anti-MPO Antibodies Research Articles

Regulatory T cells effectively downregulate the autoimmune anti-MPO response and ameliorate anti-MPO induced glomerulonephritis in mice

Regulation of autoreactive cells is key for both prevention and amelioration of autoimmune disease. A better understanding of the key cell population(s) responsible for downregulation of autoreactive cells would provide necessary foundational insight for cellular-based therapies in autoimmune disease. Utilizing a mouse model of anti-myeloperoxidase (MPO) glomerulonephritis, we sought to understand which immune cells contribute to downregulation of the anti-MPO autoimmune response. MPO−/− mice were immunized with whole MPO to induce an anti-MPO response. Anti-MPO splenocytes were then transferred into recipient mice (Rag2−/− mice or WT mice). Anti-MPO titers were followed over time. After anti-MPO splenocyte transfer, WT mice are able to downregulate the anti-MPO response while anti-MPO titers persist in Rag2−/− recipients. Reconstitution with WT splenocytes into Rag2−/− recipients prior to anti-MPO splenocyte transfer enabled mice to downregulate the anti-MPO immune response. Therefore, wildtype splenocytes contain a cellular population that is capable of downregulating the autoimmune response. Through splenocyte transfer, antibody depletion experiments, and purified cell population transfers, we confirmed that the regulatory T cell (Treg) population is responsible for the downregulation of the anti-MPO autoimmune response. Further investigation revealed that functional Tregs from WT mice are capable of downregulating anti-MPO antibody production and ameliorate anti-MPO induced glomerulonephritis. These data underscore the importance of functional Tregs for control of autoimmune responses and prevention of end-organ damage due to autoimmunity.

#2958 Can the Kidney Failure Risk Equation (KFRE) be used in ANCA associated glomerulonephritis?

Abstract Background and Aims The Kidney Failure Risk Equation (KFRE) provides a 2 and 5 year probability of treated kidney failure for a patient with chronic kidney disease (CKD) stage 3a to 5. KFRE has been validated in over 30 countries, including the United Kingdom (UK) and has become a useful tool to aid clinical decision making [1]. However there has been limited validation to determine if KFRE can be used in primary renal pathologies such as ANCA associated glomerulonephritis (AAGN). We conducted a retrospective cohort study of patients with AAGN at a single centre in Lancashire, UK to determine if KFRE accurately predicts end stage kidney disease (ESKD). Method Patients diagnosed with AAGN between 2009 and 2018 were screened for inclusion. Exclusion criteria included mortality within 6 months of diagnosis, those that remained dialysis dependent 6 months after diagnosis, transfer out of area, eGFR >60 ml/min/1.73 m2 and missing biochemical results required to calculate KFRE. Baseline and clinical characteristics were collected. The UK 4-variable KFRE was calculated at 6 months (±3 months) and 18 months (±3 months) after initial diagnosis of AAGN (1). Clinical outcomes including ESKD (dialysis or transplantation) were recorded. Statistical analysis was performed. The median and interquartile range were used for continuous variables and frequency and percentage for absolute values. The median and interquartile range were calculated for KFRE. The area under the received operator characteristic curve (AUC) was used to calculate discrimination of KFRE. Results Of the 150 patients screened, 78 patients were included in this retrospective analysis. Median age at diagnosis was 67 years (IQR 61-74 years). Forty one (53%) males and 37 (47%) females were included in the study. Forty two (53.8%) patients had positive anti-MPO antibodies, 29 (37.2%) had anti-PR3 antibodies and 7 (9.0%) had seronegative AAGN. 15 of 78 patients required dialysis or transplantation within the study period assessed. KFRE was calculated at 6 months (± 3 months) ; the median 2 year risk KFRE score was 2% (IQR 0.3-5%) and median 5 year KFRE score was 7.1% (IQR 1.1-17.6%) (Fig. 1). KFRE was calculated at 18 months (± 3 months) ; the median 2 year risk KFRE score was 0.7% (IQR 0.4–2.7%) and median 5 year KFRE score was 2.1% (IQR 0.7–9.5%). In terms of discrimination for KFRE calculated 6 months after diagnosis, in the 2 year analysis, the AUC was 0.74 (95% CI 0.55-0.92) and in the 5 year analysis, the AUC was 0.73 (95% CI 0.55-0.90). When KFRE was calculated 18 months after diagnosis, in the 2 year analysis, the AUC was 0.75 (95% CI 0.30-1) and in the 5 year analysis, the AUC was 0.81 (95% CI 0.50-1). Conclusion While KFRE is a thoroughly validated and widely adopted tool in CKD patients, its application in primary renal pathologies, such as AAGN, is yet to be determined. We have shown that KFRE provides fair discrimination, as early as 6 months after initial diagnosis and may provide future clinical utility in AAGN patients that have achieved stable remission. Current tools to risk stratify AAGN patients require histological parameters which is not always possible to achieve and further work in predicting the prognosis of glomerular diseases is needed. This study was limited by the single centre and small cohort but further analysis and validation on a larger multi-centre cohort is needed before KFRE can be considered for clinical use in AAGN patients.

E078 Long term outcomes of diffuse alveolar haemorrhage in antineutrophil cytoplasmic antibody-associated vasculitis: a single centre study

Abstract Background/Aims ANCA associated vasculitis (AAV) is a heterogeneous disease with significant mortality and morbidity. Diffuse Alveolar Hemorrhage (DAH) is a life-threatening condition and presents with hemoptysis, cough, breathlessness and respiratory failure. Studies have shown a prevalence of about 25%. This study was designed to assess the clinical features, serology, efficacy of treatment and long term outcomes in DAH patients secondary to AAV. Methods This was an observational study which included adult AAV Patients diagnosed based on Revised International Chapel Hill Consensus Conference classification criteria from January 2016 to August, 2023 after IEC approval. DAH was diagnosed based on clinical symptoms, Imaging (Diffuse alveolar infiltrates), drop in hemoglobin, hypoxia and Bronchoalveolar lavage in cases where feasible. Demographic details, clinical features, laboratory data including ANCA serology, treatment, disease activity, outcomes of patients were assessed prospectively. DAH and Non DAH groups were compared using Kruskal Wallis test for median values. Student t-test for mean values and Chi square for categorical variables were used to test association between two groups with statistical significance of P-Value <0.05. Results 133 AAV patients were included in the study. DAH was seen in 42(31.5%) patients. The mean (± SD) age of patients was 49.4(±18.9) years. Males (52.3%) were marginally higher in number then females (47.6%). Patients presented with a median of 8 weeks after AAV diagnosis. Hemoptysis (61.9%), cough (47.6%) and breathlessness(64.3%) were the presenting complaints. Renal, Sinusitis, Gastrointestinal involvement was seen in 83.3%, 61.9% and 14.2% respectively. Musculoskeletal and Ophthalmic features were seen in 33.3% each. RPRF was seen in 42.8%. c-ANCA, p-ANCA were seen in 73.8%,21.4% while Anti PR3 antibody and Anti MPO antibody were seen in 78.5%, 21.4% patients respectively. Median (IQR) BVAS score at presentation was 20(13-24) compared to 11(7-11) in Non DAH group which was statistically significant (p < 0.001). 10(23.8%) patients required ventilator support. IVIg was given in 3(7.1%), while plasmapheresis was used in 7(16.7%). Induction regimen with Cyclophosphamide was used in 73.8% and Rituximab (RTX) in 8(19%) patients. Maintenance RTX was used in 42.8% and 45.2% patients received Azathioprine. 23.8% patients relapsed and 54.6% patients were in remission. 10(23.8%) required Mechanical Ventilation. No patients were on long term oxygen therapy. 7(16.6%) patients died. Morbidity was seen in 16(38%) including CKD in 8(19%) patients. Median(IQR) duration of follow up was 10(6-24) months. Conclusion DAH is a serious manifestation of AAV and an important Rheumatological emergency. DAH was present in 31.5% with majority patients in remission. Mortality was seen in 16.6% and morbidity in 38% cases. Management with combination of Pulse steroids, Cyclophosphamide /Rituximab and Plasmapheresis is the key to better outcomes. Ventilator support may be necessary in severe cases. Timely diagnosis and aggressive management is the key to survival and prevents morbidity. Disclosure J.R. Yadavalli: None. K. Telang: None. P. Khatri: None. S. Dhuria: None. G. Ekbote: None. R. Gupta: None.

Clinical impact of ceruloplasmin levels at ANCA-associated vasculitis diagnosis.

Ceruloplasmin is an inhibitor of myeloperoxidase (MPO) activity that plays an important role in the pathophysiology of anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV). This study aimed to evaluate the prognostic impact of serum level of ceruloplasmin at diagnosis in patients with anti-MPO antibody-positive AAV. This retrospective monocentric study in Caen University Hospital involved all consecutive adult anti-MPO antibody-positive patients with microscopic polyangiitis or granulomatosis with polyangiitis, diagnosed between January 2010 and January 2022 with available serum sample at inclusion. Patients outcomes were analyzed from two subgroups constituted according to the median serum level of ceruloplasmin. The same analyses were then performed in anti-proteinase 3 (PR3) antibody-positive patients. Within the 92 patients analyzed, 50 patients had anti-MPO antibodies with a median ceruloplasmin level of 0.44 [quartiles 1-3, 0.40-0.49] g/L and a median Birmingham Vasculitis Activity Score of 19 [14-22]. After a median follow-up period of 40 [22-86] months, 13 (26%) patients had died: 10 (40%) in the low ceruloplasmin group and 3 (12%) in the high ceruloplasmin group (p = 0.03), with a significantly worse survival rate in the low ceruloplasmin group (p = 0.021). No significant differences in relapse rate or renal failure was observed between the two groups. The same analyses performed in the group of AAV patients with anti-PR3 antibody did not show any differences. In anti-MPO AAV patients, serum level of ceruloplasmin at diagnosis seems to be associated with a significant impact on survival.

Bruton’s tyrosine kinase is a possible therapeutic target in microscopic polyangiitis

BackgroundBruton’s tyrosine kinase (Btk) is an enzyme expressed in leukocytes other than T lymphocytes and plasma cells and involved in B-cell receptor- and Fcγ receptor (FcγR)-mediated signal transduction. Btk inhibitors potentially suppress autoantibody production due to the expected inhibitory ability of B lymphocyte differentiation into antibody-producing plasma cells and reduce FcγR-mediated neutrophil activation, including the release of neutrophil extracellular traps (NETs). Microscopic polyangiitis (MPA) is a systemic small-vessel vasculitis characterized by the pathogenic autoantibody, antineutrophil cytoplasmic antibody (ANCA) that reacts with myeloperoxidase (MPO). MPO and MPO-ANCA immune complex (IC)-induced FcγR-mediated NETs are critically involved in MPA pathogenesis. This study aimed to demonstrate the therapeutic efficacy of the Btk inhibitor tirabrutinib on MPA.MethodsVarious doses of tirabrutinib or vehicle were orally administered to Sprague–Dawley rats daily. Four weeks later, the number of peripheral B lymphocytes was counted, and Btk phosphorylation in B lymphocytes was evaluated by flow cytometry. Human peripheral blood neutrophils were stimulated by MPO and anti-MPO antibody ICs (MPO and anti-MPO-ICs), and Btk and its downstream Vav phosphorylation were assessed by western blotting. The effects of tirabrutinib on MPO and anti-MPO-IC-induced NET formation were examined in vitro. Wistar Kyoto rats were immunized with human MPO to induce experimental MPA and given drug-free or tirabrutinib-containing feed (0.0037% or 0.012%) from day 0 or 28. All rats were euthanized on day 42 for serological and histological evaluation.ResultsTirabrutinib inhibited Btk phosphorylation without decreasing B lymphocytes in vivo. Neutrophil Btk and Vav were phosphorylated when stimulated with MPO and anti-MPO-ICs. Tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA in a dose-dependent manner in vivo. Although MPO-ANCA production was not affected, NET-forming neutrophils in the blood were significantly reduced by tirabrutinib.ConclusionsThe Btk inhibitor tirabrutinib suppressed MPO and anti-MPO-IC-induced NET formation in vitro and ameliorated experimental MPA by reducing NET-forming neutrophils but not decreasing MPO-ANCA titer in vivo. This study suggests that Btk is a possible therapeutic target in MPA.

ANCA detection with solid phase chemiluminescence assay: diagnostic and severity association in vasculitis

ANCA-associated vasculitis (AAV) comprises a group of necrotizing vasculitis that mainly affects small- and medium-sized vessels. Serum anti-neutrophil cytoplasmic antibodies (ANCA), mainly anti-myeloperoxidase (anti-MPO) and anti-proteinase 3 (anti-PR3), levels may correlate to severity, prognosis, and recurrence of the disease. A retrospective analysis of 101 patients with MPO-positive and 54 PR3-positive vasculitis was performed, using laboratory established cut-off value, measured by chemiluminescence. Furthermore, data of renal disease and pulmonary involvement were collected at vasculitis diagnosis, as well as the progress, requiring dialysis, transplant, or mortality. For anti-MPO antibodies with a diagnosis of vasculitis (n = 77), an area under the curve (AUC) was calculated (AUC = 0.8084), and a cut-off point of 41.5 IU/ml was determined. There were significant differences in anti-MPO levels between patients with renal or pulmonary dysfunction (n = 65) versus those without them (n = 36) (p = 0.0003), and a cut-off threshold of 60 IU/ml was established. For anti-PR3 antibodies with a diagnosis of vasculitis (n = 44), an area under the curve (AUC) was calculated (AUC = 0.7318), and a cut-off point of 20.5 IU/ml was determined. Significant differences in anti-PR3 levels were observed between those patients with renal or pulmonary dysfunction (n = 30) and those without them (n = 24) (p = 0.0048), and a cut-off threshold of 41.5 IU/ml was established. No significant differences between those patients who had a worse disease progression and those who did not were found for anti-MPO and anti-PR3. Anti-MPO and anti-PR3 levels at the moment of vasculitis diagnosis are related with disease severity but not with disease outcome or vasculitis recurrence.

#2709 THE INVOLVEMENT OF NRF2-MEDIATED ANTIOXIDANT SYSTEM IN MYELOPEROXIDASE-POSITIVE ANTINEUTROPHIL CYTOPLASMIC ANTIBODY-ASSOCIATED VASCULITIS

Abstract Background and Aims Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease pathologically characterized by vascular necrosis with inflammation. The necrotic lesions consist of ANCA-mediated neutrophil extracellular traps (NETs). During AAV, activated neutrophils form NETs via reactive oxygen spices (ROS)s-mediated signaling pathway. The excessive NETs lead to endothelial vascular damage and serve as autoantigens against ANCAs in a vicious cycle. Although the role of NETs in AAV has been revealed, its treatment for targeting NETs remains unestablished. Since nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates antioxidant proteins against oxidative stress and functions as a defense system, we investigated the role of Nrf2 in the development of AAV. Method In vitro, human neutrophils treated with the Nrf2 activator bardoxolone methyl (Bard) were stimulated with ANCA, and the effects on NETs formation and signaling pathways including intracellular ROS were evaluated by cell imaging and biochemical approaches. The effects of Nrf2 activators on NETs-induced cytotoxicity were examined using human renal glomerular endothelial cells (HRGECs). Nrf2 knockout or WT mouse-derived neutrophils were stimulated with anti-MPO antibody to induce NETs. In vivo studies, Bard was administered to ANCA-transfer AAV model or spontaneous AAV developing mice models (SCG/Kj). Renal function was assessed by biochemical analysis, and the expression of ROS and antioxidant protein NAD(P)H Quinone Dehydrogenase 1 (NQO1) in tissues was evaluated by fluorescent immunostaining and immunoblotting. The formation of NETs in the glomeruli was evaluated by immunostaining and glomerular vascular endothelial damage was assessed by the expression of CD31 and TUNEL positivity. To assess the role of Nrf2 in immunological abnormality during AAV, murine splenocytes were collected and flow cytometry were conducted using CD4, CD8, B220, MCHCII, CD11c, F4/80, Ly 6G, Ly6b, and CD11b antibodies. Moreover, to assess the genetic role of Nrf2 in AAV, we established SCG/Kj mice carrying a germ-line deletion of the Nrf2-genes. A spontaneous mouse model of vasculitis genetically deficient in Nrf2 was analyzed by the same parameters to evaluate the effect of Nrf2. N1 offspring mice were generated by crossing Nrf2-/- mice with SCG/Kj mice. These backcrossed mice were obtained by crossing Nrf2-/- mice to background SCG/Kj mice for three generations. The experimental group was mated to N4 heterozygous mice, and female mice in the experimental group were analyzed at 18 weeks. Results In vitro, pharmacological activation of Nrf2 suppressed ANCA-induced NETs via the upregulation of NQO1 and inhibition of ROS. While, Nrf2-deficienet neutrophils showed the massive NETs in response to ANCA, compared to WT neutrophils. Furthermore, Nrf2 activation directly protected endothelium from cellular injury caused by the exposure of ANCA-mediated NETs. In vivo, the pharmacological activation of Nrf2 ameliorated glomerulonephritis in two kinds of AAV models through the upregulation of antioxidant and inhibition of ROS-mediated NETs. In contrast, Nrf2 genetic deficiency exacerbated vasculitis in spontaneous AAV model. The activation of Nrf2 regulated the expansion of splenocytes in AAV mice. In particular, pan-T cells in spleen and the infiltration of Th17 cells in kidney were suppressed by Nrf2 activation. Conclusion During ANCA-stimulation in neutrophils, the activation of Nrf2 initiates antioxidant system and inhibits intracellular ROS, leading to the suppression of NETs formation. Nrf2 may be a novel therapeutic target for AAV.

171 Crescentic Lupus Nephritis With Positive Anti-MPO Antibodies in a Patient With Latent Tuberculosis

171 Crescentic Lupus Nephritis With Positive Anti-MPO Antibodies in a Patient With Latent Tuberculosis

Successful treatment with rituximab and plasmapheresis of renal involvement of eosinophilic granulomatosis with polyangiitis

BackgroundEosinophilic granulomatosis with polyangiitis (EGPA) is a rare disorder characterized by asthma, eosinophilia, and systemic vasculitis. Renal involvement is not regarded as a prominent feature and the treatment is still under study.Case presentationA 68-year-old woman was admitted to our hospital because of fever, renal dysfunction, eosinophilia, and the presence of MPO-ANCA. Based on the renal pathological examination which showed extravascular eosinophilic-predominant inflammation and crescentic glomerulonephritis, EGPA was diagnosed. Considering the acute kidney injury, prominent eosinophilia, and strongly positive anti-MPO antibodies, pulse steroid therapy was administered, followed by intravenous rituximab. Plasmapheresis was also provided (9 sessions). The eosinophil count was normalized, and renal dysfunction was reversed. The patient no longer requires dialysis.ConclusionsRenal involvement of EGPA is rare, and consensus on its treatment is still lacking, because of a lack of large-scale randomized controlled trials. We treated our patient as a case with high severity. For patients with severe disease, the addition of cyclophosphamide to glucocorticoid therapy is commonly used. However, rituximab and plasmapheresis combined with systemic glucocorticoid therapy were found to be beneficial because the renal function and other clinical conditions were almost fully recovered. Thus, our treatment is highly effective against renal involvement of eosinophilic granulomatosis with polyangiitis.

Evaluation of diagnostic efficacy of digital liquid chip method for detection of specific antineutrophil cytoplasmic antibodies

To explore the clinical diagnostic efficacy of antineutrophil cytoplasmic antibody associated vasculitis (AAV) by comparing the consistency and coincidence rate of serum anti-myeloperoxidase (MPO) antibody and anti-protease 3 (PR3) antibody detected by digital liquid chip method (DLCM) and enzyme-linked immunosorbent assay (ELISA). To provide reference for the selection of detection methods of anti-MPO antibody and anti-PR3 antibody in clinical laboratory. This study is a cross-sectional study, a total of 307 cases of antineutrophil cytoplasmic antibodies were detected in the Department of Clinical Immunology, West China Hospital of Sichuan University from January to March 2021. The serum samples and related clinical information were collected. At the same time, the levels of anti-MPO antibody and anti-PR3 antibody in serum samples were detected by ELISA and DLCM, indirect immunofluorescence (IIF) was used to re-test the differential samples between the two methods. SPSS 26.0 was used to analyze the test results, Cohen's kappa coefficient analysis was used to compare the consistency of the two methods, and paired chi-square test was used to compare the sensitivity and specificity of the two methods to AAV. The results showed that the positive cases of anti-MPO antibody detected by ELISA and DLCM were 63 and 44, and the negative cases were 244 and 263; the positive cases of anti-PR3 antibody detected by ELISA and DLCM were 34 and 28, and the negative cases were 273 and 279. The results of anti-MPO antibody and anti-PR3 antibody detected by the two methods had good consistency and coincidence rate, in which the total coincidence rate of anti-MPO antibody was 92.51%, the positive coincidence rate was 66.67%, and the negative coincidence rate was 99.18%. The results of consistency analysis showed that kappa=0.741 had well consistency. The total coincidence rate of anti-PR3 antibody is 96.74%, the positive coincidence rate is 76.47%, and the negative coincidence rate is 99.27%. The consistency analysis results show that kappa=0.821 had strong consistency. The results of IIF re-test of differential samples showed that the coincidence rate between DLCM and IIF was higher. The results of comparative analysis of anti-MPO antibody and anti-PR3 antibody showed that the specificity of DLCM was better than that of ELISA, and its sensitivity was lower than that of ELISA. In conclusion, the results of anti-MPO antibody and anti-PR3 antibody detected by DLCM were consistent with those of ELISA. In the combined detection of anti-MPO antibody and anti-PR3 antibody, the specificity of DLCM is better than that of ELISA.

Anti-glomerular basement membrane disease developing 3years after the development of Sweet syndrome and 1year after the development of anti-neutrophil cytoplasmic antibody-associated vasculitis: a case report.

A 73-year-old Japanese woman, with a history of Sweet syndrome diagnosed 3years earlier and anti-myeloperoxidase (MPO) antibody anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis diagnosed 1year earlier, presented with an episode of rapidly progressive glomerulonephritis (RPGN) with anti-glomerular basement membrane (GBM) disease. At the time of diagnosis of the ANCA-associated vasculitis 1 year earlier, serological testing yielded a negative result for anti-GBM antibody. However, at the present visit, serology for anti-MPO antibody was negative, while that for anti-GBM antibody was positive. This is the first report of anti-GBM disease developing sequentially after Sweet syndrome and ANCA-associated vasculitis. This case may provide clues to the potential immunological links among these three distinct conditions.

Dialysis Dependency at Discharge in De Novo ANCA Associated Renal Limited Vasculitis: Should We Avoid Further Immunosuppression?

Introduction: ANCA associated vasculitis commonly affects the kidneys. The intense inflammatory damage disrupts the glomerular architecture. Induction immunosuppressive therapy is responsible for a large part of the morbidity and mortality of these patients, due to infectious complications. This highlights the importance of defining risk factors associated with a worse renal prognosis in order to select the patients who would benefit the most from immunosuppressive therapy when it comes to renal limited vasculitis. Methods: Retrospective collection of data from patients admitted to the Coimbra’s University Hospital Nephrology Department with the diagnosis of de novo ANCA vasculitis, between 01-06-2009 and 01-06-2019. Data were analysed with SPSS v26® using parametric and nonparametric tests, Kaplan Meyer survival and ROC curve analysis. Results: There were 81 cases of de novo vasculitis, mainly due to anti-MPO antibody associated vasculitis (N=69; 85.2%). Mean age at diagnosis was 67.4 ± 15.7 years and most patients were male (N=47; 58%). At admission, patients had a mean serum creatinine (sCr) of 6.8 ± 4.0 mg/dL. Dialysis was required in 23 (28.4%) patients at admission and 44.4% were dependent on renal replacement therapy at discharge. Among dialysis-dependent patients at discharge treated with immunosuppression for at least 3 months (N=15) we observed partial kidney recovery in 2 (13.3%). Deaths were mostly (N=8/11; 72.7%) caused by infections. Discussion: Our results call into question the need for aggressive induction treatment in dialysis-dependent patients without extrarenal manifestations.

Bisphenol A: Potential Factor of Miscarriage in Women in the Context of the Phenomenon of Neutrophil Extracellular Traps

Humans are exposed to a number of environmental pollutants every day. Among them, endocrine disruptors are particularly harmful to human health. Bisphenol A (BPA) is a xenoestrogen that has been shown to disrupt the endocrine system and cause reproductive toxicity. In this study, we aimed to verify the potential relationship between BPA and miscarriage involving the formation of neutrophil extracellular traps (NETs). Blood samples were collected from healthy women and women who had miscarriage in the first trimester of pregnancy. The serum levels of cytoplasmic anti-PR3 antibody and perinuclear anti-MPO antibody were determined using an immunoenzymatic method. The concentrations of key proinflammatory proteins TNF-α and MCP-1, as well as NADPH oxidase subunits NOX1 and NCF2, were also measured in the serum samples. The serum concentration of BPA was determined using gas chromatography. The results showed that the concentrations of BPA were significantly elevated in the serum of women who had miscarriage compared to the control group, with the highest concentration found in the “NETs-positive” group. The levels of MCP-1 and TNF-α were significantly higher in the “NETs-positive” group compared to the “NETs-negative” and control group. The levels of NOX1 and NCF2 were also higher in the “NETs-positive” group compared to the “NETs-negative” group. The study showed that BPA could play a role in the course of miscarriage through the formation of NETs. The results indicate the need to limit the exposure of women planning pregnancy to xenoestrogens, including BPA.

Clinical characteristics and outcomes of MPO-ANCA-associated glomerulonephritis with bronchiectasis: A retrospective case-control study

BackgroundThe association of bronchiectasis with myeloperoxidase (MPO) antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (MPO-AAV) has been widely described in recent studies. However, the clinical features and outcomes of MPO-ANCA-associated glomerulonephritis (MPO-ANCA GN) patients with bronchiectasis remain enigmatic. MethodsMPO-ANCA GN patients with bronchiectasis were compared to MPO-ANCA GN patients alone. Clinical imaging, pathological tests, and follow-up examination data of patients were collected retrospectively. Progression to end-stage renal disease (ESRD) and death was treated as endpoint events. Results153 cases (52 patients with bronchiectasis) were included in this study. Compared to MPO-ANCA GN patients alone, MPO-ANCA GN patients with bronchiectasis exhibited a lower level of proteinuria (p = 0.019) and relatively higher eGFR level. MPO-ANCA GN patients with bronchiectasis had less frequent incidences of interstitial lung disease (ILD) and emphysema (p<0.001, p = 0.016, respectively) but with higher rates of pulmonary infection (p<0.001). Bronchiectasis severity (the modified Reiff score) was positively correlated with MPO antibody titers (ρ=0.480, p<0.001), but not with shorter renal survival. A relatively higher remission rate was been seen in MPO-ANCA GN patients with bronchiectasis, who showed reduced susceptibility in progressing to ESRD in multivariate analysis (p = 0.043, HR=0.542, 95% CI 0.299–0.982). One-and three-year overall survival rates were 88.2% and 77.3% for MPO-ANCA GN with bronchiectasis cases versus 83.7% and 67.2% for MPO-ANCA GN patients alone (p = 0.431, p = 0.241, respectively). ConclusionThe severity of bronchiectasis was correlated with anti-MPO antibody titers in MPO-ANCA GN patients. For MPO-ANCA GN patients, bronchiectasis associated with good renal prognosis, but it did not improve overall survival.

AB0629 Glomerulonephritis associated with ANCA after COVID-19 vaccine: a systematic review

BackgroundThe COVID-19 pandemic has led to widespread vaccination with effective results and a great safety profile. However, as the vaccination rate has increased, more cases of autoimmune diseases after the COVID-19 vaccine have been described. We present a systematic review of ANCA-associated glomerulonephritis after the COVID-19 vaccine.ObjectivesTo summarize the existing evidence on ANCA-associated glomerulonephritis after the COVID-19 vaccine.MethodsWe searched all studies from inception till January 27, 2022, that described ANCA-associated biopsy-proven glomerulonephritis after COVID-19 vaccine through Embase and Medline. Methodological quality was evaluated with the 4 domains tool. We included 13 patients from 2 case series and 9 case reports. We extracted demographics, history, lab results, outcomes. We then applied descriptive statistics.Results46% of the patients were males and 54% were females. The median age was 74 years. 54% developed symptoms after the second dose of the 2-dose vaccine. The median interval between the vaccine and the symptom onset was 10.5 days. 85% had anti-MPO antibodies, and the rest – anti-PR3 antibodies. 31% of patients had persistent creatinine (Cr) elevation on follow-up, and 3 patients were requiring hemodialysis. Of those 3 patients, 1 patient had normal renal function prior to presentation, and the rest had chronic kidney disease. The summary of the cases is presented in Table 1.Table 1.Gender, ageDe novo (1) or relapse (2)AZD1222 (1), or mRNA-1273 (2), or BNT162b2 (3)OutcomeM, 75121HemodialysisM, 74121Improved CrF, 82212Improved CrF, 70312Improved CrF, 78413Improved CrF, 79513RemissionF, 54613Improved CrF, 78623HemodialysisM, 58712RemissionM, 63811Improved CrF, 29913RemissionM, 521012HemodialysisM, 841113Worsened CrConclusionAlthough the causality cannot be established on current evidence, the COVID-19 vaccine can probably trigger glomerulonephritis associated with ANCA, primarily anti-MPO type. We need a bigger cohort to identify patients predisposed for disease development or relapse after the COVID-19 vaccine.

POS-513 PLASMA EXCHANGE IN NEPHROLOGY

Plasma exchange is a therapeutic tool with broad indications in different diseases. The aim of this study was to determine its indications and possible complications in the nephrology setting. This is a retrospective study of therapeutic plasma exchange performed in our department over 15 years [2006 to 2021]. Our study included 20 patients with an average age of 46.45 years [18-77] and a sex ratio of 0.66. The initial symptomatology was variable. A glomerular syndrome was present in all patients associated with rapidly progressive renal failure in 15 cases with an average blood creatinine level of 645.05 µmol/L [144-2017], 12 cases of Haemoptysis on chest CT scan or bronchoalveolar lavage and one case of cerebellar syndrome. Immunologically, 7 cases were positive for antiGBM antibodies, 4 cases positive for anti-MPO antibodies, 3 cases positive for antiPR3 antibodies, and serum complement was consumed in 3 patients. Renal biopsy performed in 10 patients revealed 1 case of myeloma nephropathy, 2 cases of lupus nephropathy, 3 cases of vasculitis, and 4 cases of Goodpasture's disease. Indications were diverse and included rapidly progressive glomerulonephritis, acute humoral rejection in a renal transplant patient, hyperviscosity syndrome in a myeloma patient, and a case of acute polyradiculoneuritis. The products used were albumin with fresh frozen plasma in all patients and albumin alone for the renal transplant patient, with an exchange volume of 50cc/kg. The average number of sessions was 6.3 [1-16] with a frequency of 1 to 3 sessions per week. Complications ranged from 2 episodes of circuit clotting to 2 episodes of low blood pressure, one case of acute coronary syndrome, one case of seizure followed by cardiac arrest and death. The progression was marked by an improvement of the renal function in 8 patients, the use of extrarenal purification in 9 patients, and the death of 2 patients. The indications for therapeutic plasma exchange are increasingly broad in the nephrology setting, including neurological and hematological disorders in patients with various kidney diseases.

Increased Platelet S100A8/S100A9 Associated with Vasculitis in Granulomatosis with Polyangiitis (GPA)

Granulomatosis with polyangiitis (GPA), formerly known as Wegener's Granulomatosis, is characterized by vasculitis that predominantly affects small- and medium-sized blood vessels in the sinuses, lungs, and kidneys. In addition to vascular inflammation, GPA is also characterized by an increased risk of thrombosis. The role of platelets in GPA pathogenesis remains incompletely understood. We aimed to better understand the changes in platelet gene expression and function in patients with GPA. Forty-two patients diagnosed with GPA (n=9 with active GPA and n=33 with GPA in remission) and 25 healthy, age-, gender-, and race-matched donors were enrolled. Patients with GPA showed typical disease manifestations, with an average Birmingham Vasculitis Activity Score of 1.6 (Mean±SD 1.6±3.5). One sixth of GPA patients (7/42) had a history of thrombosis. When stimulated with thrombin receptor activating peptide (TRAP, 50nM), platelets from patients with GPA showed significantly increased expression of P-selectin as compared to healthy controls (P-selectin+% Mean±SEM: Healthy 15.50±1.84 vs GPA 25.71±16.05, P<0.05). This suggests increased platelet activation in GPA, consistent with previous findings of increased platelet aggregation in vitro in GPA. In addition, released chemokines sCD40L and platelet-derived growth factor (PDGF) by activated platelets were increased in patients with GPA when we measured the cytokines in the platelet poor plasma using the Miliplex human cytokine Assay [sCD40L (ng/mL) Mean±SEM: Healthy 63.05±6.63 vs GPA 100.40±11.86, P<0.05, PDGF-AA (pg/mL) Mean±SEM: Healthy 137.50±46.52 vs GPA 357.30±79.65, P=0.052]. Next, we performed RNA-sequencing on platelets from GPA patients (n=8, 3 with active GPA disease and 5 in remission) and, for comparison, 4 healthy donors. We identified 75 genes that were significantly differentially expressed between GPA patients and healthy donors. The top 30 genes are listed in Figure 1A. S100A8 and S100A9 were the top two significantly differentially expressed transcripts in patients with GPA (Fig. 1B). These two genes encode proteins that form a heterodimer S100A8/S100A9 (commonly known as calprotectin) known to be increased in the plasma of GPA patients and associated with disease activity. Interestingly, platelets have not been identified as the cellular source of plasma calprotectin in GPA previously. Significantly increased RNA and protein expression of S100A8 and S100A9 in GPA patients was independently validated by qRT-PCR and immunoblot, respectively. The mRNA expression of S100A8 and S100A9 in platelets were significantly correlated with p-ANCA and anti-MPO antibodies, indicating platelet S100A8/S100A9 promotes neutrophil activation and inflammation (Mann-Whitney nonparametric test, P<0.05). As previously reported, plasma levels of calprotectin were also increased in GPA patients. To further evaluate if plateletS100A8/S100A9 mediates endothelial inflammation and vasculitis, we co-cultured platelets activated with thrombin (which increases S100A8/S100A9 secretion) with endothelial cells in the presence or absence of an anti-S100A8/S100A9 blocking antibody. Activated platelets triggered endothelial cell inflammation (e.g., increased expression of ICAM-1) that was significantly reduced when S100A8/S100A9 was blocked. In summary, the platelet transcriptome is altered in patients with GPA, with S100A8 and S100A9 being the top upregulated genes. Platelet functional responses are enhanced in patients with GPA, and our data suggests that increased plasma calprotectin levels in GPA patients may be platelet derived. Platelets and platelet S100A8/S100A9 appear to mediate vascular inflammation and thrombosis in GPA. [Display omitted] DisclosuresRondina: Novartis: Research Funding; Platelet Biogenesis: Membership on an entity's Board of Directors or advisory committees; Acticor Biotech: Membership on an entity's Board of Directors or advisory committees; Platelet Transcriptomics: Patents & Royalties.

Interstitial Lung Disease and Anti-Myeloperoxidase Antibodies: Not a Simple Association

Anti-neutrophil cytoplasmic antibodies (ANCA), mainly anti-myeloperoxidase (MPO) antibodies, have been frequently identified in patients with idiopathic pulmonary fibrosis (IPF). However, their role remains unclear, and only 7–23% of these patients develops clinically overt vasculitis. We aimed to investigate the clinical, serological, and radiological features and prognosis of anti-MPO-positive interstitial lung disease (ILD) patients. Fifty-eight consecutive patients firstly referred for idiopathic interstitial pneumonia and showing serological positivity of anti-MPO antibodies were retrospectively enrolled. For each patient, clinical data, lung function testing, chest high-resolution computed tomography (HRCT) pattern, and survival were recorded. Thirteen patients developed a rheumatic disease during a median follow-up of 39 months. Usual interstitial pneumonia (UIP) was the most frequent ILD pattern, significantly influencing the patients’ survival. In fact, while the 52-week survival of the overall population was 71.4 ± 7.5%, significantly higher than IPF, survivals of anti-MPO patients with UIP pattern and IPF were similar. Forced vital capacity and diffusion lung capacity for CO significantly declined in 37.7 and 41.5% of cases, respectively, while disease progression at chest HRCT was observed in 45.2%. A careful clinical history and evaluation should always be performed in ILD patients with anti-MPO antibodies to quickly identify patients who are developing a systemic rheumatic disease.

Relapse rate and renal prognosis in ANCA-associated vasculitis according to long-term ANCA patterns.

Long-term observation of patients with ANCA-associated vasculitis (AAV) allows the identification of different longitudinal patterns of ANCA levels during follow-up. This study aimed to characterize these patterns and to determine their prognostic significance. All ANCA determinations performed in two university hospitals during a 2-year period were retrospectively reviewed. Patients were included in the analysis if they had high titers of anti-myeloperoxidase (anti-MPO) or anti-proteinase 3 (anti-PR3) antibodies at least once, ≥5 serial ANCA determinations and AAV diagnosed by biopsy or American College of Rheumatology (ACR) classification criteria. Patients' time-course ANCA patterns were classified as monophasic, remitting, recurrent or persistent. Associations between ANCA patterns and prognostic variables (relapse rate and renal outcome) were analysed by univariate and multivariate statistics. A total of 99 patients [55 with microscopic polyangiitis (MPA), 36 with granulomatosis with polyangiitis (GPA) and eight with eosinophilic granulomatosis with polyangiitis (EGPA)] were included. Median follow-up was 9years. Among patients diagnosed with MPA or GPA, recurrent or persistent ANCA patterns were associated with a higher risk of clinical relapse [hazard ratio (HR)=3·7, 95% confidence interval (CI)=1·5-9·1 and HR=2·9, 95% CI=1·1-8·0, respectively], independently of clinical diagnosis or ANCA specificity. In patients with anti-MPO antibodies, the recurrent ANCA pattern was associated with worsening renal function [odds ratio (OR)=5·7, 95% CI=1·2-26·0]. Recurrent or persistent ANCA patterns are associated with a higher risk of clinical relapse. A recurrent ANCA pattern was associated with worsening renal function in anti-MPO-associated vasculitis.

The performance of the chemiluminescent immunoassay for measuring serum myeloperoxidase and proteinase 3 antibodies.

BackgroundEnzyme‐linked immunosorbent assay (ELISA) has traditionally been used to detect myeloperoxidase (MPO) and proteinase 3 (PR3) antibodies, although it is time‐consuming and physically demanding. As a novel and highly effective immunoassay, we compared chemiluminescent immunoassay (CIA) with ELISA to verify the application value of CIA in MPO and PR3 antibodies detection.MethodsBy ELISA and CIA, serum levels of anti‐MPO and anti‐PR3 antibodies were measured in 63 anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV) patients (AAV group), including 47 microscopic polyangiitis (MPA) patients and 16 granulomatosis with polyangiitis (GPA) patients, in addition, 68 patients in interference control group (IC group), 19 healthy subjects in healthy control group (HC group). We compared MPO and PR3 antibodies levels and positive rates measured by these two methods among groups. Relationship and coincidence rate between ELISA and CIA were investigated. Diagnostic values for clinical outcomes for MPO and PR3 antibodies were assessed by receiver operator characteristic (ROC) curve.ResultsIn AAV patients, when detecting anti‐MPO (r = .90) and anti‐PR3 (r = .81), CIA was highly correlated with ELISA, companying with highly total (88.89%, 92.06%, respectively) and positive coincidence rates (84.78%, 77.27%, respectively). In HC group, anti‐PR3 positive rate detected by both immunoassay were 0, anti‐MPO almost were 0, which without statistically significant difference (P = .32). In IC group, the total (76.47%, 58.82, respectively) and positive coincidence rates (48.38%, 30.00%, respectively) of anti‐MPO and anti‐PR3 were the lowest, but the negative coincidence rates reached 100%. By CIA, similar to ELISA, the levels of anti‐MPO were significantly higher both in AAV patients (56.00; [4.40‐235.30]) and MPA patients (98.00; [27.90‐324.70]) compared with either IC group (3.20; [3.20‐18.55) (P < .0001) or HC group (3.20; [3.20‐3.20]) (P < .0001), yielded an area under curve (AUC) of 0.76 for AAV and 0.89 for MPA, the concentration of anti‐PR3 in GPA group (66.65; [24.43‐150.00]) was significantly higher than that in IC group (2.3; [2.3‐10.95]) (P < .0001) and HC group (2.3; [2.3‐2.3]) (P < .0001), with an AUC of 0.92.ConclusionSimilar to ELISA, CIA was competent to detect MPO and PR3 antibodies in AAV patients and healthy population, thus distinguish AAV patients from IC group and HC group and effectively diagnose MPA and GPA.