BackgroundB cell depletion with rituximab (RTX) is approved for treatment of rheumatoid arthritis (RA) and ANCA-associated vasculitides (AAV). Recently, RTX has been shown to be effective in AAV maintenance therapy, but an optimal RTX treatment schedule is unknown and the time to B cell repopulation after RTX has not been studied.MethodsRetrospective single-center analysis of B cell repopulation in patients with AAV, RA or connective tissue disease (CTD) treated with RTX.ResultsBeginning B cell repopulation within the first year after RTX treatment was observed in 93% of RA and 88% of CTD patients. Only 10% of patients with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) and no patient with eosinophilic granulomatosis with polyangiitis (EGPA) showed B cell repopulation within this time. Median time of B cell depletion was 26 months in GPA/MPA, and 21 months in EGPA compared to 9 months in RA, and 8 months in CTD (p < 0.0001). In 25 AAV-patients B cell depletion lasted for at least 44 months. There was a significant decline in serum immunoglobulin concentrations in GPA/MPA patients, but not in patients with RA or CTD. Significantly more GPA/MPA patients developed hygogammaglobulinemia (IgG <7 g/L) compared to patients with RA or CTD.ConclusionsIn contrast to RA and CTD, in AAV RTX induces long-lasting depletion of B cells that is associated with decreased antibody production. This observation points toward potential defects in the B cell compartment in AAV that are unmasked by immunosuppressive treatment and has important implications for the design of maintenance treatment schedules using RTX.

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Background:Eosinophilic granulomatosis with polyangiitis (EGPA) is a rare form of systemic vasculitis, which is characterized by bronchial asthma, hypereosinophilia, and systemic vasculitis. B-lymphocytes play a key role in EGPA as producers of IgE and anti-neutrophil cytoplasmic antibodies (ANCAs). Indeed, the neutrophils that are targeted by these antibodies are widely described as the mechanism of endothelial damage in this disease. On the other side, the therapeutic response to rituximab in EGPA patients provides evidence for a role of B-cells in the pathogenesis of EGPA. Therefore characterizing B cell subpopulations may help in understanding the disease and the treatment.Objectives:To characterize the peripheral B cell compartment in patients with EGPA and to analyze the in vivo potential of B lymphocytes to class-switch to IgE and to assess in vitro the differentiation potential of naïve B cells of EGPA patients into IgE-secreting plasmablasts.Methods:Clinical characteristics of the patients, including organ involvement and treatment regimen were evaluated. Laboratory work-up included ANCA-status, eosinophils, IgE, IgG, IgA, IgM, and peripheral CD19+B-cell count. For immunophenotyping isolated PBMCs were stained with monoclonal or polyclonal antibodies and B cells were classified into: naïve, marginal zone, class-switched memory B cells, unconventional memory B cells, transitional and plasmablasts. Furthermore, the expression of IgG+ and subclasses IgG1-4, IgA+, IgE+B cells, BAFFR and TACI was quantified. For in vitro differentiation assays magnetically isolated B lymphocytes from EGPA patients and age-matched healthy controls were stimulated with CD40L, IL-21 and IL-4. Starting the culture with equal number of B cells, the absolute number of plasmablasts, and IgE class switched cells after 9 days was determined by counting the events in the CD27highCD38high gate or the IgG/A/D-IgE+gate by flow cytometry. IgE secretion in the supernatant was measured by ELISA. JAK-STAT signalling pathway was analyzed in response to IL-4 and IL-21 stimulation and phosphorylation of STAT5 and 6 measured by flow cytometry.Results:34 patients with EGPA diagnosed according to ACR and CHC-criteria were included into the study. Ten of these patients were analysed separately because they received rituximab therapy. Peripheral B cell numbers in EGPA patients were markedly diminished. B cell subpopulation phenotyping showed in average 57.9% naïve B cells, 12.5 % marginal zone like B cells and 19.2% switched memory B cells. Plasmablasts constituted in average 1.15% of the peripheral B cell compartment, transitional B cells 2.0%. Interestingly, the expression of BAFF receptor and TACI in the memory B cell subset was significantly decreased in EGPA patients when compared with healthy donors. In vitro assays of isolated B cells from EGPA patients demonstrated an increased proportion of IgE-class-switched B cells after 9 days of culture under IL4 stimulation compared with controls. However, no differences were observed in the phosphorylation of STAT5 and STAT6 after stimulation with IL-4 or IL-21.Conclusion:In the EGPA-patients we observed markedly diminished B-cells despite of normal lymphocyte counts. B cells showed a reduced expression of BAFF-R and TACI. Class switch to IgE is enhanced in EGPA patients.Disclosure of Interests:None declared

BackgroundEosinophilic granulomatosis with polyangiitis (EGPA) belongs to the group of ANCA associated vasculitides. While the combination of asthmatic symptoms and vasculitis characterize the disease clinically, eosinophilia and increased serum IgE concentrations are serologic hallmarks. The role of B lymphocytes in EGPA has not been defined so far, but therapeutic response to rituximab in EGPA points towards a role of B-cells in the pathogenesis of EGPA.ObjectivesTo characterize the peripheral B cell compartment in patients with EGPA, to analyze the in vivo potential of B lymphocytes to class-switch to IgE, and to assess in vitro the differentiation of naïve B cells to IgE-secreting plasmablasts.MethodsLaboratory work-up included ANCA-status, eosinophils, IgE, IgG, IgA, IgM, and peripheral CD19+ B-cell count. B cell subpopulations (naïve, marginal zone, class-switched B cells and plasmablasts) were analyzed by staining PBMCs with fluorescent-labeled monoclonal antibodies against: CD27, CD20, CD38, IgD, IgG, IgA, IgE, CD21, and BAFF-R. For in vitro differentiation assays magnetically isolated B lymphocytes from EGPA patients and aged matched healthy controls were stimulated with CD40L and IL-21 and IL-4 in enriched Iscoves' medium supplemented with 10% FCS, 1 μg/mL insulin, 2.5 μg/mL apo-transferrin, 1% nonessential amino acids, 2 mmol/L glutamine, and 1 μg/mL reduced glutathione. Starting the culture with equal number of B cells, the absolute number of plasmablasts, and IgE class switched cells after 9 days was determined by counting the events in the CD27highCD38high gate or the IgG/A/D-IgE+ gate by flow cytometry. IgE secretion in the supernatant was measured by ELISA.Results18 patients (8 females, median age 59 years) with EGPA diagnosed according to ACR and CHC-criteria were included into the study. 22% of patients were ANCA-positive. Immunosuppressive therapy was azathioprine in 11 patients, methotrexate in 3 patients, and leflunomid or mycophenolate in one patient each and two patients received no immunosuppressive treatment. 7 patients had a history of a prior cyclophosphamide therapy. Median lymphocyte count was normal (1.2x103/μl; normal range 1.1 to 3.2x103/μ l) but peripheral B cell numbers were markedly diminished (37/μl, normal range: 100 – 500/μl). While the percentage of naïve B cells (53%) and marginal zone like B cells (10.5%) were within normal range, the percentage of class-switched memory B cells was high (28.2%). To assess the in vitro class switch capacity to IgE the number of IgE class-switched cells after 9 days of culture was determined by counting the events in the IgG/A/D-IgE+ gate. The percentage of IgE-switched cells was 6.8% (range: 0.3%>34%) and not statistically different from healthy controls.ConclusionsIn the EGPA-patients we report we observed markedly diminished B-cells despite of normal lymphocyte counts. Within the B cell compartment, there was a shift towards later B cell maturation stages. The in vitro B-cell development to IgE class-switched cells was not increased in EGP...

Following the recent publications of the STAR-study, the ASTRAL trial, the HERCULES trial and the CORAL trial on renal revascularization versus medical therapy in the management of atherosclerotic renovascular disease, there has been a near paradigm shift implying the nonutility of revascularization as a useful and necessary therapeutic option. Our recent experience with a patient who underwent an anastomotic bypass revascularization for worsening renal failure and uncontrolled hypertension in bilateral calcific atherosclerotic renal artery stenosis in Burlington, VT rekindled this debate. We posit that in appropriately selected patients, patients with acutely worsening renal failure, uncontrolled hypertension and/or symptomatic pulmonary edema, there is indeed a place for revascularization therapy, especially in the light of improved and safer surgical and anesthesiology techniques. It must be correctly acknowledged that the above well popularized randomized trials recruited mostly patients with otherwise stable chronic kidney disease at the time of enrollment. Similarly, only 12% of the patients in both arms of the ASTRAL trial demonstrated rapidly worsening renal failure prior to enrollment

Background In nephrotic range proteinuria of adult-onset, kidney biopsy is the diagnostic gold standard in determining the underlying cause of disease. However, in low grade or subnephrotic proteinuria the diagnostic value of kidney biopsy as first-line diagnostics is less well established. Methods We conducted a retrospective analysis of all native kidney biopsies at our institution (n = 639) between 01/2012 and 05/2021 for comparison of histological diagnoses and clinical outcomes stratified by amount of proteinuria at the time of kidney biopsy: A: <300mg/g creatinine (low grade), B: 300-3500mg/g creatinine (subnephrotic), C >3500mg/g creatinine (nephrotic). Results Nephrotic range proteinuria was associated with the highest frequency (49.3%) of primary glomerulopathies followed by subnephrotic (34.4%) and low grade proteinuria (37.7%). However, within the subnephrotic group, the amount of proteinuria at kidney biopsy was linearly associated with renal and overall survival (HR 1.05 per Δ100mg protein/g creatinine (95% CI: 1.02–1.09, p = 0.001)) independent of present histological diagnoses and erythrocyturia. Conclusion Frequency of primary glomerulopathies supports to perform kidney biopsy in patients with subnephrotic proteinuria. These patients have a substantial risk of ESKD and death upon follow-up. Therefore, diagnostic accuracy including histopathology is essential to guide personalized treatment and avert detrimental courses.

BACKGROUND AND AIMS Congenital anomalies of the kidney and urinary tract (CAKUT) represent the most common cause of chronic kidney failure in children. Despite growing knowledge of the genetic causes of CAKUT, the majority of cases remain etiologically unsolved. Genetic alterations in roundabout guidance receptor 1 (ROBO1) have been associated with neuronal and cardiac developmental defects in living individuals. Although Slit-Robo signaling is pivotal for kidney development, diagnostic ROBO1 variants have not been reported in viable CAKUT to date. METHOD We collected phenotypic data of all participants. Genetic analysis was conducted by either exome or whole genome sequencing, respectively. We used Sanger sequencing for variant confirmation and segregation analysis and performed in-silico analysis of identified missense variants. In an ENU-induced mouse model, we examined mice with the mutation Robo1Ile270Thr/Ile270Thr. RESULTS By next-generation-sequencing methods, we identified six unrelated individuals and two non-viable fetuses from Germany, France, Turkey, Japan and the USA with biallelic truncating or combined missense and truncating variants in ROBO1. Renal and genitourinary manifestation included unilateral or bilateral renal agenesis, vesicoureteral junction obstruction, vesicoureteral reflux, posterior urethral valve, genital malformation and increased renal echogenicity. Further clinical characteristics were remarkably heterogeneous, including neurodevelopmental defects, intellectual impairment, cerebral malformations, eye anomalies, and cardiac defects (Figure 1). By in-silico analysis, we determined the functional significance of identified missense variants and observed absence of renal ROBO1 expression in both human and murine mutant tissues (Figure 2). CONCLUSION This study describes six live-born and two non-viable individuals with syndromic CAKUT due to probably deleterious ROBO1 variants. While its expression in multiple tissues may explain heterogeneous organ involvement, variability of kidney disease suggests gene dosage effects due to a combination of null alleles with mild hypomorphic alleles. In conclusion, comprehensive genetic analysis in CAKUT should include ROBO1 as a new cause of recessively inherited disease. Conversely, in patients with already established ROBO1-associated cardiac or neuronal disorders, screening for renal involvement is indicated.