Clinical usefulness of urine Gram stain for diagnosing urinary tract infections at the emergency department.

BACKGROUND: Diagnosis of urinary tract infections (UTIs) is a frequent challenge at the emergency department (ED). The clinical usefulness of the urine Gram stain (GS) is uncertain. OBJECTIVE: We studied the GS performance to clarify its clinical utility at the ED. METHODS: Urine dipstick (UD), automated urinalysis (UF-1000i), GS and urine culture (UC) were performed in a cohort of consecutive adults presenting at the ED suspected of a UTI. GS performance was assessed and compared to UD and UF-1000i. RESULTS: A UTI diagnosis was established in 487/1358 (35.9%) episodes. Sensitivity and specificity for 'many' GS leucocytes was 33.7% and 95.4%; for 'many' GS bacteria 51.3% and 91.0%. GS diagnostic performance by ROC analysis was 0.796 for leucocytes and 0.823 for bacteria. GS bacteria performed better than UD nitrite comparable to UF-1000i bacteria. GS leucocytes underperformed compared to UD leucocyte esterase and UF-1000i leucocytes. UC was positive in 455 episodes. GS correctly predicted urine culture of gram-negative rods (PPV 84.6%). Prediction was poor for gram-positive bacteria (PPV 38.4% (cocci), 1.0% (rods)). CONCLUSION: With the exception of a moderate prediction of gram-negative bacteria in the UC, urine GS does not improve UTI diagnosis at the ED compared to other urine parameters.

Targeting NF-κB signaling in B cells as a potential new treatment modality for ANCA-associated vasculitis.

B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27+ memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-ß (IKKß, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement.

Kv1.3 blockade by ShK186 modulates CD4+ effector memory T-cell activity of patients with granulomatosis with polyangiitis.

OBJECTIVES: Granulomatosis with polyangiitis (GPA) is a chronic relapsing systemic autoimmune vasculitis. Current treatment of GPA is unsatisfactory, as it relies on strong immunosuppressive regimens, with either CYC or rituximab, which reduce the immunogenicity of several vaccines and are risk factors for a severe form of COVID-19. This emphasizes the need to identify new drug targets and to develop treatment strategies with less harmful side effects. Since CD4+ effector memory T cells (TEM) play a key role in the pathogenesis of GPA, we aimed in this study to modulate CD4+TEM cell activity via Kv1.3 blockade using the specific peptide inhibiter, ShK-186. METHODS: Peripheral blood samples from 27 patients with GPA in remission and 16 age- and sex-matched healthy controls (HCs) were pre-incubated in vitro in the presence or absence of ShK-186, followed by stimulation with phorbol myristate acetate, calcium ionophore and brefeldin-A. The effect of ShK-186 on the cytokine production (IFNγ, TNFα, IL-4, IL-17, IL-21) within total and subsets of CD4+ T helper (CD4+TH) cells were assessed using flow cytometry. RESULTS: ShK-186 reduced the expression level of IFNγ, TNFα, IL-4, IL-17 and IL-21 in CD4+TH cells from patients with GPA in vitro. Further analysis performed on sorted CD4+T cell subsets, revealed that ShK-186 predominantly inhibited the cytokine production of CD4+TEM cells. ShK-186 treatment reduced the production of the pro-inflammatory cytokines to the level seen in CD4+ TH cells from HCs. CONCLUSIONS: Modulation of cellular effector function by ShK-186 may constitute a novel treatment strategy for GPA with high specificity and less harmful side effects.

The elevation of the anion gap in steady state chronic kidney disease may be less prominent than generally accepted.

Background: A presumed cause of metabolic acidosis in chronic kidney disease (CKD) is accumulation of unmeasured anions, leading to a high anion gap (AG). In patients with CKD with a high AG, only minor increases are expected. The aim of this study is to evaluate the magnitude of the AG in documented steady state CKD to examine the effect of CKD on a high-AG metabolic acidosis (HAGMA). Methods: In this cross-sectional study the AG, bicarbonate, and chloride were evaluated in 1045 blood and urine samples of 501 patients with steady state CKD in the outpatient clinic. The influence of phosphate, albumin and potassium on the AG were evaluated. Results: The mean AG increased from 8.8 mEq/l (±1.57) in CKD stage 1 to 11.2 mEq/l (±2.22) in CKD stage 5 (P < 0.001). Correction for albumin or phosphate did not influence the magnitude of the AG. Correction for potassium did alter the prevalence of HAGMA, but not the severity. [HCO3-] decreased between CKD stages 1 and 5 by 5.1 mEq/l. The [Cl-] increased by 2.6 mEq/l between CKD stages 1 and 5. Conclusions: The elevation of the AG in patients with steady state CKD is limited and less pronounced than the decrease in [HCO3-]. Normal AG metabolic acidosis seems to be more important in CKD than HAGMA. The CKD stage and the magnitude of the AG should be taken into account when evaluating a patient with HAGMA. This study suggests that an AG >15 mEq/l is rarely due to renal failure alone.

Specific IgG glycosylation differences precede relapse in PR3-ANCA associated vasculitis patients with and without ANCA rise.

Introduction: Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N-linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares. Methods: Using liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV. Results: Analysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher ΔFc-bisection at diagnosis (P = 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse (P = 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse (P = 0.02). Discussion: Our data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre.

External ureteric stent versus internal double J stent in kidney transplantation: a retrospective analysis on the incidence of urological complications and urinary tract infections.

Introduction: Urologic complications (UCs) and urinary tract infections (UTIs) are common after kidney transplantation. Intraoperative stent placement at the vesicoureteric anastomosis reduces UC risk, but increases UTI risk. Methods: In 2014 our stenting protocol changed from external ureteric stent (ES) to internal double J stent (DJ). We retrospectively studied the occurrence of UCs and UTIs in relation to ES or DJ in 697 kidney recipients. Methods: An ES was used in 403 patients (57.8%), in 294 (42.2%) a DJ. ES was removed 7-12 days and DJ 3-4 weeks post-operative. Induction immunosuppression was the same in both groups. Primary outcomes at 6 months follow-up were UC (urinary leakage/ureter stenosis) and UTI; they were related to stenting procedure and clinical and transplant characteristics. The incidence of UCs was similar for ES (8.4%) and DJ (6.8%), p=0.389. ES use was a significant risk factor for UTI (OR 1.69 (1.15-2.50), p=0.008). Post-transplant hospitalization was significantly shorter in the DJ group. Despite more acute rejection episodes with ES (ES/DJ: 16.4%/6.1%, p<0.001), no clinical relevant differences in graft outcomes existed. Discussion: A DJ is, compared to ES, associated with a lower incidence of UTIs and comparable occurrence of UCs and is therefore the preferred technique for stenting the vesicoureteric anastomosis.

Oxalate nephropathy in an elderly patient with newly diagnosed celiac disease - a case report.

Oxalate nephropathy, due to secondary hyperoxaluria has widely been described in gastrointestinal diseases. However, reports of oxalate nephropathy in newly diagnosed celiac disease are rare. A 72-year-old Caucasian male presented to the hospital with abdominal discomfort and acute renal insufficiency with a creatinine of 290 µmol/L. The clinical course, laboratory results and urinalysis were suspect for tubular injury. Renal biopsy showed calcium oxalate depositions. Elevated plasma and urine oxalate levels established the diagnosis oxalate nephropathy. The abdominal complaints with steatorrhea and positive anti-tissue transglutaminase antibodies were diagnosed as celiac disease, which was confirmed after duodenal biopsies. Treatment with prednisone, and gluten-free, low oxalate and normal calcium diet, lowered the plasma oxalate levels and improved his renal function. Decreased absorption of free fatty acids can lead to increased free oxalate in the colon due to the binding of free fatty acids to calcium, preventing the formation of the less absorbable calcium oxalate in the colon. Oxalate dispositions in the kidney can lead to acute tubular injury and chronic renal insufficiency. Celiac disease is therefore one of the intestinal diseases that can lead to hyperoxaluria and oxalate nephropathy.

CD163 and CD206 expression define distinct macrophage subsets involved in active ANCA-associated glomerulonephritis.

INTRODUCTION: Macrophages are key players in the immunopathology of anti-neutrophil cytoplasmic antibody (ANCA) mediated-vasculitis (AAV) with glomerulonephritis (ANCA GN). Different macrophage phenotypes are expected to play distinct roles in ANCA GN. Macrophages expressing CD163 and CD206 are found in lesions associated with ANCA GN. Hence, we aimed to investigate the clinicopathological significance of CD206 and CD163 in ANCA GN in a multicenter retrospective cohort study. MATERIAL AND METHODS: Patients with ANCA-associated vasculitis, with clinical data, serum and urine samples were included from three cohorts. Serum soluble CD206 (ssCD206) and urinary soluble CD163 (usCD163) levels were measured. Human kidney tissue samples (n = 53) were stained for CD206 and CD163 using immunohistochemistry and immunofluorescence, and findings were correlated with clinical and pathological data. RESULTS: In total, 210 patients were included (i.e., ANCA GN, n = 134; AAV without GN, n = 24; AAV in remission n = 52). Increased levels of both ssCD206 and usCD163 were seen in ANCA GN. High levels of ssCD206 declined after reaching remission, however, ssCD206 did not improve the accuracy of usCD163 to detect ANCA GN. Soluble markers correlated with histopathological findings. CD163+CD206- macrophages were found in the glomerulus and may play pivotal roles in glomerulonephritis, whereas CD206+CD163- and CD206+CD163+ macrophages were located tubulointerstitially and likely play a more prominent role in ANCA-associated tubulointerstitial inflammation. In ANCA GN patients increasing levels of ssCD206 increased the risk for end-stage renal disease and mortality. CONCLUSIONS: Our results confirm and extend the notion that CD206+ and CD163+ macrophages are prominent components of the cellular infiltrate in ANCA GN. We found distinct macrophage phenotypes that may play distinct roles in the immunopathology of ANCA GN and elaborate on a potential mechanism underlying the findings of this study. usCD163 remains an excellent marker to detect active ANCA GN, whereas ssCD206 seems a more prominent marker for risk prediction.

Longitudinal monitoring of BKPyV miRNA levels in kidney transplant recipients with BKPyV-related pathology reflects viral DNA levels and remain high in viremia patients after clearance of viral DNA.

INTRODUCTION: It is unclear whether polyomavirus BK (BKPyV) microribonucleic acid (miRNA) measurement has additional diagnostic and predictive value in kidney transplant recipients (KTR) as compared to current methods of monitoring BKPyV DNA loads. PATIENTS AND METHODS: A retrospective, longitudinal study was performed in 30 KTR with BKPyV viruria (n = 10), BKPyV viremia (n = 10), or BKPyV-associated neuropathy (BKPyVAN) (n = 10). Bkv-miR-B1-3p and 5p and BKPyV DNA load were measured in urine and plasma and compared using receiver operating characteristic (ROC) curves. RESULTS: Levels of Bkv-miR-B1-3p and 5p and BKPyV DNA correlated strongly. Overall, mostly analog courses of urinary and plasma miRNA and DNA loads were observed. Areas under the ROC curves were not significantly different between miRNAs and DNA. Only, in contrast to BKPyV DNA load, BKPyV miRNA levels increased from 6 to 12 months in the viremia group, while in the BKPyVAN group, a decline was seen in both DNA and miRNA. CONCLUSIONS: In this study, we could not demonstrate an additional value of BKPyV miRNA detection compared to BKPyV DNA monitoring in the early phase after kidney transplantation. We did observe significant differences between the viremia and the BKPyVAN groups during follow-up. This study was performed with a small number of patients and therefore results should be verified in a larger patient cohort. Furthermore, future studies with larger patient groups are necessary to elucidate final clinical value of these data.

Predictors of renal flares and long-term renal outcome in patients with lupus nephritis: results from daily clinical practice.

OBJECTIVES: To describe renal outcomes of the lupus nephritis (LN) population of the University Medical Centre Groningen (UMCG) in the Netherlands and to identify predictors for renal flares and long-term renal outcome in daily clinical practice. METHODS: A retrospective analysis of biopsy-proven LN patients with induction and maintenance treatment in the UMCG between 1982 and 2016 was performed. Data were collected at time of diagnosis, after 6 months and every year up to 10 years after diagnosis. Outcome measures were renal relapse (biopsy proven), progression to chronic kidney disease (CKD) stage 3 or 4 and chronic renal replacement therapy. The ability of serum creatinine, proteinuria, creatinine clearance, serum anti-double stranded DNA (anti-dsDNA) antibodies, serum complement 3 (C3) and serum complement 4 (C4), as well as biographic data and histopathological class to predict long-term renal outcome was assessed. RESULTS: Seventy-one patients were included, with median follow-up of 120 months (IQR 48-120 months). During follow-up - up to 10 years - twenty-one (30%) patients experienced at least one relapse. Eleven (15%) patients had CKD stage 3 or 4, of whom eight showed persistent CKD since baseline and two (3%) patients required chronic renal replacement therapy. At baseline, low levels of serum C3 were a significant predictor of renal relapse. Low levels of C3 and C4 at 6 and 12 and proteinuria and high levels of anti-dsDNA at 12 months were significant predictors of renal relapse. At baseline, 6 months and 12 months serum creatinine and creatinine clearance were significant predictors for persistent or newly developed CKD 3 or 4, and need for chronic renal replacement therapy. CONCLUSIONS: Almost one-third of LN patients experience at least one renal relapse during long-term follow up, but only 3% need chronic renal replacement therapy. Our data suggests that early serological remission is associated with a low risk of renal relapse. Decreased renal function at onset and the first year after diagnosis is predictive for decreased renal function at a later stage.

Circulating adrenomedullin and B-type natriuretic peptide do not predict blood pressure fluctuations during pheochromocytoma resection: a cross-sectional study.

BACKGROUND: Despite adequate presurgical management, blood pressure fluctuations are common during resection of pheochromocytoma or sympathetic paraganglioma (PPGL). To a large extent, the variability in blood pressure control during PPGL resection remains unexplained. Adrenomedullin and B-type natriuretic peptide, measured as MR-proADM and NT-proBNP, respectively, are circulating biomarkers of cardiovascular dysfunction. We investigated whether plasma levels of MR-proADM and NT-proBNP are associated with blood pressure fluctuations during PPGL resection. METHODS: Study subjects participated in PRESCRIPT, a randomized controlled trial in patients undergoing PPGL resection. MR-proADM and NT-proBNP were determined in a single plasma sample drawn before surgery. Multivariable linear and logistic regression analyses were used to explore associations between these biomarkers and blood pressure fluctuations, use of vasoconstrictive agents during surgery as well as the occurrence of perioperative cardiovascular events. RESULTS: A total of 126 PPGL patients were included. Median plasma concentrations of MR-proADM and NT-proBNP were 0.51 (0.41-0.63) nmol/L and 68.7 (27.9-150.4) ng/L, respectively. Neither MR-proADM nor NT-proBNP were associated with blood pressure fluctuations. There was a positive correlation between MR-proADM concentration and the cumulative dose of vasoconstrictive agents (03B2 0.44, P =0.001). Both MR-proADM and NT-proBNP were significantly associated with perioperative cardiovascular events (OR: 5.46, P =0.013 and OR: 1.54, P =0.017, respectively). CONCLUSIONS: plasma MR-proADM or NT-proBNP should not be considered as biomarkers for the presurgical risk assessment of blood pressure fluctuations during PPGL resection. Future studies are needed to explore the potential influence of these biomarkers on the intraoperative requirement of vasoconstrictive agents and the perioperative cardiovascular risk.

A Method to Improve Continuous Renal Replacement Therapy Circuit Survival Time in Critically Ill Coronavirus Disease 2019 Patients With Acute Kidney Injury.

OBJECTIVES: Optimizing continuous renal replacement therapy circuit survival in coronavirus disease 2019 patients admitted to the ICU. DESIGN: Single-center prospective observational cohort study. SETTING: Tertiary academic teaching ICU. PATIENTS: Between March 19, 2020, and May 18, 2020, 11 out of 101 coronavirus disease 2019 patients were treated with continuous renal replacement therapy comprising 127 continuous renal replacement therapy days. INTERVENTIONS: A nonrandomized observational comparison of circuit anticoagulation modalities using standard regional citrate anticoagulation, continuous IV heparin anticoagulation, or the combination of regional citrate anticoagulation with either continuous IV heparin or therapeutic dose nadroparin. MEASUREMENTS AND MAIN RESULTS: Circuit patency was shorter than 24 hours using standard regional citrate anticoagulation or continuous IV heparin anticoagulation. Median circuit survival increased with at least 165% when the combination of regional citrate anticoagulation with either continuous IV heparin or therapeutic dose nadroparin was applied. CONCLUSIONS: Continuous renal replacement therapy circuit patency is diminished in coronavirus disease 2019 ICU patients. Combining regional citrate anticoagulation with either continuous IV heparin or therapeutic dose nadroparin increases filter survival as compared with regional citrate anticoagulation alone in this nonrandomized observational study.

Mycophenolic acid and 6-mercaptopurine both inhibit B-cell proliferation in granulomatosis with polyangiitis patients, whereas only mycophenolic acid inhibits B-cell IL-6 production.

Granulomatosis with polyangiitis (GPA) is an autoimmune disease affecting mainly small blood vessels. B-cells are important in the GPA pathogenesis as precursors of autoantibody-producing cells but likely also contribute (auto)antibody-independently. This has been underlined by the effectiveness of B-cell-depletion (with Rituximab) in inducing and maintaining disease remission. Mycophenolate-mofetil (MMF) and azathioprine (AZA) are immunosuppressive therapies frequently used in GPA-patients. Interestingly, MMF-treated GPA-patients are more prone to relapses than AZA-treated patients, while little is known about the influence of these drugs on B-cells. We investigated whether MMF or AZA treatment (or their active compounds) alters the circulating B-cell subset distribution and has differential effects on in vitro B-cell proliferation and cytokine production in GPA-patients that might underlie the different relapse rate. Circulating B-cell subset frequencies were determined in samples from AZA-treated (n = 13), MMF-treated (n = 12), untreated GPA-patients (n = 19) and matched HCs (n = 41). To determine the ex vivo effects of the active compounds of MMF and AZA, MPA and 6-MP respectively, on B-cell proliferation and cytokine production, PBMCs of untreated GPA-patients (n = 29) and matched HCs (n = 30) were cultured for 3-days in the presence of CpG-oligodeoxynucleotides (CpG) with MPA or 6-MP. After restimulation (with phorbol myristate acetate, calcium-ionophore), cytokine-positive B-cell frequencies were measured. Finally, to assess the effect of MMF or AZA treatment on in vitro B-cell proliferation and cytokine production, PBMCs of MMF-treated (n = 18), and AZA-treated patients (n = 28) and HCs (n = 41) were cultured with CpG. The memory B-cell frequency was increased in AZA- compared to MMF-treated patients, while no other subset was different. The active compounds of MMF and AZA showed in vitro that MPA decreased B-cell proliferation in GPA-patients and HCs. B-cell proliferation in MMF- and AZA-treated patients was not different. Finally, the IL-6+ B-cell frequency was decreased by MPA compared to 6-MP. No differences in IL-10+, IL-6+ or TNFα+ B-cell proportions or proliferation were found in MMF- and AZA-treated patients. Our results indicate that MMF could be superior to AZA in inhibiting B-cell cytokine production in GPA-patients. Future studies should assess the effects of these immunosuppressive drugs on other immune cells to elucidate mechanisms underlying the potential differences in relapse rates.

Gene variants and treatment outcomes in antineutrophil cytoplasmic antibody-associated vasculitis.

The introduction of immunosuppressive therapy for ANCA-associated vasculitis (AAV) has greatly improved outcomes, though patients now accumulate damage from vasculitis activity and adverse effects of treatment. Prediction of treatment outcomes using gene variants might help reduce this damage by allowing for personalized treatment. Several studies have studied genetic polymorphisms in relation to treatment outcomes of AAV. This review gives an overview of these studies, discussing both gene polymorphisms associated with inflammatory pathways (potentially influencing disease outcomes such as activity, severity, and relapse risk) and pharmacogenetics (potentially influencing drug metabolism and/or drug response). Subsequently, potential benefits of testing genetic variants for AAV and the steps needed for its implementation in clinical practice are discussed. The conclusion of this review is that measurement of most polymorphisms is currently not indicated in clinical practice.

MASP-2 Is a Heparin-Binding Protease; Identification of Blocking Oligosaccharides.

It is well-known that heparin and other glycosaminoglycans (GAGs) inhibit complement activation. It is however not known whether fractionation and/or modification of GAGs might deliver pathway-specific inhibition of the complement system. Therefore, we evaluated a library of GAGs and their derivatives for their functional pathway specific complement inhibition, including the MASP-specific C4 deposition assay. Interaction of human MASP-2 with heparan sulfate/heparin was evaluated by surface plasmon resonance, ELISA and in renal tissue. In vitro pathway-specific complement assays showed that highly sulfated GAGs inhibited all three pathways of complement. Small heparin- and heparan sulfate-derived oligosaccharides were selective inhibitors of the lectin pathway (LP). These small oligosaccharides showed identical inhibition of the ficolin-3 mediated LP activation, failed to inhibit the binding of MBL to mannan, but inhibited C4 cleavage by MASPs. Hexa- and pentasulfated tetrasaccharides represent the smallest MASP inhibitors both in the functional LP assay as well in the MASP-mediated C4 assay. Surface plasmon resonance showed MASP-2 binding with heparin and heparan sulfate, revealing high Kon and Koff rates resulted in a Kd of ~2 µM and confirmed inhibition by heparin-derived tetrasaccharide. In renal tissue, MASP-2 partially colocalized with agrin and heparan sulfate, but not with activated C3, suggesting docking, storage, and potential inactivation of MASP-2 by heparan sulfate in basement membranes. Our data show that highly sulfated GAGs mediated inhibition of all three complement pathways, whereas short heparin- and heparan sulfate-derived oligosaccharides selectively blocked the lectin pathway via MASP-2 inhibition. Binding of MASP-2 to immobilized heparan sulfate/heparin and partial co-localization of agrin/heparan sulfate with MASP, but not C3b, might suggest that in vivo heparan sulfate proteoglycans act as a docking platform for MASP-2 and possibly prevent the lectin pathway from activation.

CD27+CD38hi B Cell Frequency During Remission Predicts Relapsing Disease in Granulomatosis With Polyangiitis Patients.

Background: Granulomatosis with polyangiitis (GPA) patients are prone to disease relapses. We aimed to determine whether GPA patients at risk for relapse can be identified by differences in B cell subset frequencies. Methods: Eighty-five GPA patients were monitored for a median period of 3.1 years (range: 0.1-6.3). Circulating B cell subset frequencies were analyzed by flow cytometry determining the expression of CD19, CD38, and CD27. B cell subset frequencies at the time of inclusion of future-relapsing (F-R) and non-relapsing (N-R) patients were compared and related to relapse-free survival. Additionally, CD27+CD38hi B cells were assessed in urine and kidney biopsies from active anti-neutrophil cytoplasmic autoantibody-associated vasculitides (AAV) patients with renal involvement. Results: Within 1.6 years, 30% of patients experienced a relapse. The CD27+CD38hi B cell frequency at the time of inclusion was increased in F-R (median: 2.39%) compared to N-R patients (median: 1.03%; p = 0.0025) and a trend was found compared with the HCs (median: 1.33%; p = 0.08). This increased CD27+CD38hi B cell frequency at inclusion was correlated to decreased relapse-free survival in GPA patients. In addition, 74.7% of patients with an increased CD27+CD38hi B cell frequency (≥2.39%) relapsed during follow-up compared to 19.7% of patients with a CD27+CD38hi B cell frequency of <2.39%. No correlations were found between CD27+CD38hi B cells and ANCA levels. CD27+CD38hi B cell frequencies were increased in urine compared to the circulation, and were also detected in kidney biopsies, which may indicate CD27+CD38hi B cell migration during active disease. Conclusions: Our data suggests that having an increased frequency of circulating CD27+CD38hi B cells during remission is related to a higher relapse risk in GPA patients, and therefore might be a potential marker to identify those GPA patients at risk for relapse.

Increased miR-142-3p Expression Might Explain Reduced Regulatory T Cell Function in Granulomatosis With Polyangiitis.

Objectives: Regulatory T cells (Tregs) are frequently functionally impaired in patients with granulomatosis with polyangiitis (GPA). However, the mechanism underlying their impaired function is unknown. Here, we hypothesized that Treg dysfunction in GPA is due to altered microRNA (miRNA) expression. Methods: RNA isolated from FACS-sorted memory (M) Tregs (CD4+CD45RO+CD25+CD127-) of 8 healthy controls (HCs) and 8 GPA patients without treatment was subjected to miRNA microarray analysis. Five differentially expressed miRNAs were validated in a larger cohort by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). An miRNA target gene database search revealed targets that were tested with RT-qPCR in MTregs from patients and HCs. cAMP levels were measured using flow cytometry. Results: Microarray analysis revealed 19 differentially expressed miRNAs, of which miR-142-3p was confirmed to be significantly upregulated in MTregs from GPA patients compared to those from HCs (1.9-fold, p = 0.03). In vitro overexpression of miR-142-3p lowered the suppressive capacity of MTregs (2.1-fold, p = 0.03), and miR-142-3p expression correlated negatively with the suppressive capacity (rho = -0.446, p = 0.04). Overexpression of miR-142-3p significantly decreased cAMP levels (p = 0.02) and tended to decrease the mRNA levels of a predicted target gene, adenylate cyclase 9 (ADCY9; p = 0.06). In comparison to those from HCs, MTregs from GPA patients had lower ADCY9 mRNA levels (2-fold, p = 0.008) and produced significantly less cAMP after stimulation. Importantly, induction of cAMP production in miR-142-3p overexpressed MTregs by forskolin restored their suppressive function in vitro. Conclusion: Overexpression of miR-142-3p in MTregs from GPA patients might cause functional impairment by targeting ADCY9, which leads to the suppression of cAMP production.

A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity.

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.

Unraveling the identity of FoxP3+ regulatory T cells in Granulomatosis with Polyangiitis patients.

Human CD4+FoxP3+T-cells are heterogeneous in function and include not only suppressive cells (Tregs), but also effector cells that transiently express FoxP3 upon activation. Previous studies in Granulomatosis with Polyangiitis (GPA-)patients have demonstrated an increase in FoxP3+T-cells with impaired suppressive capacity and an increase in Th17 cells. We hypothesized that the increase in FoxP3+T-cells results from an increase in non-suppressive effector-like cells. The frequency of circulating CD4+FoxP3+T-cell subsets were determined by flow cytometry in 46 GPA-patients in remission and 22 matched healthy controls (HCs). Expression levels of FoxP3 and CD45RO were used to distinguish between CD45RO- FoxP3low resting Tregs (rTreg), CD45RO+FoxP3high activated Tregs (aTreg) and CD45RO+FoxP3low proinflammatory non-suppressive T-cells (nonTreg). Intracellular expression of IFNγ, IL-17, and IL-21 was compared within these subsets. We found a significant increase in the frequency of nonTreg cells in GPA-patients as compared with HCs. Importantly, within the nonTreg subset, antineutrophil cytoplasmic autoantibody (ANCA-)positive patients demonstrated a significantly higher percentage of IL-17+ and IL-21+ cells when compared with ANCA-negative patients and HCs. Moreover, expanded nonTregs from ANCA-positive patients induced excessive proliferation of responder cells in vitro and exhibited higher IL-21 production. Production of IL-17 and IL-21 in non-suppressive FoxP3+T-cells may point toward a pathogenic role in ANCA formation.

Evidence for enhanced Bruton's tyrosine kinase activity in transitional and naïve B cells of patients with granulomatosis with polyangiitis.

OBJECTIVES: To determine Bruton's tyrosine kinase (BTK) protein and phosphorylation levels in B cell subsets of granulomatosis with polyangiitis (GPA) patients and to investigate the effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production. METHODS: BTK protein and phosphorylation levels were determined by flow cytometry in peripheral blood B cells of 29 untreated GPA patients [9 active and 20 remission GPA patients (10 ANCA- and 10 ANCA+)], 9 age- and sex-matched healthy controls (HCs) and 9 untreated active RA patients. The effect of BTK blockade on in vitro B cell cytokine production, subset distribution and (auto)antibody production was determined in the same donors in peripheral blood mononuclear cell cultures. RESULTS: BTK protein levels were significantly increased in transitional and naïve B cells of active GPA and RA patients compared with remission GPA patients and HCs. Both B cell subsets of active patients were more sensitive to B cell receptor stimulation, as BTK and phospholipase Cγ2 phosphorylation were increased in these patients. In vitro BTK blockade had profound effects on B cell cytokine production, plasma cell formation and (auto)antibody production in both GPA patients and HCs. Interestingly, the effect of BTK blockade was less pronounced in active GPA patients, possibly due to increased activation of B cells. CONCLUSION: We show that BTK protein and phosphorylation levels are most profoundly increased in newly emerging B cells of active GPA patients compared with remission patients. BTK blockade greatly inhibits in vitro B cell effector functions in GPA patients and HCs. These promising data identify BTK as an interesting novel therapeutic target in the treatment of GPA.