Altered serum levels of autophagy proteins Beclin-1 and mTOR in patients with exudative age-related macular degeneration.

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.

Facilitated expansion of Th17 cells in lupus nephritis patients.

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)-ß; 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.

Urinary cytokines and mRNA expression as biomarkers of disease activity in lupus nephritis.

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.

Signs of impaired immunoregulation and enhanced effector T-cell responses in the primary antiphospholipid syndrome.

INTRODUCTION: We investigated whether primary antiphospholipid syndrome (PAPS) is characterized by a deficiency in immunoregulatory pathways, a phenomenon recently implicated in the pathogenesis of autoimmune diseases. METHODS: Serum levels of immunoregulatory (e.g., IL-10 and TGF-ß1) and proinflammatory (e.g., IL-17A) cytokines were measured in PAPS, systemic lupus erythematosus (SLE) with secondary APS (SAPS), or without APS, and in healthy controls (n = 40 in each group). In a subgroup of PAPS patients we also compared phenotype and function (flow cytometry) of regulatory T-cells (Treg) and cytokine production by effector T-cells. RESULTS: Our major finding was decreased levels of TGF-ß1 in PAPS and SAPS as compared to SLE without APS and controls. TGF-ß1 was the lowest in PAPS patients showing high levels of aPL IgG with significant negative correlation with the titer. SLE patients were characterized by lower serum levels of IL-2 and increased IL-17A, as compared to the other groups. The numbers of circulating Treg cells and their phenotype (e.g., FoxP3 isoforms) were not disturbed in PAPS. However, surface expression of latency associated peptide (binds TGF-ß) in activated FoxP3 + cells and in vitro production of TGF-ß1 were decreased in PAPS patients with high titers of aPL IgG. Moreover, frequencies of cytokine producing effector T-helper cells (including Th17) were significantly elevated in this group. CONCLUSIONS: PAPS patients with high titers of aPL IgG antibodies were characterized by decreased systemic levels of TGF-ß1 and its impaired production in vitro, suggesting impaired immunoregulation and enhanced adaptive autoimmune responses leading to the production of aPL antibodies.

Leukotrienes biosynthesis in vascular surgery patients during perioperative period.

Leukotrienes (LTs), highly bioactive lipid mediators play a major role in inflammation, wound healing and in the development of atherosclerosis. LTs biosynthesis have been suggested to be increased in myocardial infarction (MI) and in surgical patients with abdominal aortic aneurysms. Among LTs, Cysteinyl-LTs have the most potent biological properties and their production is well reflected by LTE4 concentration in urine (uLTE4). Aim of the study was to evaluate perioperative biosynthesis of uLTE4 in noncardiac vascular surgery patients, and its impact on patients' outcomes. Twenty eight consecutive patients aged 61.5 (59.0-72.5) that undergone an elective surgery for abdominal aortic aneurysm (AAA; n=6) or peripheral artery disease (PAD; n=22) were studied. uLTE4 was measured in urine samples using ELISA: before surgery (LT0), 6 hours postoperatively (LT1), and on three following days (LT2-LT4), and the results were adjusted for the urinary creatinine concentration. Patients were followed-up for 30-days for cardio-vascular complications including myocardial infarction (MI) with active post-surgery troponin T screening. One way analysis of variance (ANOVA) for repeated measurements and logistic regression tests were used to analyse the data with P<05 considered significant. Excretion of uLTE4 raised in the first two urine sample (LT1 and LT2) after surgery as compared to preoperative baseline value (LT0) (P=0.008) and returned to normal values on the second day (LT3). Patients that suffered MI during postoperative period had increased uLTE4 levels when compared to the no-MI patients (P=0.006). In conclusion we state that uLTE4 biosynthesis is increased shortly after surgery and returns to the preoperative level on the second day. The increase in uLTE4 biosynthesis is higher in patients that suffer MI after surgery, however this warrants further investigations.

Collagen receptors α(1)ß(1) and α(2)ß(1) integrins are involved in transmigration of peripheral blood eosinophils, but not mononuclear cells through human microvascular endothelial cells monolayer.

UNLABELLED: Asthma development may be driven by T helper lymphocytes with eosinophils playing the role of major effector cells. Recruitment of the inflammatory cells from blood to the airways is mediated by adhesive molecules, e.g. selectins and integrins. The most important in cell trafficking are integrins containing α(4) and ß(2) subunits. We hypothesized that also collagen receptors: α(1)ß(1) and α(2)ß(1), may be involved in cell migration to the inflammatory site in asthma. The aim of the study was to determine whether the inhibition of α(1)ß(1) or α(2)ß(1) integrins, affects transmigration of eosinophils and peripheral blood mononuclear cells (PBMC) through human microvascular endothelial cells monolayer (HMVEC) seeded on collagen IV coated wells in moderate persistent atopic asthmatics. METHODS: PBMC from 9 asthmatics were separated by gradient centrifugation followed by negative magnetic separation of eosinophils. Snake venom derived anti-adhesive proteins: viperistatin and VP12 (potent and selective inhibitors of α(1)ß(1) and α(2)ß(1) integrins, respectively) as well as VLO4 (a non-selective inhibitor of α(4)ß(1), α(5)ß(1) and α(v)ß(3) - used as a positive control), were used for inhibition studies. All anti-adhesive proteins studied, inhibited eosinophils, but only VLO4 affected PBMC transmigration through HMVEC. In bronchial asthma both collagen receptors α(1)ß(1) and α(2)ß(1) are likely to be involved in eosinophil transmigration to the inflammatory site. The role of α(2)ß(1) on lymphocytes is probably different. As the α(2)ß(1) integrin has been described as a stimulator of collagen accumulation, it might be, at least in part, responsible for asthma airway remodelling.