, collagen (col)Iα1, mmp-1, toll-like receptor (tlr)2, tlr3, tlr4, tlr7, tlr8, tlr9, interferon (ifn)-α, ifn-β and endothelin-1 mRNA, and NFκB, p38MAPK and SAPK-JNK activation rate. Experiments were also performed after pretreatment with DNase I/RNase and NFκB/p38MAPK inhibitors.

Results: The antigenic reactivity for each SSc-IC mirrored the corresponding serum autoantibody specificity, while no positivity was observed in NHS-ICs or sera. SSc-ICs but not NHS-ICs increased ICAM-1 expression, stimulated IL-6, IL-8, MMP-2, MCP-1, TGF-β1 and Pro-CollagenIα1 secretion, upregulated et-1, ifn-α, ifn-β, tlr2, tlr3 and tlr4, and activated NFκB, p38MAPK and SAPK-JNK. tlr9 was significantly upregulated by ARA-ICs, mmp-1 was significantly induced by ACA-ICs whereas colIα1 was not modulated by any SSc-ICs. SLE-ICs and PAPS-ICs significantly upregulated MMP-2 and activated NFκB, p38MAPK and SAPK-JNK. SLE-ICs and PAPS-ICs did not affect colIα1, mmp-1 and Pro-CollagenIα1. DNase I and RNase treatment significantly reduced the upregulation of study mediators induced by SSc-ICs. Pretreatment with NFκB/p38MAPK inhibitors suggested that response to anti-Th/To-ICs was preferentially mediated by p38MAPK whereas ATA-ICs, ACA-ICs and ARA-ICs engaged both mediators. In dcSSc fibroblasts, stimulation with SSc-ICs and NHS-ICs upregulated IL-6 and IL-8.

Conclusions: These data provide the first demonstration of the proinflammatory and profibrotic effects of SSc-ICs on fibroblasts, suggesting the potential pathogenicity of SSc autoantibodies. These effects might be mediated by Toll-like receptors via the interaction with nucleic acid fragments embedded in SSc-ICs.
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http://dx.doi.org/10.1186/s13075-018-1689-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6116570PMC
August 2018

Pathogenic Role of Complement in Antiphospholipid Syndrome and Therapeutic Implications.

Front Immunol 2018 19;9:1388. Epub 2018 Jun 19.

Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, Milan, Italy.

Antiphospholipid syndrome (APS) is an acquired autoimmune disease characterized by thromboembolic events, pregnancy morbidity, and the presence of antiphospholipid (aPL) antibodies. There is sound evidence that aPL act as pathogenic autoantibodies being responsible for vascular clots and miscarriages. However, the exact mechanisms involved in the clinical manifestations of the syndrome are still a matter of investigation. In particular, while vascular thrombosis is apparently not associated with inflammation, the pathogenesis of miscarriages can be explained only in part by the aPL-mediated hypercoagulable state and additional non-thrombotic effects, including placental inflammation, have been described. Despite this difference, evidence obtained from animal models and studies in APS patients support the conclusion that complement activation is a common denominator in both vascular and obstetric APS. Tissue-bound aPL rather than circulating aPL-beta2 glycoprotein I immune complexes seem to be responsible for the activation of the classical and the alternative complement pathways. The critical role of complement is supported by the finding that complement-deficient animals are protected from the pathogenic effect of passively infused aPL and similar results have been obtained blocking complement activation. Moreover, elevated levels of complement activation products in the absence of abnormalities in regulatory molecules have been found in the plasma of APS patients, strongly suggesting that the activation of complement cascade is the result of aPL binding to the target antigen rather than of a defective regulation. Placental complement deposits represent a further marker of complement activation both in animals and in patients, and there is also some suggestive evidence that complement activation products are deposited in the affected vessels. The aim of this review is to analyze the state of the art of complement involvement in the pathogenesis of APS in order to provide insights into the role of this system as predictive biomarker for the clinical manifestations and as therapeutic target.
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http://dx.doi.org/10.3389/fimmu.2018.01388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018396PMC
June 2018

Obstetric and vascular antiphospholipid syndrome: same antibodies but different diseases?

Nat Rev Rheumatol 2018 07;14(7):433-440

Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, Milan, Italy.

Recurrent thrombosis and miscarriages are the main clinical manifestations of antiphospholipid syndrome (APS). Although most patients display both clinical signs, some patients can have isolated vascular or obstetric variants. Emerging data raise the question of whether obstetric and vascular APS are the same or different diseases. An important difference between the two conditions is that a thrombophilic state is a common feature in vascular APS, whereas clot occlusions of the decidual spiral arteries are seldom observed in obstetric APS, and infarctions are found in only one-third of APS placentae. Conversely, inflammation, which is undetectable in vascular APS, is frequently observed in the placentae of patients with obstetric APS and has been documented in the placentae of pregnant mice with fetal loss mediated by antiphospholipid antibodies. Attempts to identify different antibodies or epitopes responsible for the two clinical manifestations of APS have so far been unsuccessful. Possible mechanisms exist that might explain the development of the two clinical presentations, including the tissue distribution and expression level of the main target antigen of antiphospholipid antibodies, β2 glycoprotein I (β2GPI). The identification of the factors that promote the onset of either obstetric or vascular APS has important diagnostic and therapeutic implications.
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http://dx.doi.org/10.1038/s41584-018-0032-6DOI Listing
July 2018

Diagnostic laboratory tests for systemic autoimmune rheumatic diseases: unmet needs towards harmonization.

Clin Chem Lab Med 2018 09;56(10):1743-1748

Experimental Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Via Zucchi 18, 20095 Cusano Milanino, Milan, Italy.

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http://dx.doi.org/10.1515/cclm-2018-0066DOI Listing
September 2018

Pitfalls of antinuclear antibody detection in systemic lupus erythematosus: the positive experience of a national multicentre study.

Ann Rheum Dis 2019 06 25;78(6):e50. Epub 2018 Apr 25.

Immunorheumatology Research Laboratory, IRCCS Istituto Auxologico Italiano, Milan, Italy.

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http://dx.doi.org/10.1136/annrheumdis-2018-213516DOI Listing
June 2019

Successful sequential therapy with rituximab and belimumab in patients with active systemic lupus erythematosus: a case series.

Clin Exp Rheumatol 2018 Jul-Aug;36(4):643-647. Epub 2018 Feb 27.

Lupus Clinic, Division of Rheumatology, ASST Pini, Department of Clinical Sciences and Community Health, University of Milan; Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, IRCCS, Milan, Italy.

Objectives: B cells play an important role in the initiation and progression of systemic lupus erythematosus (SLE). Accordingly, B cell-targeted therapy has been suggested as a new rational approach for treating lupus. Belimumab, a human monoclonal antibody directed against B lymphocyte stimulator (BLyS), was reported as the first biological treatment effective in reducing mild-to-moderate SLE disease activity by using different scoring systems and endpoints. Conversely clinical trials with rituximab, a chimeric monoclonal antibody directed against the CD20 expressed by B cells, have failed to achieve primary endpoints in spite of a number of reports showing its beneficial effects. Anecdotal reports have described the sequential use of rituximab and belimumab as a more effective treatment than using the individual drugs alone, without compromising safety.

Methods: We report a case series of three patients with active SLE refractory to conventional therapies, who underwent treatment with rituximab followed by belimumab as maintenance therapy.

Results: We observed a beneficial effect after sequential treatment with rituximab and belimumab. All patients achieved long-standing remission and could reduce or discontinue corticosteroids. Concomitantly, after rituximab administration we observed a rise in BLyS levels, which were dramatically reduced after belimumab introduction.

Conclusions: The modulation of plasma BLyS kinetics in patients undergoing sequential treatment with rituximab and belimumab may represent a possible rationale behind the effectiveness of this combined therapy.
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September 2018

Beyond thrombosis: Anti-β2GPI domain 1 antibodies identify late pregnancy morbidity in anti-phospholipid syndrome.

J Autoimmun 2018 06 14;90:76-83. Epub 2018 Feb 14.

Department of Clinical Sciences and Community Health, University of Milan, Via Festa del Perdono 7, 20122 Milan, Italy; Experimental Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Via Zucchi 18, 20095 Cusano Milanino, Milan, Italy; Department of Rheumatology, ASST Istituto Gaetano Pini & CTO, Piazza Cardinal Ferrari, 1 20122 Milan, Italy. Electronic address:

Antibodies against β2 glycoprotein I (anti-β2GPI) have been identified as the main pathogenic autoantibody subset in anti-phospholipid syndrome (APS); the most relevant epitope is a cryptic and conformation-dependent structure on β2GPI domain (D) 1. Anti-β2GPI domain profiling has been investigated in thrombotic APS, leading to the identification of antibodies targeting D1 as the main subpopulation. In contrast, scarce attention has been paid to obstetric APS, hence this study aimed at characterizing the domain reactivity with regards to pregnancy morbidity (PM). To this end, 135 women with persistently positive, medium/high titre anti-β2GPI IgG, without any associated systemic autoimmune diseases and at least one previous pregnancy were included: 27 asymptomatic carriers; 53 women with obstetric APS; 20 women with thrombotic APS; and 35 women with both thrombotic and obstetric complications. Anti-D1 and anti-D4/5 antibodies were tested using a chemiluminescent immunoassay and a research ELISA assay, respectively (QUANTA Flash β2GPI Domain 1 IgG and QUANTA Lite β2GPI D4/5 IgG, Inova Diagnostics). Positivity for anti-D1 antibodies, but not anti-D4/5 antibodies, was differently distributed across the 4 subgroups of patients (p < 0.0001) and significantly correlated with thrombosis (χ2 = 17.28, p < 0.0001) and PM (χ2 = 4.28, p = 0.039). Patients with triple positivity for anti-phospholipid antibodies displayed higher anti-D1 titres and lower anti-D4/5 titres compared to women with one or two positive tests (p < 0.0001 and p = 0.005, respectively). Reactivity against D1 was identified as a predictor for PM (OR 2.4, 95% confidence interval [CI] 1.2-5.0, p = 0.017); in particular, anti-D1 antibodies were predictive of late PM, conveying an odds ratio of 7.3 (95% CI 2.1-25.5, p = 0.022). Positivity for anti-D1 antibodies was not associated with early pregnancy loss. Anti-D4/5 antibodies were not associated with clinical APS manifestations. As a whole, our data suggest that anti-D1 antibodies are significantly associated not only with thrombosis, but also with late PM, while positive anti-D4/5 antibodies are not predictive of thrombosis or PM.
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http://dx.doi.org/10.1016/j.jaut.2018.02.002DOI Listing
June 2018