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.
We evaluated functional subsets of T‐helper cells in granulomatosis with polyangiitis (GPA) patients. Disease flare was characterized by expansion of Th17 and marked reduction in Treg cells. We showed suppression of Th17 responses early after induction therapy, while sustained remission was linked with additional expansion of Treg and Th2 subsets.
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.
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/β
(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/β 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.
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.
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.
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.
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.
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 antineutrophil
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.
Myocardial injury after non-cardiac surgery (MINS) increases the risk of cardiovascular events and deaths, which anticoagulation therapy could prevent. Dabigatran prevents perioperative venous ...thromboembolism, but whether this drug can prevent a broader range of vascular complications in patients with MINS is unknown. The MANAGE trial assessed the potential of dabigatran to prevent major vascular complications among such patients.
In this international, randomised, placebo-controlled trial, we recruited patients from 84 hospitals in 19 countries. Eligible patients were aged at least 45 years, had undergone non-cardiac surgery, and were within 35 days of MINS. Patients were randomly assigned (1:1) to receive dabigatran 110 mg orally twice daily or matched placebo for a maximum of 2 years or until termination of the trial and, using a partial 2-by-2 factorial design, patients not taking a proton-pump inhibitor were also randomly assigned (1:1) to omeprazole 20 mg once daily, for which results will be reported elsewhere, or matched placebo to measure its effect on major upper gastrointestinal complications. Research personnel randomised patients through a central 24 h computerised randomisation system using block randomisation, stratified by centre. Patients, health-care providers, data collectors, and outcome adjudicators were masked to treatment allocation. The primary efficacy outcome was the occurrence of a major vascular complication, a composite of vascular mortality and non-fatal myocardial infarction, non-haemorrhagic stroke, peripheral arterial thrombosis, amputation, and symptomatic venous thromboembolism. The primary safety outcome was a composite of life-threatening, major, and critical organ bleeding. Analyses were done according to the intention-to-treat principle. This trial is registered with ClinicalTrials.gov, number NCT01661101.
Between Jan 10, 2013, and July 17, 2017, we randomly assigned 1754 patients to receive dabigatran (n=877) or placebo (n=877); 556 patients were also randomised in the omeprazole partial factorial component. Study drug was permanently discontinued in 401 (46%) of 877 patients allocated to dabigatran and 380 (43%) of 877 patients allocated to placebo. The composite primary efficacy outcome occurred in fewer patients randomised to dabigatran than placebo (97 11% of 877 patients assigned to dabigatran vs 133 15% of 877 patients assigned to placebo; hazard ratio HR 0·72, 95% CI 0·55–0·93; p=0·0115). The primary safety composite outcome occurred in 29 patients (3%) randomised to dabigatran and 31 patients (4%) randomised to placebo (HR 0·92, 95% CI 0·55–1·53; p=0·76).
Among patients who had MINS, dabigatran 110 mg twice daily lowered the risk of major vascular complications, with no significant increase in major bleeding. Patients with MINS have a poor prognosis; dabigatran 110 mg twice daily has the potential to help many of the 8 million adults globally who have MINS to reduce their risk of a major vascular complication.
Boehringer Ingelheim and Canadian Institutes of Health Research.
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.
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