Pier Luigi Meroni

Scopus Publications

Antibodies Against Anti-Oxidant Enzymes in Autoimmune Glomerulonephritis and in Antibody-Mediated Graft Rejection
Maurizio Bruschi, Giovanni Candiano, Andrea Petretto, Andrea Angeletti, Pier Luigi Meroni, Marco Prunotto, and Gian Marco Ghiggeri
MDPI AG
Historically, oxidants have been considered mechanisms of glomerulonephritis, but a direct cause–effect correlation has never been demonstrated. Several findings in the experimental model of autoimmune conditions with renal manifestations point to the up-regulation of an oxidant/anti-oxidant system after the initial deposition of autoantibodies in glomeruli. Traces of oxidants in glomeruli cannot be directly measured for their rapid metabolism, while indirect proof of their implications is derived from the observation that Superoxide Oxidase 2 (SOD2) is generated by podocytes after autoimmune stress. The up-regulation of other anti-oxidant systems takes place as well. Here, we discuss the concept that a second wave of antibodies targeting SOD2 is generated in autoimmune glomerulonephritis and may negatively influence the clinical outcome. Circulating and renal deposits of anti-SOD2 antibodies have been detected in patients with membranous nephropathy and lupus nephritis, two main examples of autoimmune disease of the kidney, which correlate with the clinical outcome. The presence of anti-SOD2 antibodies in circulation and in the kidney has been interpreted as a mechanism which modifies the normal tissue response to oxidative stress. Overall, these findings repropose the role of the oxidant/anti-oxidant balance in autoimmune glomerulonephritis. The same conclusion on the oxidant/anti-oxidant balance may be proposed in renal transplant. Patients receiving a renal graft may develop antibodies specific for Glutathione Synthetase (GST), which modulates the amount of GST disposable for rapid scavenging of reactive oxygen species (ROS). The presence of anti-GST antibodies in serum is a major cause of rejection. The perspective is to utilize molecules with known anti-oxidant effects to modulate the anti-oxidative response in autoimmune pathology of the kidney. A lot of molecules with known anti-oxidant effects can be utilized, many of which have already been proven effective in animal models of autoimmune glomerulonephritis. Many molecules with anti-oxidant activity are natural products; in some cases, they are constituents of diets. Owing to the simplicity of these drugs and the absence of important adverse effects, many anti-oxidants could be directly utilized in human beings.

Technology Innovation for Discovering Renal Autoantibodies in Autoimmune Conditions
Maurizio Bruschi, Giovanni Candiano, Andrea Petretto, Andrea Angeletti, Pier Luigi Meroni, Marco Prunotto, and Gian Marco Ghiggeri
MDPI AG
Autoimmune glomerulonephritis is a homogeneous area of renal pathology with clinical relevance in terms of its numerical impact and difficulties in its treatment. Systemic lupus erythematosus/lupus nephritis and membranous nephropathy are the two most frequent autoimmune conditions with clinical relevance. They are characterized by glomerular deposition of circulating autoantibodies that recognize glomerular antigens. Technologies for studying renal tissue and circulating antibodies have evolved over the years and have culminated with the direct analysis of antigen–antibody complexes in renal bioptic fragments. Initial studies utilized renal microdissection to obtain glomerular tissue. Obtaining immunoprecipitates after partial proteolysis of renal tissue is a recent evolution that eliminates the need for tissue microdissection. New technologies based on ‘super-resolution microscopy’ have added the possibility of a direct analysis of the interaction between circulating autoantibodies and their target antigens in glomeruli. Peptide and protein arrays represent the new frontier for identifying new autoantibodies in circulation. Peptide arrays consist of 7.5 million aligned peptides with 16 amino acids each, which cover the whole human proteome; protein arrays utilize, instead, a chip containing structured proteins, with 26.000 overall. An example of the application of the peptide array is the discovery in membranous nephropathy of many new circulating autoantibodies including formin-like-1, a protein of podosomes that is implicated in macrophage movements. Studies that utilize protein arrays are now in progress and will soon be published. The contribution of new technologies is expected to be relevant for extending our knowledge of the mechanisms involved in the pathogenesis of several autoimmune conditions. They may also add significant tools in clinical settings and modify the therapeutic handling of conditions that are not considered to be autoimmune.

Molecular subtypes explain lupus epigenomic heterogeneity unveiling new regulatory genetic risk variants
Olivia Castellini-Pérez, Elena Povedano, Guillermo Barturen, Manuel Martínez-Bueno, Andrii Iakovliev, Martin Kerick, Raúl López-Domínguez, Concepción Marañón, Javier Martín, Esteban Ballestar,et al.
Springer Science and Business Media LLC
AbstractThe heterogeneity of systemic lupus erythematosus (SLE) can be explained by epigenetic alterations that disrupt transcriptional programs mediating environmental and genetic risk. This study evaluated the epigenetic contribution to SLE heterogeneity considering molecular and serological subtypes, genetics and transcriptional status, followed by drug target discovery. We performed a stratified epigenome-wide association studies of whole blood DNA methylation from 213 SLE patients and 221 controls. Methylation quantitative trait loci analyses, cytokine and transcription factor activity - epigenetic associations and methylation-expression correlations were conducted. New drug targets were searched for based on differentially methylated genes. In a stratified approach, a total of 974 differential methylation CpG sites with dependency on molecular subtypes and autoantibody profiles were found. Mediation analyses suggested that SLE-associated SNPs in the HLA region exert their risk through DNA methylation changes. Novel genetic variants regulating DNAm in disease or in specific molecular contexts were identified. The epigenetic landscapes showed strong association with transcription factor activity and cytokine levels, conditioned by the molecular context. Epigenetic signals were enriched in known and novel drug targets for SLE. This study reveals possible genetic drivers and consequences of epigenetic variability on SLE heterogeneity and disentangles the DNAm mediation role on SLE genetic risk and novel disease-specific meQTLs. Finally, novel targets for drug development were discovered.

The role of complement in kidney disease: conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference
Marina Vivarelli, Jonathan Barratt, Laurence H. Beck, Fadi Fakhouri, Daniel P. Gale, Elena Goicoechea de Jorge, Marta Mosca, Marina Noris, Matthew C. Pickering, Katalin Susztak,et al.
Elsevier BV

Interferon and B-cell Signatures Inform Precision Medicine in Lupus Nephritis
Ioannis Parodis, Julius Lindblom, Daniel Toro-Domínguez, Lorenzo Beretta, Maria O. Borghi, Jessica Castillo, Elena Carnero-Montoro, Yvonne Enman, Chandra Mohan, Marta E. Alarcón-Riquelme,et al.
Elsevier BV

Tracking the immune response profiles elicited by the BNT162b2 vaccine in COVID-19 unexperienced and experienced individuals
Eugenia Galeota, Valeria Bevilacqua, Andrea Gobbini, Paola Gruarin, Mauro Bombaci, Elisa Pesce, Andrea Favalli, Andrea Lombardi, Francesca Vincenti, Jessica Ongaro,et al.
Elsevier BV

Fibroblasts and Endothelial Cells in Three-Dimensional Models: A New Tool for Addressing the Pathogenesis of Systemic Sclerosis as a Prototype of Fibrotic Vasculopathies
Caterina Bodio, Alessandra Milesi, Paola Adele Lonati, Cecilia Beatrice Chighizola, Alessandro Mauro, Luca Guglielmo Pradotto, Pier Luigi Meroni, Maria Orietta Borghi, and Elena Raschi
MDPI AG
Two-dimensional in vitro cultures have represented a milestone in biomedical and pharmacological research. However, they cannot replicate the architecture and interactions of in vivo tissues. Moreover, ethical issues regarding the use of animals have triggered strategies alternative to animal models. The development of three-dimensional (3D) models offers a relevant tool to investigate disease pathogenesis and treatment, modeling in vitro the in vivo environment. We aimed to develop a dynamic 3D in vitro model for culturing human endothelial cells (ECs) and skin fibroblasts, simulating the structure of the tissues mainly affected in systemic sclerosis (SSc), a prototypical autoimmune fibrotic vasculopathy. Dermal fibroblasts and umbilical vein ECs grown in scaffold or hydrogel, respectively, were housed in bioreactors under flow. Fibroblasts formed a tissue-like texture with the deposition of a new extracellular matrix (ECM) and ECs assembled tube-shaped structures with cell polarization. The fine-tuned dynamic modular system allowing 3D fibroblast/EC culture connection represents a valuable model of the in vivo interplay between the main players in fibrotic vasculopathy as SSc. This model can lead to a more accurate study of the disease’s pathogenesis, avoiding the use of animals, and to the development of novel therapies, possibly resulting in improved patient management.

Antiphospholipid syndrome pathogenesis in 2023: an update of new mechanisms or just a reconsideration of the old ones?
Elena Raschi, Maria Orietta Borghi, Francesco Tedesco, and Pier Luigi Meroni
Oxford University Press (OUP)
Abstract Antibodies against phospholipid (aPL)-binding proteins, in particular, beta 2 glycoprotein I (β2GPI), are diagnostic/classification and pathogenic antibodies in antiphospholipid syndrome (APS). β2GPI-aPL recognize their target on endothelium and trigger a pro-thrombotic phenotype which is amplified by circulating monocytes, platelets and neutrophils. Complement activation is required as supported by the lack of aPL-mediated effects in animal models when the complement cascade is blocked. The final result is a localized clot. A strong generalized inflammatory response is associated with catastrophic APS, the clinical variant characterized by systemic thrombotic microangiopathy. A two-hit hypothesis was suggested to explain why persistent aPL are associated with acute events only when a second hit allows antibody/complement binding by modulating β2GPI tissue presentation. β2GPI/β2GPI-aPL are also responsible for obstetric APS, being the molecule physiologically present in placental/decidual tissues. Additional mechanisms mediated by aPL with different characteristics have been reported, but their diagnostic/prognostic value is still a matter of research.

Stratification according to autoantibody status in systemic sclerosis reveals distinct molecular signatures
Bénedicte Rouvière, Christelle Le Dantec, Eleonore Bettacchioli, Lorenzo Beretta, Nathan Foulquier, Celine Cao, Christophe Jamin, Jacques-Olivier Pers, Martin Kerick, Javier Martin,et al.

OBJECTIVES Systemic sclerosis (SSc) is a heterogeneous disease, complicating its management. Its complexity and the insufficiency of clinical manifestations alone to delineate homogeneous patient groups further challenge this task. However, autoantibodies could serve as relevant markers for the pathophysiological mechanisms driving the disease. Identifying specific immunological mechanisms based on patients' serological statuses might facilitate a deeper understanding of the diversity of the disease. METHODS A cohort of 206 patients with SSc enrolled in the PRECISESADS cross-sectional study was examined. Patients were stratified based on their anti-centromere (ACA) and anti-SCL70 (SCL70) antibody statuses. Comprehensive omics analyses including transcriptomic, flow cytometric, cytokine and metabolomic data were analysed to characterise the differences between these patient groups. RESULTS Patients with SCL70 antibodies showed severe clinical features such as diffuse cutaneous sclerosis and pulmonary fibrosis and were biologically distinguished by unique transcriptomic profiles. They exhibit a pro-inflammatory and fibrotic signature associated with impaired tissue remodelling and increased carnitine metabolism. Conversely, ACA-positive patients exhibited an immunomodulation and tissue homeostasis signature and increased phospholipid metabolism. CONCLUSIONS Patients with SSc display varying biological profiles based on their serological status. The findings highlight the potential utility of serological status as a discriminating factor in disease severity and suggest its relevance in tailoring treatment strategies and future research directions.

Antiphospholipid Antibody Syndrome: Clinical Manifestations and Laboratory Diagnosis
Pier Luigi Meroni, Caterina Bodio, Claudia Grossi, and Maria Orietta Borghi
Wiley

2023 ACR/EULAR antiphospholipid syndrome classification criteria
Medha Barbhaiya, Stephane Zuily, Ray Naden, Alison Hendry, Florian Manneville, Mary-Carmen Amigo, Zahir Amoura, Danieli Andrade, Laura Andreoli, Bahar Artim-Esen,et al.
BMJ
ObjectiveTo develop new antiphospholipid syndrome (APS) classification criteria with high specificity for use in observational studies and trials, jointly supported by the American College of Rheumatology (ACR) and EULAR.MethodsThis international multidisciplinary initiative included four phases: (1) Phase I, criteria generation by surveys and literature review; (2) Phase II, criteria reduction by modified Delphi and nominal group technique exercises; (3) Phase III, criteria definition, further reduction with the guidance of real-world patient scenarios, and weighting via consensus-based multicriteria decision analysis, and threshold identification; and (4) Phase IV, validation using independent adjudicators’ consensus as the gold standard.ResultsThe 2023 ACR/EULAR APS classification criteria include an entry criterion of at least one positive antiphospholipid antibody (aPL) test within 3 years of identification of an aPL-associated clinical criterion, followed by additive weighted criteria (score range 1–7 points each) clustered into six clinical domains (macrovascular venous thromboembolism, macrovascular arterial thrombosis, microvascular, obstetric, cardiac valve, and hematologic) and two laboratory domains (lupus anticoagulant functional coagulation assays, and solid-phase enzyme-linked immunosorbent assays for IgG/IgM anticardiolipin and/or IgG/IgM anti–β2-glycoprotein I antibodies). Patients accumulating at least three points each from the clinical and laboratory domains are classified as having APS. In the validation cohort, the new APS criteria vs the 2006 revised Sapporo classification criteria had a specificity of 99% vs 86%, and a sensitivity of 84% vs 99%.ConclusionThese new ACR/EULAR APS classification criteria were developed using rigorous methodology with multidisciplinary international input. Hierarchically clustered, weighted, and risk-stratified criteria reflect the current thinking about APS, providing high specificity and a strong foundation for future APS research.

The 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria
Medha Barbhaiya, Stephane Zuily, Ray Naden, Alison Hendry, Florian Manneville, Mary‐Carmen Amigo, Zahir Amoura, Danieli Andrade, Laura Andreoli, Bahar Artim‐Esen,et al.
Wiley
ObjectiveTo develop new antiphospholipid syndrome (APS) classification criteria with high specificity for use in observational studies and trials, jointly supported by the American College of Rheumatology (ACR) and EULAR.MethodsThis international multidisciplinary initiative included 4 phases: 1) Phase I, criteria generation by surveys and literature review; 2) Phase II, criteria reduction by modified Delphi and nominal group technique exercises; 3) Phase III, criteria definition, further reduction with the guidance of real‐world patient scenarios, and weighting via consensus‐based multicriteria decision analysis, and threshold identification; and 4) Phase IV, validation using independent adjudicators’ consensus as the gold standard.ResultsThe 2023 ACR/EULAR APS classification criteria include an entry criterion of at least one positive antiphospholipid antibody (aPL) test within 3 years of identification of an aPL‐associated clinical criterion, followed by additive weighted criteria (score range 1–7 points each) clustered into 6 clinical domains (macrovascular venous thromboembolism, macrovascular arterial thrombosis, microvascular, obstetric, cardiac valve, and hematologic) and 2 laboratory domains (lupus anticoagulant functional coagulation assays, and solid‐phase enzyme‐linked immunosorbent assays for IgG/IgM anticardiolipin and/or IgG/IgM anti–β2‐glycoprotein I antibodies). Patients accumulating at least 3 points each from the clinical and laboratory domains are classified as having APS. In the validation cohort, the new APS criteria versus the 2006 revised Sapporo classification criteria had a specificity of 99% versus 86%, and a sensitivity of 84% versus 99%.ConclusionThese new ACR/EULAR APS classification criteria were developed using rigorous methodology with multidisciplinary international input. Hierarchically clustered, weighted, and risk‐stratified criteria reflect the current thinking about APS, providing high specificity and a strong foundation for future APS research.

Effect of the JAK/STAT Inhibitor Tofacitinib on Macrophage Cholesterol Metabolism
Maria Pia Adorni, Bianca Papotti, Maria Orietta Borghi, Elena Raschi, Francesca Zimetti, Franco Bernini, Pier Luigi Meroni, and Nicoletta Ronda
MDPI AG
The impact of JAK/STAT inhibitors, which are used in various inflammatory diseases, on cardiovascular risk is controversial and has recently raised safety concerns. Our study investigates the direct effects of tofacitinib on macrophage cholesterol metabolism, which is crucial for atherosclerosis plaque development and stability. Cultured human macrophages THP-1 were used to assess the impact of tofacitinib on cell cholesterol efflux and synthesis via radioisotopic methods, and on cholesterol uptake by measuring the cell cholesterol content with a fluorometric assay. The cholesterol acceptors and donors were either standard lipoproteins or sera from patients with juvenile idiopathic arthritis (JIA) and from control subjects. Tofacitinib significantly increased the macrophage cholesterol efflux to all acceptors; it reduced cholesterol uptake from both the normal and hypercholesterolemic sera; and it reduced cholesterol synthesis. The treatment of macrophages with tofacitinib was able to increase the cholesterol efflux and decrease cholesterol uptake when using sera from untreated JIA patients with active disease as cholesterol acceptors and donors, respectively. In conclusion, our in vitro data support the concept that tofacitinib has a favorable impact on macrophage cholesterol metabolism, even in the presence of sera from rheumatologic patients, and suggest that other mechanisms may be responsible for the cardiovascular risk associated with tofacitinib use in selected patient populations.

The SPROUT study: A survey on current management practice of reproductive aspects in women of childbearing age with systemic autoimmune rheumatic diseases
Cecilia Beatrice Chighizola, Megan Clowse, Pier Luigi Meroni, Laura Andreoli, Angela Tincani, Marianne Wallenius, and Catherine Nelson-Piercy
Elsevier BV

Correspondence on “European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) SLE classification criteria item performance”
Xavier Bossuyt, Walter Fierz, and Pier Luigi Meroni
BMJ
Antinuclear antibodies (ANA) are important laboratory markers for the diagnosis and classification of systemic lupus erythematosus (SLE). In the 2019 European League Against Rheumatism (EULAR)/American College of Rheumatology (ACR) classification criteria for SLE, ANA with tit re ≥1:80 are an entry criterion. Even though ANA 1:80 are highly sensitive for SLE, they have a low specificity. This has recently been reinforced by Aringer et al who analysed the performance of the individual items included in the 2019 EULAR/ACR classification criteria for SLE on a large group of patients with SLE (n=1197) and nonSLE disease controls (n=1074), including patients with other connective tissue diseases (twothirds of the controls). In this study, ANA with tit re ≥1:80 were highly sensitive (99.5%), but only 19.4% specific for SLE. As ANA are an entry criterion for the 2019 EULAR/ACR classification criteria, the low specificity of ANA for SLE does not affect the specificity of the 2019 SLE classification criteria. An important item that conferred specificity to the 2019 SLE classification was the attribution rule. The authors stressed that ANA are a useful screening test, but not specific and that the classification criteria should not be used as diagnostic criteria. As ANA are employed in the context of both classification and diagnosis, it is important to get better insights into the interpretation of ANA test results. We here argue that interpretation of ANA test results can be improved by assigning titrespecific likelihood ratios (LR)s. The LR is a good indicator of the clinical value of a test result. It is the ratio of the fraction of patients with a particular test results to the fraction of controls with the same test result. For example, a test result with an LR of 5 indicates that such result is 5 times more likely in patients than in controls. Positioning of ANA as an entry criterion in the 2019 EULAR/ ACR classification was based on a literature review and metaregression analysis on the performance of indirect immunofluorescence (IIF) on HEp2 substrates. In the paper that describes this metaregression analysis (including 13 080 patients with SLE and 7539 controls), data on sensitivity, specificity and negative and positive LR of ANA for SLE are given for different cutoffs (1:40, 1:80, 1:160 and 1:320). Figure 1 (left hand part) graphically recapitulates the results of the metaregression analysis. ANA at a cutoff titre of 1:80 had a high sensitivity (97.8%). The specificity and positive LR were 74.7 and 3.87, respectively. Because of the high sensitivity, ANA at 1:80 cutoff by IIF was positioned as an entry criterion for the SLE classification. The LRs reported by Leuchten et al are for single cutoff values, implying that all values higher than the cutoff value are considered equal. For example, for a 1:80 cutoff, all patients with ANA ≥1:80 are grouped (ie, 1:80, 1:160, 1:320, 1:640, ...). Such approach does not take into account the fact that the likelihood for disease increases with increasing antibody levels. Conversely, titrespecific information gives more detailed information on the clinical value of a specific result. Based on the data provided by Leuchten et al, we calculated the titrespecific LRs for a negative, 1:40, 1:80 and 1:160 test result (figure 1, righthand panel). The titrespecific LRs were 0.02, 0.08, 0.17 and 0.94, respectively. These titrespecific LRs are clearly lower than the LRs calculated using a dichotomous approach as reported by Leuchten et al, the reason being that a dichotomous approach groups all values exceeding the cutoff. For example, the titrespecific LR for 1:80 was 0.17, whereas the positive LR associated with a single 1:80 cutoff was 3.87. Of note, the LR associated with a single 1:80 cutoff reported in the recent study by Aringer et al was 1.23. Correspondence

Anti–Neutrophil Extracellular Trap Antibodies in Antiphospholipid Antibody–Positive Patients: Results From the Antiphospholipid Syndrome Alliance for Clinical Trials and InternatiOnal Networking Clinical Database and Repository
Yu Zuo, Sherwin Navaz, Alex Tsodikov, Katarina Kmetova, Lyndsay Kluge, Amala Ambati, Claire K. Hoy, Srilakshmi Yalavarthi, Danieli de Andrade, Maria G. Tektonidou,et al.
Wiley
OBJECTIVE Our study aimed to elucidate the presence, antigen specificities, and potential clinical association of anti-neutrophil extracellular trap (anti-NET) antibodies in a multinational cohort of antiphospholipid antibody (aPL)-positive patients who did not have lupus. METHODS Anti-NET IgG/IgM were measured in sera of 389 aPL-positive patients; 308 met the classification criteria for APS. Multivariate logistic regression with best variable model selection was used to determine clinical associations. For a subset of the patients (n=214), we profiled autoantibodies with an autoantigen microarray platform. RESULTS We found elevated levels of anti-NET IgG and/or IgM in 45% of aPL-positive patients. High anti-NET antibody levels are associated with more circulating myeloperoxidase (MPO)-DNA complexes, a biomarker of NETs. When considering clinical manifestations, positive anti-NET IgG was associated with brain white matter lesions even after adjusting for demographic variables and aPL profiles. Anti-NET IgM tracked with complement consumption after controlling for aPL profiles; furthermore, patient serum containing high levels of anti-NET IgM efficiently deposited complement C3d on NETs. As determined by autoantigen microarray, positive testing for anti-NET IgG was significantly associated with several autoantibodies, including those recognizing citrullinated histones, heparan sulfate proteoglycan, laminin, MPO-DNA complexes, and nucleosomes. Anti-NET IgM positivity associated with autoantibodies targeting single-stranded DNA, double-stranded DNA, and proliferating cell nuclear antigen. CONCLUSION These data reveal high levels of anti-NET antibodies in 45% of aPL-positive patients, where they potentially activate the complement cascade. While anti-NET IgM may especially recognize DNA in NETs, anti-NET IgG species appear more likely to target NET-associated protein antigens. This article is protected by copyright. All rights reserved.

Autoantibodies testing in autoimmunity: Diagnostic, prognostic and classification value
Savino Sciascia, Nicola Bizzaro, Pier Luigi Meroni, Bogdanos Dimitrios, Maria Orietta Borghi, Xavier Bossuyt, Claudia Grossi, Dávid Tornai, Maria Papp, Yehuda Shoenfeld,et al.
Elsevier BV

Plasminogen activator-coated nanobubbles targeting cell-bound β2-glycoprotein I as a novel thrombus-specific thrombolytic strategy
Paolo Macor, Paolo Durigutto, Monica Argenziano, Kate Smith-Jackson, Sara Capolla, Valeria Di Leonardo, Kevin Marchbank, Valerio Stefano Tolva, Fabrizio Semeraro, Concetta T. Ammollo,et al.
Ferrata Storti Foundation (Haematologica)
β2-glycoprotein I (β2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with antiphospholipid syndrome (APS). β2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NB) coated with recombinant tissue plasminogen activator (rtPA) and a recombinant antibody specific for cell-bound β2-GPI. The therapeutic efficacy of targeted NB was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: i) thrombosis developed in a rat model of APS; ii) ferric chloride-induced mesenteric thrombosis in rats, and iii) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted NB bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through β2-GPI expressed on activated cells. In vitro, rtPA-targeted NB (rtPA-tNB) induced greater lysis of platelet-rich blood clots than untargeted NB. In a rat model of APS, administration of rtPA-tNB caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNB, but not untargeted NB and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNB, that target β2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NB. Our findings suggest that targeting cell-bound β2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APS-unrelated thrombotic conditions.

Nailfold Videocapillaroscopic Alterations as Markers of Microangiopathy in COVID-19 Patients
Roberta Gualtierotti, Sharon Fox, Fernanda Da Silva Lameira, Andrea Giachi, Luca Valenti, Maria Borghi, Pier Meroni, Massimo Cugno, Flora Peyvandi, and
MDPI AG
Nailfold videocapillaroscopic alterations have been described in COVID-19, but their correlations with biomarkers of inflammation, coagulation and endothelial perturbation are still unclear, and no information is available on nailfold histopathology. Nailfold videocapillaroscopy was performed on fifteen patients with COVID-19 in Milan, Italy and the signs of microangiopathy were correlated with plasma biomarkers of inflammation (C reactive protein [CRP], ferritin), coagulation (D-dimer, fibrinogen), endothelial perturbation (Von Willebrand factor [VWF]) and angiogenesis (vascular endothelial growth factor [VEGF]) along with genetic drivers of COVID-19 susceptibility. Histopathological analysis of autoptic nailfold excisions was performed on fifteen patients who died for COVID-19 in New Orleans, United States. All COVID-19 patients studied with videocapillaroscopy showed alterations rarely seen in healthy individuals consistent with microangiopathy, such as hemosiderin deposits (sign of microthrombosis and microhemorrhages) and enlarged loops (sign of endotheliopathy). The number of hemosiderin deposits correlated both with ferritin and CRP levels (r = 0.67, p = 0.008 for both) and the number of enlarged loops significantly correlated with the levels of VWF (r = 0.67, p = 0.006). Ferritin levels were higher in non-O groups, determined by the rs657152 C > A cluster, (median 619, min–max 551–3266 mg/dL) than in the O group (373, 44–581 mg/dL, p = 0.006). Nailfold histology revealed microvascular damage, i.e., mild perivascular lymphocyte and macrophage infiltration and microvascular ectasia in the dermal vessels of all cases, and microthrombi within vessels in five cases. Alterations in nailfold videocapillaroscopy and elevated biomarkers of endothelial perturbation that match histopathologic findings open new perspectives in the possibility of non-invasively demonstrating microangiopathy in COVID-19.

Fluctuation of Anti–Domain 1 and Anti–β<inf>2</inf>-Glycoprotein I Antibody Titers Over Time in Patients With Persistently Positive Antiphospholipid Antibodies
Cecilia B. Chighizola, Francesca Pregnolato, Danieli Andrade, Maria Tektonidou, Vittorio Pengo, Guillermo Ruiz‐Irastorza, H. Michael Belmont, Maria Gerosa, Paul Fortin, D. Ware Branch,et al.
Wiley
OBJECTIVE This work aims at evaluating longitudinally titers of antibodies against β2-glycoprotein I (β2GPI) and domain 1 (anti-D1), identifying predictors of the variation of anti-D1 and anti-β2GPI antibody titers and clarifying whether antibody titer fluctuations predict thrombosis in a large international cohort of patients persistently positive for antiphospholipid antibodies (aPL), the "APS ACTION Registry". METHODS Patients with available blood samples from at least 4 time points were included. Anti-β2GPI and anti-D1 IgG were tested by chemiluminescence (BioFlash, INOVA Diagnostics). RESULTS In a cohort of 230 patients, anti-D1 and anti-β2GPI titers decreased significantly over time (p<0.0001 and p=0.010, respectively). After adjustment for age, gender, and number of positive aPL tests, the fluctuation of anti-D1 and anti-β2GPI titers was associated with treatment with hydroxychloroquine (HCQ) at each time-point. Treatment with HCQ, but not immunosuppressors, was associated with 1.3-fold and 1.4-fold decrease in anti-D1 and anti-β2GPI titers, respectively. Incident vascular events were associated with 1.9-fold and 2.1-fold increase of anti-D1 and anti-β2GPI titers, respectively. Anti-D1 and anti-β2GPI titers at the time of thrombosis were lower compared to the other time-points: 1.6-fold decrease in anti-D1 titers and 2-fold decrease in anti-β2GPI titers conferred an OR for incident thrombosis of 6.0 (95%CI 0.62-59.3) and 9.4 (95%CI 1.1-80.2), respectively. CONCLUSIONS Treatment with HCQ and incident vascular events significant predicted anti-D1 and anti-β2GPI titer fluctuation over time. Both anti-D1 and anti-β2GPI titers drop around the time of thrombosis, with potential clinical relevance. This article is protected by copyright. All rights reserved.

Anti-SSA/Ro positivity and congenital heart block: obstetric and foetal outcome in a cohort of anti-SSA/Ro positive pregnant patients with and without autoimmune diseases
Micaela Fredi, Lorenza Maria Argolini, Fabrizio Angeli, Laura Trespidi, Veronique Ramoni, Sonia Zatti, Tamara Vojinovic, Davide Donzelli, Federica Giulia Gazzola, Blerina Xoxi,et al.
Clinical and Experimental Rheumatology
OBJECTIVES Neonatal lupus (NL) is an acquired disease caused by the transplacental passage of anti-SSA/Ro antibodies. The rate of congenital heart block (CHB), its most serious manifestation, ranges from 1 to 5%. The aim of this study was to retrospectively assess the prevalence of CHB in anti-SSA/Ro positive pregnant women with or without systemic autoimmune diseases from 2010 to 2020. METHODS Patients underwent monthly visit and a shared follow-up programme of weekly (16th-24th week) foetal heart rate assessment by obstetric ultrasound. RESULTS 322 pregnancies in 258 anti-SSA/Ro patients were included; 314 were followed from the beginning of pregnancy because of the known presence of anti-SSA/Ro autoantibodies and 1 case of CHB occurred in an anti-SSA/Ro+ asymptomatic subject (0.3%). In the same period, 8 additional patients were referred to our clinics after in utero CHB diagnosis and subsequent discovery of anti-SSA/Ro without a disease diagnosis. Globally, 9 cases of congenital CHB (2.8%) occurred: 7 complete, 1 II-III degree and 1 rst degree CHB. Anti-SSB/La positivity was associated with a higher risk of CHB (7.8% vs. 1.2%; p=0.0071). No differences in maternal or foetal outcomes were found in comparison with a large cohort of unselected pregnancies except for caesarian section. Hydroxychloroquine (HCQ) was used in 58.3% pregnancies, with a different prevalence according with maternal diagnosis. CONCLUSIONS Our data suggest that anti-SSA/Ro positive patents with a de ned systemic autoimmune disease undergoing a strict follow-up since positive pregnancy test display a low risk of pregnancy complications, including but not limited to NL.

Head-to-head comparison of four COVID-19 vaccines on platelet activation, coagulation and inflammation. The TREASURE study
Marta Brambilla, Paola Canzano, Patrizia Della Valle, Alessia Becchetti, Maria Conti, Mariangela Alberti, Arianna Galotta, Maria Luisa Biondi, Paola Adele Lonati, Fabrizio Veglia,et al.
Elsevier BV

Immunology of pregnancy and reproductive health in autoimmune rheumatic diseases. Update from the 11<sup>th</sup> International Conference on Reproduction, Pregnancy and Rheumatic Diseases
Laura Andreoli, Cecilia B. Chighizola, Luca Iaccarino, Angela Botta, Maria Gerosa, Véronique Ramoni, Chiara Tani, Bonnie Bermas, Antonio Brucato, Jill Buyon,et al.
Elsevier BV

Beta 2 glycoprotein I and neutrophil extracellular traps: Potential bridge between innate and adaptive immunity in anti-phospholipid syndrome
Claudia Grossi, Nagaja Capitani, Marisa Benagiano, Cosima Tatiana Baldari, Chiara Della Bella, Paolo Macor, Francesco Tedesco, Maria Orietta Borghi, Norma Maugeri, Mario Milco D’Elios,et al.
Frontiers Media SA
Antiphospholipid syndrome (APS) is a systemic autoimmune disorder characterized by recurrent vascular thrombosis and miscarriages in the absence of known causes. Antibodies against phospholipid-binding proteins (aPL) are pathogenic players in both clotting and pregnancy APS manifestations. There is sound evidence that antibodies specific for beta2 glycoprotein I (β2GPI) trigger thrombotic and pregnancy complications by interacting with the molecule on the membranes of different cell types of the coagulation cascade, and in placenta tissues. In addition to the humoral response against β2GPI, both peripheral and tissue CD4+ β2GPI-specific T cells have been reported in primary APS as well as in systemic lupus erythematosus (SLE)-associated APS. While adaptive immunity plays a clear role in APS, it is still debated whether innate immunity is involved as well. Acute systemic inflammation does not seem to be present in the syndrome, however, there is sound evidence that complement activation is crucial in animal models and can be found also in patients. Furthermore, neutrophil extracellular traps (NETs) have been documented in arterial and venous thrombi with different etiology, including clots in APS models. Keeping in mind that β2GPI is a pleiotropic glycoprotein, acting as scavenger molecule for infectious agents and apoptotic/damaged body constituents and that self-molecules externalized through NETs formation may become immunogenic autoantigens, we demonstrated β2GPI on NETs, and its ability to stimulate CD4+β2GPI-specific T cells. The aim of this review is to elucidate the role of β2GPI in the cross-talk between the innate and adaptive immunity in APS.

Complement activation predicts negative outcomes in COVID-19: The experience from Northen Italian patients.
Pier Luigi Meroni, Stefania Croci, Paola Adele Lonati, Francesca Pregnolato, Lucia Spaggiari, Giulia Besutti, Martina Bonacini, Ilaria Ferrigno, Alessandro Rossi, Geir Hetland,et al.
Elsevier BV

Pier Luigi Meroni

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