Search

Natalia Celejewska-Wójcik,1 Aleksander Kania,1 Karolina Górka,1 Paweł Nastałek,1 Krzysztof Wójcik,2 Anna Gielicz,3 Lucyna Mastalerz,1 Marek Sanak,3 Krzysztof Sładek1 1Department of Pulmonology, 2nd Department of Internal Medicine, Faculty of Medicine, Jagiellonian University Medical College, Kraków, Poland; 2 2nd Department of Internal Medicine, Faculty of Medicine, Jagiellonian University Medical College, Kraków, Poland; 3Department of Molecular Biology and Clinical Genetics, Faculty of Medicine, Jagiellonian University Medical College, Kraków, PolandCorrespondence: Natalia Celejewska-Wójcik 2nd Department of Internal Medicine, Faculty of Medicine, Jagiellonian University Medical College, ul. Jakubowskiego 2, Krakow, 30-688, PolandTel +48 12 400 30 53Fax +48 12 400 30 67Email natalia.celejewska@gmail.comPurpose: Lipid mediators, particularly eicosanoids, are associated with airway inflammation, especially with the eosinophilic influx. This study aimed to measure lipid mediators and cells in induced sputum, that could possibly reflect the inflammatory process in the bronchial tree of COPD subjects.Patients and Methods: Eighty patients diagnosed with COPD and 37 healthy controls participated in the study. Induced sputum samples were ascertained for differential cell count and induced sputum supernatant concentrations of selected eicosanoids by the means of gas chromatography/mass spectrometry and high-performance liquid chromatography/tandem mass spectrometry.Results: Increased sputum eosinophilia was associated with higher concentrations of selected proinflammatory eicosanoids. In COPD subjects prostaglandin D2 and 11-dehydro-thromboxane B2 correlated negatively with airway obstruction measured by FEV1 and FEV1/FVC values. COPD subjects with disease exacerbations during past 12 months had significantly higher concentrations of prostaglandin D2, 12-oxo-eicosatetraenoic acid and 5-oxo-eicosatetraenoic acid.Conclusion: Stable COPD is often associated with eosinophil influx in the lower airways and elevated concentrations of eicosanoids that is reflected by some disease characteristics.Keywords: chronic obstructive pulmonary disease, eosinophils, eicosanoids

Summary: In vasculitis disorders, inflammation affects blood vessels. Granulomatosis with polyangiitis (GPA) is a chronic systemic vasculitis distinguished by the presence of anti‐proteinase‐3 autoantibodies (anti‐PR3). In this study we analyzed the molecular signature of human umbilical endothelial cells (HUVECs) in response to neutrophil‐derived extracellular vesicles (EVs). EVs were obtained from anti‐PR3‐activated neutrophils, purified and characterized by flow cytometry, nanoparticle tracking and miRNA screening. HUVECs were stimulated with EVs and miRNA/mRNA expression was measured. Cell culture media proteins were identified by antibody microarrays and selected cytokines were measured. Comparison of differentially expressed miRNAs/mRNAs between non‐stimulated and EV‐stimulated HUVECs revealed two regulatory patterns. Significant up‐regulation of 14 mRNA transcripts (including CXCL8, DKK1, IL1RL1, ANGPT‐2, THBS1 and VCAM‐1) was accompanied by 11 miRNAs silencing (including miR‐661, miR‐664a‐3p, miR‐377‐3p, miR‐30d‐5p). Significant down‐regulation was observed for nine mRNA transcripts (including FASLG, CASP8, STAT3, GATA3, IRAK1 and IL6) and accompanied by up‐regulation of 10 miRNAs (including miR‐223‐3p, miR‐142‐3p, miR‐211‐5p). Stimulated HUVECs released IL‐8, Dickkopf‐related protein 1 (DKK‐1), soluble interleukin (IL)‐1 like receptor‐1 (ST2), growth differentiation factor 15 (GDF‐15), angiopoietin‐2, endoglin, thrombospondin‐1 and vascular adhesion molecule‐1 (VCAM‐1). Moreover, transfection of HUVECs with mimics of highly expressed in EVs miR‐223‐3p or miR‐142‐3p, stimulated production of IL‐8, ST2 and endoglin. Cytokines released by HUVECs were also elevated in blood of patients with GPA. The most increased were IL‐8, DKK‐1, ST2, angiopoietin‐2 and IL‐33. In‐vitro stimulation of HUVECs by neutrophil‐derived EVs recapitulates contribution of endothelium in autoimmune vasculitis. Proinflammatory phenotype of released cytokines corresponds with the regulatory network of miRNAs/mRNAs comprising both EVs miRNA and endothelial cell transcripts. [ABSTRACT FROM AUTHOR]

Autophagy is a key process in the maintenance of cellular survival and homeostasis. Inhibition of autophagy results in degenerative changes resembling ageing. We wondered if autophagy can contribute to the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate the serum concentrations of two key autophagy regulators, Beclin-1 and mechanistic target of rapamycin (mTOR), in patients with exudative AMD. This retrospective case-control study included 38 patients with exudative AMD and 36 sex- and age-matched controls selected among senile cataract patients. Circulating Beclin-1 and mTOR were assessed using an enzyme-linked immunosorbent assay. The proteins levels were correlated with age, sex, duration of ocular symptoms, as well as angiographic and optical coherence tomography findings. Serum Beclin-1 levels were much lower in patients with AMD than in controls (median, 0.100 ng/ml versus 1.123 ng/ml; p = 0.0033), while mTOR levels did not differ (median, 4.377 ng/ml versus 3.608 ng/ml; p = 0.4522). Participants of the study older than 70 years had lower Beclin-1 levels than younger ones (p = 0.0444). However, this difference was the most evident in patients with AMD (p = 0.0024). Serum mTOR levels increased with age. In patients with AMD, lower mTOR levels were associated with drusen, while higher levels were observed in those with a fibrous scar in the contralateral eye (p = 0.0212). Our findings suggest that circulating Beclin-1 decreases with age and that is downregulated in patients with AMD. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the prevalence of serum anti-retinal (ARAs) and anti-endothelial cell antibodies (ACEAs) in patients with acute and chronic central serous chorioretinopathy (CSC). We enrolled 28 patients with acute CSC, 42 patients with chronic CSC, and 40 healthy controls. The presence of ARAs was determined by indirect immunofluorescence using monkey retina as an antigen substrate, while the presence of AECAs was determined using cultivated human umbilical vein endothelial cells (HUVECs) and primate skeletal muscle according to the manufacturer's instructions (Euroimmun AG). There were no differences in the prevalence of antibodies against rods, cones, cytoplasmic components of retinal nuclear layer cells, and retinal vessels between the acute and chronic CSC groups and the control group (P = 0.27, P = 0.16, P = 0.71, and P = 0.06, respectively). However, AECAs reactive with HUVECs were observed in 46% of patients with acute CSC, 45% of those with chronic CSC, and 22% of controls, whereas AECAs reactive with the skeletal muscle were present in 46%, 45%, and 15%, respectively (difference between groups: P = 0.045 for HUVECs and P = 0.005 for the skeletal muscle). Furthermore, AECA titers were higher in CSC patients than in controls (P = 0.004). This study provides evidence for the possible involvement of an autoimmune process directed against vessel antigens in the pathogenesis of CSC. AECAs may be more important than ARAs in this disease and may be involved in endothelial damage in the choroidal vessels and choriocapillaris, leading to hyperpermeability, which is central to the pathophysiology of CSC. [ABSTRACT FROM AUTHOR]

Objectives: Neutrophil apoptosis is mandatory for resolving inflammation and is regulated by expression of pro- and anti-apoptotic genes. We studied neutrophils isolated from patients with granulomatosis with polyangiitis (GPA) to investigate apoptosis alterations and to identify transcriptional and circulating factors affecting this process.Method: We enrolled 36 patients (18 in active stage, 18 in remission) and 18 healthy controls. Circulating levels of tumour necrosis factor-α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage migration inhibitory factor, plasminogen activator inhibitor-1, interferon-γ, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, platelet endothelial cell adhesion molecule-1, soluble Fas (sFas), sFas ligand, survivin, and pentraxin-3 (PTX3) were evaluated by enzyme-linked immunosorbent assay/Luminex; circulating apoptotic neutrophils by flow cytometry; and apoptosis-related gene transcripts by real-time polymerase chain reaction.Results: Patients had decreased fractions of circulating apoptotic neutrophils and delayed neutrophil apoptosis was present in vitro. Circulating levels of TNF-α, GM-CSF, sFas, and PTX3 were higher in GPA. Delayed neutrophil apoptosis was accompanied by decreased mRNA of pro-apoptotic genes and transcription factors (DIABLO, PMAIP1, BAX, CASP3, CASP7, RUNX3, E2F1, TP53) and increased anti-apoptotic CFLAR and BCL2A1 mRNA. TNF-α and sFas levels correlated with circulating apoptotic neutrophils and expression of apoptosis genes. Stimulation with TNF-α of neutrophils from controls significantly down-regulated E2F1 and CASP3 expression.Conclusions: Circulating neutrophils in GPA have anti-apoptotic phenotype involving both intrinsic and extrinsic pathways of apoptosis. This is accompanied by increased levels of circulating pro-survival factors (GM-CSF, TNF-α, sFas), independent of disease activity. Anti-apoptotic phenotype of neutrophils in GPA is reproduced by exposure to low concentrations of TNF-α. [ABSTRACT FROM AUTHOR]

Summary: The objective of this study was to investigate the mechanisms of T helper type 17 (Th17) expansion in lupus nephritis (LN) patients, and to determine whether or not it is associated with impaired function of regulatory T cells (Treg). Major effector subsets of peripheral blood CD4+ T cells were assessed by flow cytometry in 33 LN patients with different activity of the disease and 19 healthy controls. The percentage of circulating Th17 cells was increased in LN (median = 1·2% of CD4+ compared to 0·6% in the control group, P < 0·01), while Treg cells remained unchanged (12·3 versus 12·1% in controls), resulting in a significantly lower Treg/Th17 ratio. Th17 expansion in the patient group was not related to LN activity, renal histology or blood and urine inflammatory biomarkers, but has been associated with a higher cumulative dose of cyclophosphamide. Treg cells in LN displayed mainly effector memory phenotype and expressed higher levels of transforming growth factor (TGF)‐β; however, their suppressant activity in lymphocyte proliferation assay was diminished compared to controls (~fourfold, P < 0·05). Co‐culture of Treg and conventional CD4+ T cells resulted in marked suppression of the Th1 subset in both of the groups studied, but also in a potent expansion of Th17 cells, which in LN was twofold higher, as in controls (P < 0·05). In conclusion, our results demonstrate that Th17 expansion in LN is not increased during disease exacerbation, but is related to chronic immunosuppressive therapy. This immune signature is probably linked to the abnormal function of Treg cells, which were less suppressive in LN patients and even facilitated differentiation of Th17 cells. Lupus nephritis (LN) is a serious manifestation of systemic lupus erythematosus associated with an imbalance between T‐regulatory (Treg) and Th17 responses. Here, we show that Th17 expansion develops in a subgroup of LN patients, but is not related to disease activity, renal histology, or blood and urine inflammatory biomarkers. This immune signature is likely linked to the abnormal function of Treg cells, which were less suppressive in LN patients and even facilitated expansion of Th17 cells. [ABSTRACT FROM AUTHOR]

Introduction Renal involvement is one of the most serious manifestations of systemic lupus erythematosus, but non-invasive assessment of inflammatory response in kidneys is challenging. In this study we aimed to validate markers of active lupus nephritis (LN) using urine immune profiling. Methods Urine and serum cytokines (17-plex array) and urine mRNA expression (∼40 immune and glomerular injury genes) were measured in LN patients with active disease (n = 17) during remission (n = 16) and in healthy subjects (n = 18). Results Urine and serum levels of CCL2, CCL5 and CXCL10 were elevated in active LN as compared with disease remission (best discrimination for urine CXCL10 and CCL2) and correlated with LN activity. In the active disease, urinary cell transcriptome showed marked upregulation of proinflammatory cytokines (e.g. TNF, CCL2, CCL5, CXCL10), and type-1 immunity-related genes (e.g. CD3G, CD4, TBX21, IFNG). An active pattern of gene expression was also observed in four patients in remission, who had moderately increased urinary leucocyte count. Two patients from this group developed renal exacerbation during the following 3 months. Markers of type-17 immune axis (e.g. IL-17A) were not significantly increased in active LN. Conclusions Active LN patients were characterized by marked increase of proinflammatory mediators in the urine. Urine cytokines (CCL2 and CXCL10) and type-1 T-cell-related gene markers in the urine sediment had similar diagnostic performance in detection of active LN. [ABSTRACT FROM AUTHOR]