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OBJECTIVE: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays a role in blastocyst implantation and is down-regulated in preeclampsia and in hypertensive pregnancy disorders associated with defective extravillous trophoblast invasion. Defective placentation and severe preeclampsia are also features of the antiphospholipid syndrome (APS). The purpose of this study was to investigate whether abnormal HB-EGF expression plays a pathogenic role in antiphospholipid antibody (aPL)-mediated defective placentation. METHODS: HB-EGF expression in placental tissue was evaluated by Western blotting and messenger RNA analysis in normal and APS placentae. Polyclonal IgG fractions or monoclonal beta(2)-glycoprotein I-dependent aPL and their respective controls were investigated for the following 4 features: their binding to human trophoblast monolayers, as determined by cell enzyme-linked immunosorbent assay (ELISA); their effect on HB-EGF expression by Western blotting in trophoblast cell extracts as well as by ELISA as a protein secreted in the culture supernatants; their inhibitory effect on in vitro trophoblast invasiveness, as evaluated by Matrigel assay; and their inhibitory effect on matrix metalloproteinase (MMP) levels, as measured by gelatin zymography. Experiments were also performed in the presence of serial concentrations of heparin or recombinant HB-EGF. RESULTS: Placental APS tissue displayed reduced expression of HB-EGF. Polyclonal and monoclonal aPL bound to trophoblast monolayers and significantly reduced the in vitro synthesis and secretion of HB-EGF. Heparin inhibited aPL binding and restored HB-EGF expression in a dose-dependent manner. Addition of recombinant HB-EGF reduced the in vitro aPL-induced inhibition of Matrigel invasiveness as well as MMP-2 levels. CONCLUSION: These preliminary findings suggest that the reduction of aPL-mediated HB-EGF represents an additional mechanism that is responsible for the defective placentation associated with APS and that heparin protects from aPL-induced damage by inhibiting antibody binding. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Clinical evidence and experimental evidence suggest that anti-Ro/La autoantibodies are necessary but not sufficient for the development of congenital complete heart block (CCHB). Maternal, fetal, and environmental factors may also contribute to heart damage in CCHB. The aim of our study was to investigate polymorphisms of transforming growth factor beta1 (TGFbeta1) and tumor necrosis factor alpha (TNFalpha) genes in twins and triplets discordant for CCHB whose mothers are anti-Ro/La positive. METHODS: We studied 2 families in which 1 of the mothers gave birth to triplets and the other gave birth to twins. Only 1 child in each family was affected by CCHB, although 1 of the triplets had incomplete heart block. We analyzed TNFalpha and TGFbeta1 polymorphisms in the 2 babies with CCHB and their siblings. TNFalpha polymorphisms at the promoter region and TGFbeta1 polymorphisms at codons 10 and 25 were determined using polymerase chain reaction-restriction fragment length polymorphism analysis. In addition, the production of TGFbeta1 and TNFalpha by resting or mitogen-stimulated peripheral blood mononuclear cells in cell culture supernatants was evaluated by enzyme-linked immunosorbent assay. RESULTS: The profibrotic TGFbeta1 genotype was detected in the twin with CCHB but not in the healthy twin, while all of the triplets displayed the same TGFbeta1 genotype at codon 10. Peripheral blood mononuclear cells from the children with CCHB displayed higher spontaneous and mitogen-stimulated TGFbeta1 secretion than was observed in their siblings. No differences regarding TNFalpha polymorphisms and secretion of TNFalpha were observed. CONCLUSION: The results of this study suggest that, besides anti-Ro/La autoantibodies, a fetal profibrotic response might contribute to the development of CCHB, but additional pathogenic mechanism(s) are also likely to play a role. [ABSTRACT FROM AUTHOR]

β2-glycoprotein I (β2GPI) is a cofactor for anti-phospholipid (aPL) binding to cardiolipin (CL)- coated plates. β2GPI is also able to bind to endothelial cell (EC) membranes as supported by in-vivo as well as by in-vitro studies. The PL-binding site in the fifth domain of the molecule is involved in the adhesion to endothelium. Actually, specific mutations in this molecular portion abolish endothelium binding and a synthetic peptide spanning the sequence Glu274-Cys288 of the CL binding site displays comparable adhesion to EC monolayers. Heparan sulphate appears to be one of the anionic EC membrane structures with which cationic β2GPI interacts, as supported by studies with heparitinase-treated EC. β2GPI binding to EC might be related to its activity as endothelial growth factor or as a lipid-carrying glycoprotein. Adhesion of β2GPI to endothelial membranes offers suitable epitopes for circulating aPL that, once bound, can induce cell activation [ABSTRACT FROM PUBLISHER]

Objective: Primary antiphospholipid syndrome (PAPS) is a rare autoimmune disease characterized by the presence of antiphospholipid antibodies and the occurrence of thrombotic events and pregnancy complications. Our study aimed to identify novel genetic susceptibility loci associated with PAPS., Methods: We performed a genome-wide association study comprising 5,485 individuals (482 affected individuals) of European ancestry. Significant and suggestive independent variants from a meta-analysis of approximately 7 million variants were evaluated for functional and biological process enrichment. The genetic risk variability for PAPS in different populations was also assessed. Hierarchical clustering, Mahalanobis distance, and Dirichlet Process Mixtures with uncertainty clustering methods were used to assess genetic similarities between PAPS and other immune-mediated diseases., Results: We revealed genetic associations with PAPS in a regulatory locus within the HLA class II region near HLA-DRA and in STAT1-STAT4 with a genome-wide level of significance; 34 additional suggestive genetic susceptibility loci for PAPS were also identified. The disease risk allele near HLA-DRA is associated with overexpression of HLA-DRB6, HLA-DRB9, HLA-DQA2, and HLA-DQB2 in immune cells, vascular tissue, and nervous tissue. This association is independent of the association between PAPS and HLA-DRB1*1302. Functional analyses highlighted immune-related pathways in PAPS-associated loci. The comparison with other immune-mediated diseases revealed a close genetic relatedness to neuromyelitis optica, systemic sclerosis, and Sjögren syndrome, suggesting co-localized causal variations close to STAT1-STAT4, TNPO3, and BLK., Conclusion: This study represents a comprehensive large-scale genetic analysis for PAPS and provides new insights into the genetic basis and pathophysiology of this rare disease., (© 2024 The Author(s). Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Competing Interests: Declaration of competing interests There are no competing interests to disclose.

The present review reports the history of our scientific collaboration with Professor Shoenfeld's group. The collaboration started at the end of the 80s and was mainly focused on studies on the pathogenetic mechanisms of the anti-phospholipid syndrome (APS). Following the initial collaborative studies on antibodies against endothelium in systemic autoimmune vasculitis, we were able to use a similar strategy in APS. This line of research has resulted in the characterization of beta 2 glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) as mechanisms capable of mediating an endothelial perturbation crucial for the pathogenesis of APS. Thanks to these studies, the collaboration has led to the characterization of the membrane receptors for β2GPI and the cellular signaling resulting from antibody binding. This mechanism has also been shown to mediate the aPL effect on other cell types involved in APS pathogenesis. Finally, the exchange of information made it possible to replicate and extend the setting of animal models of the syndrome, which proved to be valuable tools for understanding the pathogenesis of the syndrome. It has been a long story recently refueled by common studies on the similarity of pro-inflammatory and pro-coagulant endotheliopathy in APS and in COVID-19., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Pier Luigi Meroni reports financial support was provided by IRCCS Istituto Auxologico Italiano. Pier Luigi Meroni reports a relationship with IRCCS Istituto Auxologico Italiano that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The 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., (© 2024. The Author(s).)

Introduction: Lung carcinoids (LCs) are a type of neuroendocrine tumor (NET) that originate in the bronchopulmonary tract. LCs account for 20-25% of all NETs and approximately 1-2% of lung cancers. Given the highly vascularized nature of NETs and their tendency to overexpress vascular growth factor receptors (VEGFR), inhibiting angiogenesis appears as a potential therapeutic target in slowing down tumor growth and spread. This study evaluated the long-term antitumor activity and related mechanisms of axitinib (AXI), a VEGFR-targeting drug, in LC cell lines., Methods: Three LC cell lines (NCI-H727, UMC-11 and NCI-H835) were incubated with their respective EC 50 AXI concentrations for 6 days. At the end of the incubation, FACS experiments and Western blot analyses were performed to examine changes in the cell cycle and the activation of apoptosis. Microscopy analyses were added to describe the mechanisms of senescence and mitotic catastrophe when present., Results: The primary effect of AXI on LC cell lines is to arrest tumor growth through an indirect DNA damage. Notably, AXI triggers this response in diverse manners among the cell lines, such as inducing senescence or mitotic catastrophe. The drug seems to lose its efficacy when the DNA damage is mitigated, as observed in NCI-H835 cells., Conclusion: The ability of AXI to affect cell viability and proliferation in LC tumor cells highlights its potential as a therapeutic agent. The role of DNA damage and the consequent activation of senescence seem to be a prerequisite for AXI to exert its function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Oldani, Cantone, Gaudenzi, Carra, Dicitore, Saronni, Borghi, Lombardi, Caraglia, Persani and Vitale.)

Objectives: Giant cell arteritis (GCA) is a common vasculitis affecting patients aged 50 and older. GCA leads to chronic inflammation of large/medium-sized vessel walls with complications such as permanent vision loss and risk of stroke and aortic aneurysms. Early diagnosis is crucial and relies on temporal artery biopsy (TAB) and ultrasound imaging of temporal and axillary arteries. However, these methods have limitations. Serum biomarkers as autoantibodies have been reported but with inconclusive data for their use in the clinical setting. Additionally, C-reactive protein and erythrocyte sedimentation rate are non-specific and limited in reflecting disease activity, particularly in patients treated with IL-6 inhibitors. This study aimed to identify serum autoantibodies as new diagnostic biomarkers for GCA using a human protein array., Methods: One commercial and one proprietary human protein array were used for antibody profiling of sera from patients with GCA (n=55), Takayasu (TAK n=7), and Healthy Controls (HC n=28). The identified candidate autoantigens were purified and tested for specific autoantibodies by ELISA., Results: Antibodies against two proteins, VSIG10L (V-Set and Immunoglobulin Domain Containing 10 Like) and DCBLD1 (discoidin), were identified and found to be associated with GCA, with an overall prevalence of 43-57%, respectively, and high specificity as individual antibodies. A control series of TAK sera tested negative., Conclusions: Detecting GCA-specific autoantibodies may offer a new, non-invasive tool for improving our diagnostic power in GCA. Even though cell-mediated immune responses are crucial for GCA pathogenesis, this finding opens the way for investigating the additional role of humoral immune responses in the disease.

Multiple vaccines have been approved to control COVID-19 pandemic, with Pfizer/BioNTech (BNT162b2) being widely used. We conducted a longitudinal analysis of the immune response elicited after three doses of the BNT162b2 vaccine in individuals who have previously experienced SARS-CoV-2 infection and in unexperienced ones. We conducted immunological analyses and single-cell transcriptomics of circulating T and B lymphocytes, combined to CITE-seq or LIBRA-seq, and VDJ-seq. We found that antibody levels against SARS-CoV-2 Spike, NTD and RBD from wild-type, delta and omicron VoCs show comparable dynamics in both vaccination groups, with a peak after the second dose, a decline after six months and a restoration after the booster dose. The antibody neutralization activity was maintained, with lower titers against the omicron variant. Spike-specific memory B cell response was sustained over the vaccination schedule. Clonal analysis revealed that Spike-specific B cells were polyclonal, with a partial clone conservation from natural infection to vaccination. Spike-specific T cell responses were oriented towards effector and effector memory phenotypes, with similar trends in unexperienced and experienced individuals. The CD8 T cell compartment showed a higher clonal expansion and persistence than CD4 T cells. The first two vaccinations doses tended to induce new clones rather than promoting expansion of pre-existing clones. However, we identified a fraction of Spike-specific CD8 T cell clones persisting from natural infection that were boosted by vaccination and clones specifically induced by vaccination. Collectively, our observations revealed a moderate effect of the second dose in enhancing the immune responses elicited after the first vaccination. Differently, we found that a third dose was necessary to restore comparable levels of neutralizing antibodies and Spike-specific T and B cell responses in individuals who experienced a natural SARS-CoV-2 infection., Competing Interests: Declaration of competing interest Author Renata Grifantini is currently employed by CheckmAb Srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Coronavirus disease 2019 (COVID-19) displays clinical heterogeneity, but little information is available for patients with mild or very early disease. We aimed to characterize biomarkers that are useful for discriminating the hospitalization risk in a COVID-19 cohort from Northern Italy during the first pandemic wave. We enrolled and followed for four weeks 76 symptomatic SARS-CoV-2 positive patients and age/sex-matched healthy controls. Patients with mild disease were discharged (n.42), and the remaining patients were hospitalized (n.34). Blood was collected before any anti-inflammatory/immunosuppressive therapy and assessed for soluble C5b-9/C5a, H3-neutrophil extracellular traps (NETs), calprotectin, and DNase plasma levels via ELISA and a panel of proinflammatory cytokines via ELLA. Calprotectin and NET levels discriminate between hospitalized and non-hospitalized patients, while DNase negatively correlates with NET levels; there are positive correlations between calprotectin and both NET and neopterin levels. Neopterin levels increase in patients at the beginning of the disease and do so more in hospitalized than non-hospitalized patients. C5a and sC5b-9, and other acute phase proteins, correlate with neopterin, calprotectin, and DNase. Both NET and neopterin levels negatively correlate with platelet count. We show that calprotectin, NETs, and neopterin are important proinflammatory parameters potentially useful for discriminating between COVID-19 patients at risk of hospitalization.

Introduction: Current therapeutic management of lupus nephritis (LN) fails to induce long-term remission in over 50% of patients, highlighting the urgent need for additional options., Methods: We analyzed differentially expressed genes (DEGs) in peripheral blood from patients with active LN ( n  = 41) and active nonrenal lupus ( n  = 62) versus healthy controls (HCs) ( n  = 497) from the European PRECISESADS project (NTC02890121), and dysregulated gene modules in a discovery ( n  = 26) and a replication ( n  = 15) set of active LN cases., Results: Replicated gene modules qualified for correlation analyses with serologic markers, and regulatory network and druggability analysis. Unsupervised coexpression network analysis revealed 20 dysregulated gene modules and stratified the active LN population into 3 distinct subgroups. These subgroups were characterized by low, intermediate, and high interferon (IFN) signatures, with differential dysregulation of the "B cell" and "plasma cells/Ig" modules. Drugs annotated to the IFN network included CC-motif chemokine receptor 1 (CCR1) inhibitors, programmed death-ligand 1 (PD-L1) inhibitors, and irinotecan; whereas the anti-CD38 daratumumab and proteasome inhibitor bortezomib showed potential for counteracting the "plasma cells/Ig" signature. In silico analysis demonstrated the low-IFN subgroup to benefit from calcineurin inhibition and the intermediate-IFN subgroup from B-cell targeted therapies. High-IFN patients exhibited greater anticipated response to anifrolumab whereas daratumumab appeared beneficial to the intermediate-IFN and high-IFN subgroups., Conclusion: IFN upregulation and B and plasma cell gene dysregulation patterns revealed 3 subgroups of LN, which may not necessarily represent distinct disease phenotypes but rather phases of the inflammatory processes during a renal flare, providing a conceptual framework for precision medicine in LN., (© 2024 International Society of Nephrology. Published by Elsevier Inc.)

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.

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease that presents a challenge for clinicians. To identify potential biomarkers for diagnosis and disease activity in SLE, we investigated a selected yet broad panel of cytokines and autoantibodies in patients with SLE, healthy controls (HC), and patients with other autoimmune diseases (AIDs)., Methods: Serum samples from 422 SLE patients, 546 HC, and 1223 other AIDs were analysed within the frame of the European PRECISESADS project (NTC02890121). Cytokine levels were determined using Luminex panels, and autoantibodies using different immunoassays., Results: Of the 83 cytokines analysed, 29 differed significantly between patients with SLE and HC. Specifically, CCL8, CXCL13, and IL-1RA levels were elevated in patients with active, but not inactive, SLE versus HC, as well as in patients with SLE versus other AIDs. The levels of these cytokines also correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores, among five other cytokines. Overall, the occurrence of autoantibodies was similar across SLEDAI-2K organ domains, and the correlations between autoantibodies and activity in different organ domains were weak., Discussion: Our findings suggest that, upon validation, CCL8, CXCL13, and IL-1RA could serve as promising serum biomarkers of activity in SLE., Competing Interests: IP has received research funding and/or honoraria from Amgen, AstraZeneca, Aurinia, Elli Lilly, Gilead, GlaxoSmithKline, Janssen, Novartis, Otsuka, and Roche. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Lindblom, Beretta, Borghi and PRECISESADS Clinical Consortium, Alarcón-Riquelme and Parodis.)

Lung carcinoids (LCs) comprise well-differentiated neuroendocrine tumors classified as typical (TCs) and atypical (ACs) carcinoids. Unfortunately, curative therapies remain elusive for metastatic LCs, which account for 25-30% of cases. This study evaluated the antitumor activity of axitinib (AXI), a second-generation tyrosine kinase inhibitor selectively targeting vascular endothelial growth factor receptors (VEGFR-1, VEGFR-2, VEGFR-3) in human lung TC (NCI-H727, UMC-11, NCI-H835) and AC (NCI-H720) cell lines. In vitro and in vivo (zebrafish) assays were performed following AXI treatment to gather several read-outs about cell viability, cell cycle, the secretion of proangiogenic factors, apoptosis, tumor-induced angiogenesis and migration. AXI demonstrated relevant antitumor activity in human LC cells, with pronounced effects observed in UMC-11 and NCI-H720, characterized by cell cycle perturbation and apoptosis induction. AXI significantly hindered tumor induced-angiogenesis in Tg(fli1a:EGFP) y1 zebrafish embryos implanted with all LC cell lines and also reduced the invasiveness of NCI-H720 cells, as well as the secretion of several proangiogenic factors. In conclusion, our study provides initial evidence supporting the potential anti-tumor activity of AXI in LC, offering a promising basis for future investigations in mammalian animal models and, eventually, progressing to clinical trials.