Clinical Characteristics of EGPA Patients in Comparison to GPA Subgroup with Increased Blood Eosinophilia from POLVAS Registry.

Objective: To characterize the eosinophilic granulomatosis with polyangiitis (EGPA) population from the POLVAS registry depending on ANCA status and diagnosis onset, including their comparison with the granulomatosis with polyangiitis (GPA) subset with elevated blood eosinophilia (min. 400/µl) (GPA HE) to develop a differentiating strategy. Methods: A retrospective analysis of the POLVAS registry. Results: The EGPA group comprised 111 patients. The ANCA-positive subset (n = 45 [40.54%]) did not differ from the ANCA-negative one in clinics. Nevertheless, cardiovascular manifestations were more common in ANCA-negative patients than in those with anti-myeloperoxidase (MPO) antibodies (46.97% vs. 26.92%, p = 0.045). Patients diagnosed before 2012 (n = 70 [63.06%]) were younger (median 41 vs. 49 years, p < 0.01), had higher blood eosinophilia at diagnosis (median 4,946 vs. 3,200/µl, p < 0.01), and more often ear/nose/throat (ENT) and cardiovascular involvement. GPA HE comprised 42 (13.00%) out of 323 GPA cases with reported blood eosinophil count. Both GPA subsets had a lower prevalence of respiratory, cardiovascular, and neurologic manifestations but more often renal and ocular involvement than EGPA. EGPA also had cutaneous and gastrointestinal signs more often than GPA with normal blood eosinophilia (GPA NE) but not GPA HE. The model differentiating EGPA from GPA HE, using ANCA status and clinical manifestations, had an AUC of 0.92, sensitivity of 96%, and specificity of 95%. Conclusion: Cardiovascular symptoms were more prevalent in the ANCA-negative subset than in the MPO-ANCA-positive one. Since EGPA and GPE HE share similarities in clinics, diagnostic misleading may result in an inappropriate therapeutic approach. Further studies are needed to optimize their differentiation and tailored therapy, including biologics.

Activity of granulomatosis with polyangiitis and its correlation with mTOR phosphoproteomics in neutrophils.

Introduction: Granulomatosis with polyangiitis (GPA) is a small vessel vasculitis with a complex pathomechanism. Organ damage in GPA is also mediated by extracellular trap formation (NETosis). We analyzed the functional status of phosphoproteins modulating NETosis in neutrophils by the mammalian target of rapamycin (mTOR) pathway in GPA along with NETosis biomarkers. Methods: Phosphoproteins levels measured in isolated neutrophils from 42 patients with GPA (exacerbation n=21; remission n=21) and 21 healthy controls were compared to serum biomarkers of the disease. Results: Neutrophils in active disease manifested lowered levels of phosphorylated mTOR(Ser2448), PTEN(Ser380) and ULK1(Ser555), whereas phosphorylated GSK-3α/ß(Ser21/Ser9) was elevated. Exacerbation of GPA was characterized by elevated neutrophil dsDNA in serum, circulating mitochondrial DNA, and DNA-MPO complexes. A significant negative correlation between mTOR or PTEN phosphoproteins and biomarkers of GPA activity was also present, reflecting the clinical activity score of GPA. Positive correlations between phosphorylated GSK-3 α/ß and circulating mtDNA, DNA-MPO complexes, neutrophil-released dsDNA, or circulating proteins were also significant. Increased serum levels of IGFBP-2, TFF-3, CD147, and CHI3L1 accompanied GPA exacerbation, whereas DPP-IV levels were the lowest in active GPA. Using a principal component analysis basigin, PTEN and mTOR had the highest loadings on the discrimination function, allowing classification between active, remission, and control subjects with 98% performance. Conclusions: We present evidence that inhibited mTOR signaling accompanies NETosis in patients with GPA. The functional status of phosphoproteins suggests simultaneous activation of NETosis and autophagy. These results give rise to the study of autophagy as a mechanism underlying granuloma formation in GPA.

Functional magnetic resonance imaging for the assessment of autonomic dysfunction in patients with antineutrophil cytoplasmic antibody-associated vasculitides.

INTRODUCTION: Nervous system involvement is common in antineutrophil cytoplasmic antibody-associated vasculitides (AAV). While the involvement of the peripheral and central nervous system is well described, it is still unclear how and to what extent the autonomic nervous system (ANS) is affected. Functional magnetic resonance imaging (fMRI) can provide information on both structure and potential damage of the brain, as well as on the function of selected brain centers. OBJECTIVES: The aim of this study was to investigate the ANS dysfunction in AAV patients and its correlation with the results of fMRI performed during the Valsalva maneuver. PATIENTS AND METHODS: A total of 31 patients with AAV and 30 healthy controls were enrolled in the study. Each participant completed the Composite Autonomic Symptom Score (COMPASS)-31 questionnaire. MRI was performed using a 3T scanner. The participants were asked to perform the Valsalva maneuver according to the fixed protocol, and their airway pressure was monitored. During the maneuver, fMRI data were collected. The generalized least­ squares time series analysis and the region of interest (ROI) analysis were subsequently performed. RESULTS: The patients with AAV had a higher median COMPASS­ 31 score than the controls (12.86 vs 2.99, respectively; P <0.01). Structural MRI investigation did not reveal any significant differences between the groups. The brain centers involved in ANS function were detected during fMRI; however, the ROI analysis showed no differences between the study patients and controls. CONCLUSIONS: The patients with AAV reported symptoms related to the ANS dysfunction; however, no differences with respect to the functioning of the ANS brain centers were demonstrated between these patients and healthy controls in the fMRI study during the Valsalva maneuver.

Profile of circulating extracellular vesicles microRNA correlates with the disease activity in granulomatosis with polyangiitis.

Granulomatosis with polyangiitis is a chronic systemic inflammation of small vessels characterized by circulating anti-proteinase 3 antibodies. MicroRNAs are short transcripts specifically inhibiting protein translation. Neutrophils can release extracellular vesicles (EVs). In this study, we characterized profile of microRNA trafficked by EVs in GPA. Fifty patients with GPA were enrolled in the study, 25 at acute phase and 25 in remission. EVs were isolated from the blood serum, characterized by their number, size distribution. Following unbiased screening for microRNA expression, differentially expressed candidates were measured by quantitative real-time PCR. Circulating DNA-myeloperoxidase complexes and apoptosis-related transcripts in peripheral blood neutrophils were quantified. We identified four differentially expressed microRNAs from EVs in granulomatosis with polyangiitis (GPA). MirRs-223-3p, 664a-3p, and 200b-3p were overexpressed and miR-769-5p suppressed in the disease. A distinction between GPA and healthy controls was the best for miR-223-3p, whereas miR-664a-3p discriminated between active vs. remission of GPA. Correct classification of the disease based on multivariate discriminant analysis was between 92% for acute phase and 85% for all study participants. Bioinformatics tools identified genes transcripts potentially targeted by the microRNAs belonging to pathways of focal adhesion, mTOR signaling and neutrophil extracellular traps formation. Two microRNAs positively correlating with the disease activity were involved in neutrophil extracellular traps formation and apoptosis inhibition. A comprehensive characteristics of microRNAs trafficked in bloodstream inside EVs correlates well with our understanding of the mechanisms of GPA and suggests the importance of EVs in progression of the disease.

Respiratory involvement in antineutrophil cytoplasmic antibody-associated vasculitides: a retrospective study based on POLVAS registry.

OBJECTIVES: The study aimed to characterise the Polish population of (ANCA)-associated vasculitides (AAV) with respiratory involvement (RI), in comparison to the subgroup without lung manifestations and the other cohorts. METHODS: Retrospective analysis of the Polish population of AAV with RI was conducted, based on data from the POLVAS registry. Standard descriptive statistics, χ2 test, and Mann-Whitney U test were used to perform comparisons. RESULTS: Among 461 cases qualified to this study, there were 316 cases with RI (68.5%), 206 with granulomatosis with polyangiitis (GPA) (65.2%), 80 with eosinophilic granulomatosis with polyangiitis (EGPA) (25.3%) and 30 with microscopic polyangiitis (MPA) (9.5%). Proportion of RI in GPA, MPA, and EGPA accounted for 67.8%; 40.0%; 97.6%, respectively. The number of relapses was higher in the RI group (median 1.0 vs. 0.0; p=0.01). In the subgroup of combined GPA and MPA with RI, the trends toward higher proportion of deaths (11.7% vs. 5.7%; p=0.07), relapses requiring hospitalisation (52.2% vs. 42.4%, p=0.07) and relapses requiring admission to the intensive care unit (5.6% vs. 1.4%, p=0.09) were observed, median maximal concentration of CRP was higher (46 vs. 25 mg/l; p=0.01) and more aggressive treatment was administered. CONCLUSIONS: Prevalence of RI in the Polish population of AAV is similar to the values reported in the literature, however, the proportion observed in GPA is closer to those presented in Asian than Western European cohorts. RI seems to be associated with a more severe course of disease and its presence prompts more aggressive treatment.

Chronic prothrombotic tendency in patients with granulomatosis with polyangiitis.

INTRODUCTION: Patients with granulomatosis with polyangiitis (GPA) show increased tendency toward thromboembolic phenomena in exacerbation of their disease. OBJECTIVES: The aim of the study was to evaluate thrombin generation potential and fibrinolytic plasma activity in patients with GPA, both in exacerbation and in remission. PATIENTS AND METHODS: We included 38 patients with GPA: 18 with exacerbated GPA and 20 in remission. The control group included 39 healthy participants matched for age and sex. Plasma thrombogenic potential was assessed using calibrated automated thrombography. Plasma fibrinolytic potential was assessed using clot lysis time (CLT). We also measured levels of inflammatory markers, thrombomodulin, and fibrinolysis proteins in all participants. RESULTS: In the whole group of patients with GPA, endogenous thrombin potential was higher by about 25% (P <0.001), while CLT was lower by about 20% (P = 0.02) when compared with controls. The endogenous thrombin potential was higher, the CLT lower, and the levels of thrombomodulin and inflammation markers (C­reactive protein, fibrinogen, factor VIII) higher both in patients with exacerbation and in remission than in the control group; no such differences were noted when comparing those with exacerbation and those in remission, however. The only parameter that differentiated patients with GPA exacerbation from those in remission was the D­dimer level (median [interquartile range], 1151 [597.2-2468.7] ng/ml vs 340.4 [255.1-500.7] ng/ml; P <0.001), a marker of lysis of intravascularly formed fibrin. CONCLUSIONS: Patients with GPA show an increased prothrombotic state, regardless of the disease phase. This is probably related to ongoing low-grade inflammation and endothelial injury. Large clinical studies are required to address the need for, and appropriate type of, antithrombotic prophylaxis during the course of GPA.

Subphenotypes of ANCA-associated vasculitis identified by latent class analysis.

OBJECTIVES: ANCA-associated vasculitides (AAV) are a heterogeneous group of rare diseases with unknown aetiology and the clinical spectrum ranging from life-threatening systemic disease, through single organ involvement to minor isolated skin changes. Thus, there is an unmet need for phenotype identification, especially among patients with granulomatosis with polyangiitis (GPA). Patients with microscopic polyangiitis (MPA) seem to be clinically much more uniform. Recently, three subcategories of AAV have been proposed and described as non-severe AAV, severe PR3-AAV, and severe MPO-AAV. METHODS: In line with these attempts, we decided to use an unbiased approach offered by latent class analysis (LCA) to subcategorise GPA and MPA in a large cohort of Polish AAV patients included in a multicentre POLVAS registry. RESULTS: LCA of our AAV group identified a four-class model of AAV, including previously proposed three subphenotypes and revealing a fourth (previously not described) clinically relevant subphenotype. This new subphenotype includes only GPA patients, usually diagnosed at a younger age as compared to other groups, and characterised by multiorgan involvement, high relapse rate, relatively high risk of death, but no end-stage kidney disease. CONCLUSIONS: Based on multiple clinical and serological variables, LCA methodology identified 4-class model of AAV. This newly described fourth class of AAV may be of clinical relevance and may require prompt diagnosis and aggressive treatment due to the multiorgan involvement, high risk of relapse and marked mortality among these relatively young GPA subjects.

Treatment and its side effects in ANCA-associated vasculitides - Study based on POLVAS registry data.

PURPOSE: The aim of this study is to present the treatment modalities and associated side effects in a Polish nation-wide ANCA-associated vasculitides (AAV) patients' cohort. MATERIALS AND METHODS: Retrospective analysis of patients diagnosed with AAV between 1990 and 2016, included in the POLVAS registry was performed. Standard descriptive statistic methods were used with an emphasis on the treatment modalities. RESULTS: There were 625 patients diagnosed with AAV included in this study: 417 cases of granulomatosis with polyangiitis (GPA; 66.7%), 106 cases of microscopic polyangiitis (MPA; 17.0%) and 102 cases of eosinophilic granulomatosis with polyangiitis (EGPA; 16.3%). The mean age at the date of diagnosis was 50.4 (±15.7) years and the median observational period amounted to 4.0 (2.0-8.0) years. Glucocorticosteroids (GCs) were the medicaments most frequently used for remission induction (593/622; 95.3%), followed by cyclophosphamide (487/622; 78.3%), rituximab (44/622; 7.1%), and methotrexate (39/622; 6.3%). GCs were also most frequently administered for maintenance therapy (499/592; 84.3%), followed by azathioprine (224/592; 37.8%), methotrexate (136/592; 23.0%) and mycophenolate mofetil (99/592; 16.7%). The median cumulative doses of cyclophosphamide and rituximab equalled 7.99 g (4.18-14.0) and 2000 mg (1500-2800), respectively. The most commonly observed adverse events included: infections - 214/551 cases (38.8%), which were associated with the time of observation (OR = 1.05; 95% CI 1.01-1.10), the use of GCs intravenous pulses (OR = 2.76; 95% CI 1.68-4.54) and need for haemodialysis (OR = 1.73; 95% CI 1.10-2.71). CONCLUSIONS: Polish patients with AAV were predominantly treated according to appropriate guidelines. The most frequent adverse events were typical for usually administered immunosuppressive treatment.

LTB4 and 5-oxo-ETE from extracellular vesicles stimulate neutrophils in granulomatosis with polyangiitis.

Activation of neutrophils is an important mechanism in the pathology of granulomatosis with polyangiitis (GPA). In this study, we evaluated whether extracellular vesicles (EVs) circulating in the plasma of GPA patients could contribute to this process. EVs from the plasma of GPA patients in the active stage of the disease (n = 10) and healthy controls (n = 10) were isolated by ultracentrifugation and characterized by flow cytometry (CD63, CD8) and nanoparticle tracking analysis. Targeted oxylipin lipidomics of EVs was performed by HPLC-MS/MS. EV/oxylipin-induced neutrophil extracellular traps (NETs) were analyzed by confocal microscopy, and released double-stranded DNA (dsDNA) was quantified by PicoGreen fluorescent dye. Reactive oxygen species (ROS) production and neutrophils' EV binding/uptake were evaluated by flow cytometry. Brief priming with granulocyte-macrophage colony-stimulating factor was required for EV-mediated ROS production and dsDNA release. It was observed that priming also increased EV binding/uptake by neutrophils only for EVs from GPA patients. EVs from GPA patients had higher concentrations of leukotriene (LT)B4 and 5-oxo-eicosatetraenoic acid (5-oxo-ETE) as compared with EVs from healthy controls. Moreover, neutrophils stimulated with LTB4 or 5-oxo-ETE produced ROS and released dsDNA in a concentration-dependent manner. These results reveal the potential role of EVs containing oxylipin cargo on ROS production and NET formation by activated neutrophils.

Oral manifestations of granulomatosis with polyangiitis - Clinical and radiological assessment.

BACKGROUND/PURPOSE: Granulomatosis with polyangiitis (GPA) is a type of primary systemic vasculitis associated with the presence of anti-neutrophil cytoplasmic antibodies (ANCA). GPA mainly affects medium and small vessels and may manifest in different organs, most commonly upper respiratory tract. Oral lesions occur in 6-13% of GPA patients and might be the first symptom observed by the patient. This study presents the evaluation of orofacial manifestations of patients with GPA. MATERIALS AND METHODS: Prospective study was conducted between June 2014 and November 2017 in Department of Allergy and Immunology at University Hospital in Krakow. Patients diagnosed with GPA, after medical assessment, were examined including precise dental inspection, bacterial and fungal flora of oral cavity evaluation and Cone Beam Computed Tomography (CBCT) imaging. RESULTS: Nine patients were enrolled in the study. Characteristic for GPA strawberry gingivitis was observed in one patient. Bone destruction and inflammatory lesions in paranasal sinuses was confirmed by CBCT in 55.6% of patients. Fungal infection was revealed in 66.7% of patients. CONCLUSION: These findings oblige dentists to consult patient with laryngologist or internal medicine physician to establish further diagnostic approach, because early diagnosis of GPA is crucial for implementing appropriate treatment and preventing chronic organ damage.

Skewing toward Treg and Th2 responses is a characteristic feature of sustained remission in ANCA-positive granulomatosis with polyangiitis.

The objective of our study was to evaluate the T-helper (Th) and regulatory T (Treg) cell profile in ANCA-positive granulomatosis with polyangiitis (GPA) and its relation to disease activity. In a prospective study, we studied two groups of GPA patients: (i) disease flare (active-GPA, BVAS>6, n = 19), (ii) sustained remission (≥ 1-year prior enrollment, inactive-GPA, BVAS = 0, n = 18). 24 age-sex matched healthy subjects served as controls. Active-GPA patients were followed for 6 months and reevaluated during remission (early remission; n = 13). We analyzed subsets of Th-cells (flow cytometry), production of signature cytokines by in vitro stimulated lymphocytes, and broad spectrum of serum cytokines (Luminex). In all GPA patients we observed expansion of effector Th17 cells, and increased production of IL-17A by in vitro stimulated T cells, as compared to controls. Disease flare was characterized by marked reduction in Treg cells, whereas in sustained remission we showed expansion of both Treg and Th2 subset. Finally, analyzing the cytokine profile, we identified CCL23 and LIGHT, as potential biomarkers of active disease. We conclude that in GPA, expansion of Treg and Th2 lymphocytes in parallel to increased Th17 response is a characteristic feature of sustained remission. In contrast, Treg cells are markedly decreased in disease flare.

Cough-induced lung intercostal hernia.

A 73-year-old male with marked emphysema was admitted to the 2nd Department of Internal Medicine, University Hospital in Krakow because of chronic obstructive pulmonary disease (COPD) exacerbation. His medical history was significant for total laryngectomy due to laryngeal cancer in 2010.

Neutrophil-related and serum biomarkers in granulomatosis with polyangiitis support extracellular traps mechanism of the disease.

OBJECTIVES: Granulomatosis with polyangiitis (GPA) is an autoimmune disease with still unknown etiology. Recent studies indicate that neutrophils extra-cellular traps participate in the pathophysiology of GPA. This study investigates the levels of circulating NET formation markers and neutrophil-platelet interaction in patients with GPA. METHODS: We enrolled 40 GPA patients (20 in the active stage of the disease and 20 in remission). Twenty sex- and age-matched healthy subjects served as a control group. Serum/plasma levels of serine proteases, and histone-, myeloperoxidase-, proteinase-3 DNA complexes and sP-selectin were measured using ELISA or Luminex assays. Circulating platelet-neutrophil aggregates and neutrophils activation markers expression was measured by flow cytometry. RESULTS: Patients in active stage of GPA had higher circulating levels of serine proteases, DNA-histone and myeloperoxidase -DNA complexes. In addition, platelet-neutrophil aggregates and sP-selectin were also elevated in this group. Platelet-neutrophil aggregates and myeloperoxidase -DNA complexes correlated positively with the disease activity score (BVAS). CONCLUSIONS: NETs production and activation of platelets in GPA is supported by elevated myeloperoxidase-DNA complexes and platelet-neutrophil aggregates correlating positively with the disease activity score. This mechanism justifies laboratory measurements of myeloperoxidase-DNA complexes and plasma sP-selectin as biomarkers for studying GPA activity.

Neutrophil MiRNA-128-3p is Decreased During Active Phase of Granulo-matosis with Polyangiitis.

Granulomatosis with polyangiitis is a rare chronic inflammatory disease. In this multisystem autoimmune disorder neutrophils cause small vessels necrosis and infiltrate perivascular tissue to form granulomas. Progression of the disease is evaluated by the symptoms score and by a titer of anti-neutrophil cytoplasm antibodies. Despite glucocorticoid and immunosuppressive therapy, prognosis is complicated by chronic renal insufficiency, hearing loss and skin ulceration. In this preliminary study we tested the hypothesis that altered neutrophil expression of miRNAs can contribute to the cell activation, extracellular traps formation and decreased apoptosis. First we compared a profile of 728 miRNAs expressed in circulating neutrophils of patients with active disease and matched healthy donors. Subsequently, candidate miRNAs were quantified in neutrophils from 16 subjects with active disease, 16 asymptomatic patients at the remission and in 16 healthy controls. Out of 11 candidate miRNAs, only miR-128-3p was both biologically (relative quantity < 30% control or remission patients) and statistically (p<0.01) decreased in the cells during active stage of the disease. This miRNA correlated with a clinical score of the disease well. A set of 10 transcripts involved in the mechanism of the disease was quantified from the same neutrophils RNA. Relative expression of MMP9 was higher in neutrophils from the patients with active disease and correlated negatively with miR-128-3p. The opposite finding was present for MTA1 transcripts. Despite surprisingly scarce changes in the expression of neutrophil miRNAs, miR-128-3p is the best candidate for deciphering etiology of granulomatosis with polyangiitis.

Lower doses of rituximab in remission induction for refractory granulomatosis with polyangiitis.

INTRODUCTION: Recently, rituximab (RTX)--monoclonal antibody against the CD20 molecule on the surface of B-lymphocytes is used in the treatment of antineutrophil cytoplasmic antibody (ANCA) associated vasculitides. Efficacy of the drug administered in so-called lymphoma treatment protocol (4 x 375 mg/m2/week) has been shown not to be inferior to cyclophosphamide. However, some data published lately suggest that rituximab could also be effective in much lower doses, which could lead to reducing side effects, but above all, the cost of the therapy. OBJECTIVES: Analysis of efficacy of lower doses of rituximab in remission induction in GPA patients. PATIENTS AND METHODS: We retrospectively analyzed the course, the efficacy and safety of rituximab administered at a dose lower than average in lymphoma treatment protocol (median = 1.0 g). The drug was used only in patients who presented resistance to the standard treatment with cyclophosphamide, or in whom such treatment was impossible. Disease activity was evaluated using Birmingham Vasculitis Activity Score and disease remission was defined as score 0. RESULTS: Out of the twelve patients who received RTX induction doses (period 07. 2009-07.2014), remission was achieved in the eleven (92%). Averaged observation period was 7.5 months (median). The total B-cell depletion was observed in all treated with induction scheme. During further follow-up, disease relapse in 2 patients was observed. One patient achieved remission again after re use of rituximab. The second patient died in the course of diffuse alveolar hemorrhage. In these patients, recurrence was observed respectively after 42 and 56 months of follow-up. CONCLUSIONS: The efficacy of lower doses of rituximab for the induction of remission in refractory granulomatosis with polyangiitis was confirmed. Ava- ilability of the drug in the treatment of primary vasculitis is currently limited mainly due to economic issues.