Type I interferon pathway activation across the antiphospholipid syndrome spectrum: associations with disease subsets and systemic antiphospholipid syndrome presentation.

Introduction: While the type I interferon (IFN-I) pathway is crucial in autoimmunity, its role in antiphospholipid antibody (aPL)-positive subjects, including aPL carriers and antiphospholipid syndrome (APS) patients, is poorly understood. This study aims at characterizing IFN-I pathway activation within the spectrum of aPL-positive subsets. Methods: A total of 112 patients [29 aPL carriers, 31 primary APS (PAPS), 25 secondary APS (SAPS), 27 systemic lupus erythematosus (SLE) patients without aPL, and 44 healthy controls (HCs)] were recruited. IFI6, IFI44, IFI44L, MX1, IFI27, OAS1, and RSAD2 gene expression was evaluated in whole blood, and a composite index (IFN score) was calculated. Results: An overall activation of the IFN-I pathway was observed across the entire APS spectrum, with differences among genes based on the specific disease subset. The composite score revealed quantitative differences across subsets, being elevated in aPL carriers and PAPS patients compared to HCs (both p < 0.050) and increasing in SAPS (p < 0.010) and SLE patients (p < 0.001). An unsupervised cluster analysis identified three clusters, and correspondence analyses revealed differences in clusters usage across APS subsets (p < 0.001). A network analysis revealed different patterns characterizing different subsets. The associations between IFN-I pathway activation and clinical outcomes differed across APS subsets. Although no differences in gene expression were observed in systemic APS, the network analyses revealed specific gene-gene patterns, and a distinct distribution of the clusters previously identified was noted (p = 0.002). Conclusion: IFN-I pathway activation is a common hallmark among aPL-positive individuals. Qualitative and quantitative differences across the APS spectrum can be identified, leading to the identification of distinct IFN-I signatures with different clinical values beyond traditional categorization.

Assessing the steroid-sparing effect of biological agents in randomized controlled trials for lupus: a scoping review.

Prompt disease control of flares in patients with systemic lupus erythematosus (SLE) is a priority in treatment strategy planning. However, the long-term dosage-related collateral effects of glucocorticoids (GCs) have pushed researchers towards the identification and utilization of novel biological agents that could both induce and maintain low disease activity and remission, especially in the context of lupus nephritis (LN). This scoping review aims at assessing the current evidence of the potential steroid-sparing effect of biologic therapies by reviewing phase II and phase III randomized, placebo-controlled trials involving SLE/LN patients. A scoping review of the literature was carried out in accordance with PRISMA-ScR recommendations. Risk of bias was assessed through the utilization of the Cochrane Collaboration's tool for randomized controlled trials (RCTs). Eight RCTs met the inclusion criteria and were included in this analysis (treatment drug, 7 belimumab; 1 anifrolumab). Four studies showed a definite steroid-sparing effect (treatment drug, 3 belimumab; 1 anifrolumab), while in the remaining four RCTs, the steroid-sparing effect was not observed. When focusing on phase III trials, the overall quality of the studies resulted fair or good considering the risk of bias. However, a degree of heterogeneity of steroid regimen protocol (considering initial dosage, tapering and rescue treatment allowance) was observed. While a growing body of evidence is supporting the safety and efficacy of biological treatment in SLE, the evidence on their steroid-sparing effect remains scattered. Future research needs to pursue the identification of precise SLE clusters of patients who would benefit most from a specific treatment protocol with a definite steroid-sparing effect.

2023 ACR/EULAR classification criteria in existing research cohorts: an international study.

OBJECTIVE: Assessing the impact of the updated ACR/EULAR APS classification criteria to our research cohort. METHODS: Consecutive patients who tested persistently positive for at least one aPL in the last three years were enrolled. The first APS Sydney index event was considered and computed for the comparison between Sydney and 2023 APS criteria. When computing the 2023 APS criteria, additional manifestations were also considered. RESULTS: The cohort comprised 249 patients (185 with APS and 64 aPL carriers according to Sydney criteria). The 185 patients had as first index event VT in 55 cases (29.8%) AT in 63 (34%) and PM in 67 (36.2%). When applying the updated criteria, 90 subjects (48.7%) failed to reach the composite score of the new criteria. The percentage of thrombotic APS per Sydney criteria decreased from 47.3% to 34.9% because of high cardiovascular risk in 23 cases, IgM aPL profile in 6 cases and in 2 patients for both reasons. Patients with PM decreased from 26.9-3.2% (39 cases of recurrent early pregnancy loss and 20 of fetal losses). Consequently, the percentage of aPL carriers increased from 26% to 61%. When looking at the disease evolution at follow-up, 32 additional patients out of 90 (35.6%) fulfilled the new APS criteria, after developing additional clinical manifestation following index event. CONCLUSION: When applying the new APS criteria to our research cohort, not negligible differences exist in patients' classification. A multidisciplinary approach will be mandatory to assess the impact into research and, ultimately, patient's care of new criteria.

Pediatric Presentation of Antiphospholipid Syndrome: A Review of Recent Literature With Estimation of Local Prevalence.

We aimed to investigate the epidemiology, the clinical and laboratory characteristics of the pediatric involvement of antiphospholipid syndrome (APS), by performing a review of the current evidence and reviewing local experience in the Northwest Italy. To achieve this, we performed a detailed literature search to identify articles describing clinical and laboratory characteristics of pediatric APS. In concomitance, we conducted a registry-based study collecting data from the Piedmont and Aosta Valley Rare Disease Registry including pediatric patients diagnosed with APS in the last 11 years. The literature review led to inclusion of six articles with a total of 386 pediatric patients (65% females, 50% with systemic lupus erythematosus (SLE) as concomitant diagnosis). Rates of venous and arterial thrombosis were 57 and 35%, respectively. "Extra-criteria manifestations" included mostly hematologic and neurologic involvement. Almost one-quarter of patients (19%) reported recurrent events and 13% manifested as catastrophic APS. A total of 17 pediatric patients (mean age 15.1 ± 2.8, 76% female) developed APS in the Northwest of Italy. In 29% of cases, SLE was a concomitant diagnosis. Deep vein thrombosis was the most frequent manifestation (28%) followed by catastrophic APS (6%). The estimated prevalence of pediatric APS in Piedmont and Aosta Valley Region is 2.5/100,000 people, whereas the estimated annual incidence is 0.2/100,000 inhabitants. In conclusion, clinical manifestations of pediatric APS seem to be more severe and with a high prevalence of noncriteria manifestations. International efforts are needed to better characterize this condition and to develop new specific diagnostic criteria to avoid missed/delayed diagnosis in children with APS.

Thrombosis and APS: Lessons Learned from Another Black Swan Tale.

Antiphospholipid syndrome (APS) is a chronic systemic autoimmune disease characterized by venous, arterial, and microvascular thromboses and/or recurrent pregnancy morbidity, that occur in the persistent presence of antiphospholipid antibodies (aPL). APS can present with a wide range of clinical manifestations often reffered as "extra-criteria". These features, although apparently less common, can severely impact patients' outcome. Here, we report the case of a patient with a newly diagnosed APS. He previously experienced a recurrence of venous thrombosis after discontinuation of anticoagulant therapy in association with cutaneous ulcerations as presenting symptoms. Interestingly, skin lesions did not improve with full anticoagulant treatment. Due to concomitant presence of thrombotic and microvascular involvement, immunomodulatory therapy with steroid pulses followed by intravenous injections of belimumab was started, with progressive and significant amelioration, leading to complete recovery. Following the presentation of the current case report, we highlight the importance of suspecting APS in young patients experiencing unprovoked thrombosis. We also emphasized the critical issue of testing aPL during anticoagulant treatment and focused on the need of aPL retesting in patients with positivity at high titers. We also highlight the double nature of aPL-mediated clinical manifestations. While most patients presented with pure thrombotic complications, one should always remember that APS is an autoimmune-mediated disease, which can benefit from alternative therapeutic approaches beyond anticoagulation.

Complement levels during the first trimester predict disease flare and adverse pregnancy outcomes in systemic lupus erythematosus: A network meta-analysis on 532 pregnancies.

BACKGROUND: Complement levels have been proposed as candidate biomarkers of disease activity and obstetric risk in systemic lupus erythematosus (SLE) pregnancies, but their reliability has been questioned due to the physiologic fluctuations of complement during gestation. Thus, this network meta-analysis aimed at assessing the clinical significance of complement fluctuations in lupus pregnant women. METHODS: Corresponding authors of 19 studies meeting inclusion criteria were invited to contribute with additional data including C3 and C4 levels [before pregnancy, at conception, in every trimester (T) and 3 months after delivery]; data were pooled together in a network meta-analysis. RESULTS: A total of 532 lupus women from four studies were included in the analysis. In SLE women, C3 and C4 increased progressively during gestation: levels remained stable during T1 and peaked in T2 to decrease in T3. Patients with previous lupus nephritis (LN) and those who experienced flares during pregnancy had significantly lower mean levels of C3 and C4 at all timepoints. The lowest levels of complement were observed, particularly during T1, in patients with LN and gestational flare. Both reduction and the lack of increase of C3 and C4 levels at T1 versus conception were associated with gestational flares, particularly in LN patients. Pregnancies with flare had a statistically significant higher rate of maternal and fetal complications(60% versus 50.3%; p = 0.03). CONCLUSIONS: Low complement levels, particularly in T1, were associated with a higher frequency of gestational flare. Either reduction or smaller increase of C3 and/or C4 levels, even within normal range, might predict flares especially in early gestation.

Safety and efficacy of pre-exposure prophylaxis with tixagevimab/cilgavimab (Evusheld) in patients with glomerular diseases who received rituximab.

Background: Patients on B-cell-depleting agents may have a suboptimal response to vaccination, placing them at a higher risk of contracting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or suffering from a more severe prognosis. Indeed, available data on pre-exposure prophylaxis with tixagevimab/cilgavimab (Evusheld) in subjects with glomerular diseases (GDs) who received rituximab are limited. Methods: We conducted a prospective study analysing the safety and efficacy of tixagevimab/cilgavimab for pre-exposure prophylaxis in patients with GDs who received rituximab in the previous 12 months. The rates of symptomatic infections and hospitalizations were compared with those for patients with GD treated with rituximab who refused to receive tixagevimab/cilgavimab. Results: Tixagevimab/cilgavimab was administered to 22 patients (12 females, mean age 58.4 ± 19.6 years) with GD diagnoses including membranous nephropathy, lupus nephritis, anti-neutrophil cytoplasmic antibody-associated vasculitis and focal segmental glomerulosclerosis. No patient treated with tixagevimab/cilgavimab experienced symptomatic infection with SARS-CoV-2 during the follow-up (mean observation time of follow-up was 112 ± 23 days), while 11 of 28 controls (39.3%) reported a symptomatic infection (P = .0001), requiring hospitalization in 2 cases. Reported adverse events were mild, namely self-limiting headache [4], discomfort at the injection site [3], flu-like symptoms/myalgia [3] and fever [1]. No serious adverse events (e.g. cardiac events, anaphylaxis) were reported. Conclusion: Pre-exposure prophylaxis with tixagevimab/cilgavimab seems safe and lowered the risk of symptomatic SARS-CoV-2 infection by ≈40% in vaccinated subjects with GD who received anti-CD20 therapy. Possible applications in the subset of patients who need immunosuppressive therapy, especially with rituximab, in a pandemic setting might be envisaged.

Daratumumab monotherapy for refractory lupus nephritis.

Treatment-refractory lupus nephritis (LN) has a high risk of a poor outcome and is often life-threatening. Here we report a case series of six patients (one male and five females) with a median age of 41.3 years (range, 20-61 years) with refractory LN who received renal biopsies and were subsequently treated with intravenous daratumumab, an anti-CD38 monoclonal antibody (weekly for 8 weeks, followed by eight biweekly infusions and up to eight monthly infusions). One patient did not show any improvement after 6 months of therapy, and daratumumab was discontinued. In five patients, the mean disease activity, as assessed by the Systemic Lupus Erythematosus Disease Activity 2000 index, decreased from 10.8 before treatment to 3.6 at 12 months after treatment. Mean proteinuria (5.6 g per 24 h to 0.8 g per 24 h) and mean serum creatinine (2.3 mg dl-1 to 1.5 mg dl-1) also decreased after 12 months. Improvement of clinical symptoms was accompanied by seroconversion of anti-double-stranded DNA antibodies; decreases in median interferon-gamma levels, B cell maturation antigen and soluble CD163 levels; and increases in C4 and interleukin-10 levels. These data suggest that daratumumab monotherapy warrants further exploration as a potential treatment for refractory LN.

New Frontiers in Autoimmune Diagnostics: A Systematic Review on Saliva Testing.

(1) Background: Immunological laboratory testing is known to be complex, and it is usually performed in tertiary referral centers. Many criticalities affect diagnostic immunological testing, such as limited availability, the need for specifically trained laboratory staff, and potential difficulties in collecting blood samples, especially in the most vulnerable patients, i.e., the elderly and children. For this reason, the identification of a new feasible and reliable methodology for autoantibody detection is urgently needed. (2) Methods: We designed a systematic review to investigate the available literature on the utilization of saliva samples for immunological testing. (3) Results: A total of 170 articles were identified. Eighteen studies met the inclusion criteria, accounting for 1059 patients and 671 controls. The saliva collection method was mostly represented by passive drooling (11/18, 61%), and the most frequently described methodology for antibody detection was ELISA (12/18, 67%). The analysis included 392 patients with rheumatoid arthritis, 161 with systemic lupus erythematosus, 131 with type 1 diabetes mellitus, 116 with primary biliary cholangitis, 100 with pemphigus vulgaris, 50 with bullous pemphigoids, 49 with Sjogren syndrome, 39 with celiac disease, 10 with primary antiphospholipid syndromes, 8 with undifferentiated connective tissue disease, 2 with systemic sclerosis, and 1 with autoimmune thyroiditis. The majority of the reviewed studies involved adequate controls, and saliva testing allowed for a clear distinction of patients (10/12 studies, 83%). More than half of the papers showed a correlation between saliva and serum results (10/18, 55%) for autoantibody detection, with varying rates of correlation, sensitivity, and specificity. Interestingly, many papers showed a correlation between saliva antibody results and clinical manifestations. (4) Conclusions: Saliva testing might represent an appealing alternative to serum-based testing for autoantibody detection, considering the correspondence with serum testing results and the correlation with clinical manifestations. Nonetheless, standardization of sample collection processing, maintenance, and detection methodology has yet to be fully addressed.

Clinical-Pathological Characteristics of Renal Injuries Identify Different Clusters in Patients With Antiphospholipid Antibodies.

Introduction: Significant heterogeneity still exists in the nomenclature of renal involvement in antiphospholipid syndrome (APS). Methods: We applied a hierarchical cluster analysis to determine subgroups of patients according to clinical, laboratory, and renal histology characteristics in a cohort of subjects with confirmed antiphospholipid antibodies (aPL) positivity and biopsy proven aPL-related renal injuries. Kidney outcomes were then assessed at 12 months. Results: A total of 123 aPL-positive patients were included in the study (101 [82%] female, 109 [88.6%] with systemic lupus erythematosus [SLE], 14 (11.4%) with primary APS [PAPS]). Three clusters were identified. Twenty-three patients (18.7%) were included in the first cluster (cluster 1), characterized by a higher prevalence of glomerular capillary and arteriolar thrombi and fragmented red blood cells in the subendothelial space. Cluster 2 included 33 patients (26.8%) and showed a higher prevalence of fibromyointimal proliferative lesions as seen in hyperplastic vasculopathy. Cluster 3 was the largest (67 patients, mainly with SLE) and was characterized by higher prevalence of subendothelial edema, of both glomerular capillaries and arterioles. Conclusion: Three different clusters of patients with aPL and renal injuries emerged from our study as follows: the first, with the worst renal prognosis, was associated with features of thrombotic microangiopathy (TMA), thrombosis, triple aPL positivity and higher adjusted Global APS Score (aGAPSS) values; the second, characterized by hyperplastic vasculopathy with an intermediate prognosis, was seen more frequently in patients with cerebrovascular manifestations; and the third, more benign in terms of outcomes and with no overt association with thrombotic features, was characterized by endothelial swelling in concomitant lupus nephritis (LN).

Chasing the Zebra: a case of membranous-like Glomerulopathy with SSA/RO52 deposits and no overt connective tissue disease.

BACKGROUND: The nature of the deposits in immune-mediated glomerulonephritis with a membranous pattern and masked IgG-Kappa deposits (MGMID) remains still to be elucidated. CASE PRESENTATION: We present a case of 33-year-old woman developing a continuous asymptomatic proteinuria (0.8-1 g/24 h) with no overt connective tissue diseases. She tested positive at high titers for SSA antibodies (Ro52 838 UI/mL, Ro60 2716 UI/mL) and at the kidney biopsy histological findings were compatible with an immune-mediated glomerulonephritis with a membranous pattern and masked IgG-Kappa deposits. Also, we demonstrated a positive immunohistochemistry staining for anti-Ro52-SSA antibodies, with a granular positivity in mesangium and along rare glomerular capillaries. To date, only one case of a patient with overt diagnosis of Sjögren's syndrome with MGMID has been described but a pathogenic role for SSA and SSB antibodies has never been proven. CONCLUSIONS: In this case, we described for the first time by immunohistochemistry a Ro52+ granular positivity in the mesangium and glomerular capillaries, potentially paving the way for a better understanding of MGMID.

Finding the Needle in the Haystack: Serological and Urinary Biomarkers in Behçet's Disease: A Systematic Review.

Urinary and serological markers play an essential role in the diagnostic process of autoimmune diseases. However, to date, specific and reliable biomarkers for diagnosing Behçet's disease (BD) are still lacking, negatively affecting the management of these patients. To analyze the currently available literature on serological and urinary BD biomarkers investigated in the last 25 years, we performed a systematic literature review using the Population, Intervention, Comparison, and Outcomes (PICO) strategy. One hundred eleven studies met the eligibility criteria (6301 BD patients, 5163 controls). Most of them were retrospective, while five (5%) were prospective. One hundred ten studies (99%) investigated serological biomarkers and only two (2%) focused on urinary biomarkers. One hundred three studies (93%) explored the diagnostic potential of the biomolecules, whereas sixty-two (56%) tested their effect on disease activity monitoring. Most articles reported an increase in inflammatory markers and pro-oxidant molecules, with a decrease in antioxidants. Promising results have been shown by the omics sciences, offering a more holistic approach. Despite the vast number of investigated markers, existing evidence indicates a persistent gap in BD diagnostic/prognostic indices. While new steps have been taken in the direction of pathogenesis and disease monitoring, international efforts for the search of a diagnostic marker for BD are still needed.

"How we treat" clinical dilemmas in antiphospholipid syndrome: A case-based approach.

Antiphospholipid syndrome (APS) is an autoimmune condition characterized by thrombosis (arterial, venous, and microvascular) and/or pregnancy morbidity occurring in subjects persistently positive for antiphospholipid antibodies (aPL). While the APS classification criteria are being currently updated to improve homogeneity in clinical research, patients who seek medical treatment often have a variety of laboratory and clinical characteristics that may not completely fulfill the classification criteria for overt APS. Those cases might represent a challenge in terms of treatment and overall management. We aim to present a collection of challenging scenarios of patients who tested positive for aPL and to discuss available literature to guide the therapeutic strategies.

Open-label, prospective, phase II descriptive pilot trial of belimumab therapy for refractory and/or non-criteria manifestations of antiphospholipid syndrome: study protocol.

OBJECTIVES: To evaluate the safety and tolerability of belimumab given for 24 months in patients persistently positive for antiphospholipid antibodies (aPL) with clinical features attributable to aPL [refractory and/or non-criteria manifestations of the antiphospholipid syndrome (APS)]. METHODS: In this investigator-initiated, single-centre, open-label, prospective, phase II descriptive pilot trial, belimumab will be administered in 15 patients attending San Giovanni Bosco Hospital (Turin) showing refractory and/or non-criteria manifestations of APS. Subjects will receive belimumab 10 mg/kg intravenously (in addition to their ongoing APS treatment) with regimen at 0, 2, 4 weeks and every 4 weeks thereafter (up to week 104). Study endpoints determined at 4, 16, 24, 36, 52 and 104 weeks will include: primary (safety and adverse events) and secondary outcomes, such as changes in clinical outcomes (recurrent thromboses, thrombocytopenia, haemolytic anaemia, cardiovascular events, skin ulcer, aPL-related nephropathy and cognitive dysfunction), laboratory outcomes (routine tests, aPL, ENA and anti-dsDNA tests, thrombin generation assay, interferon-signature analysis, lymphocytes immunophenotyping, BLyS determination) and QoL evaluation. RESULTS: Study endpoints determined at 4, 16, 24, 36, 52 and 104 weeks will include: primary (safety and adverse events) and secondary outcomes, such as changes in clinical outcomes (recurrent thromboses, thrombocytopenia, haemolytic anaemia, cardiovascular events, skin ulcer, aPL-related nephropathy and cognitive dysfunction), laboratory outcomes (routine tests, aPL, ENA and anti-dsDNA tests, thrombin generation assay, interferon-signature analysis, lymphocytes immunophenotyping, BLyS determination) and QoL evaluation. CONCLUSIONS: Targeting B-cells is emerging as an appealing strategy for patients with APS. Preliminary observations showed aPL negativisation after starting therapy with belimumab. The clinical relevance of these findings will be investigated in this prospective study. If confirmed, the current 'anti-thrombotic' approach to APS patients could be complemented, at least in selected cases, with an 'immunomodulatory' strategy.

Thrombin generation assay and lupus anticoagulant synergically distinguish populations of patients with antiphospholipid antibodies.

AIM: To apply thrombin generation assay (TGA) in a large cohort of antiphospholipid antibodies (aPL)-positive patients. MATERIAL AND METHODS: 108 patients were tested with TGA and lupus anticoagulant (LA) testing and divided according to their aPL profile. Briefly, 21 patients were positive for anti-phosphatidylserine (aPS)/prothrombin (PT) IgG/IgM (group1), 29 for anti-ß2-glycoprotein-I (aß2GPI) and anti-cardiolipin (aCL) IgG/IgM (group2), 31 for aPS/PT, aß2GPI and aCL IgG/IgM (group3), 27 for aPS/PT and/or aß2GPI+aCL IgM at low-titres (group4). 31 healthy donors (HDs) and 24 controls treated with vitamin K antagonists (VKA) were included. RESULTS: The most deranged TGA and LA profiles were observed in tetra-positive patients (group3) that differed significantly to the other groups, thus those with isolated, double or triple aPL-positivity. Moreover, when comparing the TGA profile of all antiphospholipid syndrome (APS) patients, aPL-carriers, HDs and VKA controls, we observed that the aPL+ patients (especially APS) showed a distinctive profile that allowed to distinguish them from the other groups with significantly higher tLag and tPeak, and lower Peak and area under the curve.When focusing on APS clinical manifestations, patients with a high-risk profile (group3) showed significant differences from those presenting low-titres aPL (group 4) regarding the number of venous events (p=0.04), recurrence of any thrombotic event (p=0.01), of arterial events (5 vs 0, p=0.02), the occurrence of TIA (p=0.04), DVT (p=0.02) and, when analysing extracriteria manifestations, of peripheral artery disease (p=0.04). CONCLUSIONS: TGA seems a valuable approach to stratify aPL+ patients according to their risk profile. The differences among different populations of autoantibodies specificities could be considered a translational validation of the increased thrombotic risk of patients with triple or tetra aPL-positivity.

Serum Biomarkers of Renal Fibrosis: A Systematic Review.

Chronic kidney disease (CKD) is a widely diffuse pathological condition which deeply impacts upon an affected patient's quality of life and its worldwide rate is predicted to further rise. The main biological mechanism underlying CKD is renal fibrosis, a non-reversible process representing, for the affected system, a point of no return of tissue damage and dysfunction, deeply reducing the possible therapeutic strategies at the disposal of physicians. The best tool clinicians can use to address the extent of renal fibrosis at any level (glomeruli, tubule-interstitium, vasculature) is kidney biopsy that, despite its overall safety, remains an invasive procedure showing some shortcomings. Thus, the identification of novel non-invasive renal fibrosis biomarkers would be of fundamental importance. Here, when systematically reviewing the available evidence on serological biomarkers associated with renal fibrosis evaluated in patients suffering from CKD in the last five years, we found that despite the presence of several promising biomarkers, the level of observed evidence is still very scattered. Probably, the use of multiple measures capable of addressing different aspects involved in this condition would be the most suitable way to capture the high complexity characterizing the renal fibrotic process, having consequently a great impact on clinical practice by maximizing prevention, diagnosis, and management.