11 research outputs found

    DataSheet_1_Cytokine dysregulation despite immunoglobulin replacement therapy in common variable immunodeficiency (CVID).docx

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    IntroductionCommon variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency. CVID is a heterogeneous disorder with a presumed multifactorial etiology. Intravenous or subcutaneous immunoglobulin replacement therapy (IgRT) can prevent severe infections but not underlying immune dysregulation.MethodsIn this study, we evaluated the serum concentrations of proinflammatory (TNF-α, IL-1β, IL-6) and immunoregulatory cytokines (IL-10), as well as lipopolysaccharide (LPS) and soluble CD14 (sCD14) in CVID individuals with infectious only (INF-CVID), and those with additional systemic autoimmune and inflammatory disorders (NIC-CVID), and healthy donors (HD).ResultsOur results showed increased serum concentrations of TNF-α, IL-1β, IL-6, and IL-10 in both INF-CVID and NIC-CVID subjects compared to HD. However, elevations of TNF-α, IL-1β, IL-6, and IL-10 were significantly more marked in NIC-CVID than INF-CVID. Additionally, LPS concentrations were increased only in NIC-CVID but not in INF-CVID compared to HD. Circulating levels of sCD14 were significantly increased in NIC-CVID compared to both INF-CVID and HD.DiscussionThese findings indicate persistent cytokine dysregulation despite IgRT in individuals with CVID. Moreover, the circulating cytokine profile reveals the heterogeneity of immune dysregulation in different subgroups of CVID subjects.</p

    The number of tumor-infiltrating neutrophils positively correlated with tumor size in human TC specimens.

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    <p><b>A and B.</b> Histological analysis of TC specimens stained with a monoclonal anti-CD66b antibody. Whole-tumor section density of CD66b<sup>+</sup> neutrophils was scored at 200× magnification. Representative cases of papillary thyroid carcinomas with a high <b>(A)</b> and low <b>(B)</b> CD66b<sup>+</sup> neutrophil count (arrows; hematoxylin counterstaining, 200×). <b>C.</b> CD66b<sup>+</sup> neutrophil counts in tumor specimens were distributed according to the tumor size. The median value of tumor size served as a cutoff level. Results are shown as the median, the 25th and 75th percentiles (boxes), and 5th and 95th percentiles (whiskers); *p < 0.05, according to the two-tailed Mann–Whitney <i>U</i> test. <b>D.</b> Neutrophil density positively correlated with larger tumor size in TC patients (r = 0.43; p = 0.01; Pearson’s correlation test).</p

    TC-derived soluble factors modified neutrophils’ kinetic properties.

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    <p>Neutrophils were stimulated with a TC-CM or the control medium for 18 hours. Within this time window, digital phase contrast images of 15 fields/well were captured every 15 minutes via a 20× objective in the Operetta high-content imaging system. PhenoLOGIC (PerkinElmer) was employed for image segmentation and to calculate the single-cell kinetic properties <b>(A)</b>, accumulated distance, <b>(B)</b> speed and straightness <b>(C)</b> in dedicated analysis sequence. The results were expressed as mean ± SEM of six independent experiments; ***p < 0.005; **p < 0.01; *p < 0.05. <b>D-E.</b> Timepoint analyses of time-dependent properties such as current step size (D) and current speed (E) illustrating dynamic changes of the behavior of neutrophils (in presence or absence of TC-CMs) in function of the elapsed time from treatment. The results were expressed as mean ± SEM of six independent experiments; ****p < 0.001.</p

    TC-CMs induced morphological changes in neutrophils.

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    <p>Neutrophils were stimulated with a TC-CM or the control medium for 16 hours and then were imaged by means of an Operetta high-content imaging system at 20× magnification. The images were analyzed in the Harmony software with PhenoLOGIC (PerkinElmer) and a dedicated analysis sequence (morphological properties, method STAR) to evaluate cell area <b>(A)</b>, roundness <b>(B)</b>, radial mean <b>(C)</b>, symmetry <b>(D)</b>, width-to-length ratio <b>(E)</b>, and axial length <b>(F)</b>. The results were expressed as an increase or decrease compared to the control (mean ± SEM of five independent experiments); ***p < 0.005.</p

    TC-derived soluble mediators induced neutrophil chemotaxis.

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    <p><b>A.</b> Neutrophil chemotaxis toward TC-CM or the control medium was evaluated using 3 μm cell culture inserts in 96-well companion plates. Neutrophils (2.5 × 10<sup>6</sup> cells/ml per 75 μl) were allowed to migrate (37°C, 60 minutes) toward a TC-CM or the control medium (235 μl per well). At the end of the incubation, the cells were centrifuged and resuspended in PBS (100 μl) and counted by flow cytometry. Data are expressed as migratory cells relative to the control (mean ± SEM of five independent experiments), **p < 0.01. <b>B.</b> The CXCL8/IL-8 release by TPC1 and 8505c cells was evaluated by an ELISA in a TC-CM or in the control medium. Results are expressed as mean ± SEM of seven independent experiments; ****p < 0.001. <b>C and D</b>. Chemotactic activity of neutrophils <i>via</i> a TPC1-derived <b>(C)</b> or 8505c-derived <b>(D)</b> conditioned medium was analyzed in the presence of blocking antibodies directed against CXCR1 and/or CXCR2 (10 μg/ml) or the related isotype control. Migratory neutrophils were counted by flow cytometry. The results are expressed as a percentage of isotype control (mean ± SEM of eight independent experiments); ***p < 0.005; **p < 0.01; *p < 0.05.</p

    TC-derived soluble factors promoted neutrophil survival.

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    <p><b>A.</b> Neutrophils were cultured in a TC-CM or the control medium. At the indicated time points, live cells were evaluated by flow cytometry with FITC-conjugated annexin V and PI. Results were expressed as percentages of live cells (mean ± SEM of five independent experiments); ***p < 0.005; **p < 0.01; *p < 0.05. <b>B.</b> Representative flow cytometric panels of dot plots of PMNs cultured in a TC-CM or control medium and stained with FITC-conjugated annexin V and propidium iodide (PI) at 24 (upper panels) and 48 (lower panels) hours. <b>C</b>. The GM-CSF release by TPC1 and 8505c cells was evaluated by an ELISA in a TC-CM or in the control medium. Results were expressed as mean ± SEM of seven independent experiments; ****p < 0.001; ***p < 0.005. <b>D-F.</b> Neutrophil survival in a TPC1-derived <b>(D-E)</b> or 8505c-derived <b>(F-G)</b> conditioned medium was evaluated in the presence of an anti-GM-CSF blocking antibody or the relative isotype control (10 μg/ml). At 24 hours, live cells were stained with FITC-conjugated annexin V and PI and analyzed by flow cytometry. <b>Figs E and G</b> illustrate representative flow cytometric panels of one out of five independent experiments. The results were expressed as mean ± SEM of five independent experiments; **p < 0.01; *p < 0.05.</p

    TC-derived soluble factors induced activation of neutrophils.

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    <p><b>A–C.</b> Neutrophils were stimulated with a TC-CM or the control medium for 90 minutes, stained for neutrophil activation markers CD11b <b>(A)</b>, CD66b <b>(B)</b>, and CD62L <b>(C)</b> and subjected to cytofluorimetric analysis. The results were expressed as mean fluorescence intensity or percentages of positive cells gated on neutrophils (mean ± SEM of five independent experiments); ***p < 0.005, **p < 0.01, *p < 0.05. <b>D-F.</b> Representative flow cytometric panels with respect to the gating strategy of total cells <b>(D)</b>, singlets <b>(E)</b> and CD15+ CCR3- neutrophils <b>(F)</b>. <b>G-I.</b> Representative histograms illustrating mean fluorescence intensity (MFI) and cell counts for CD11b <b>(G)</b>, CD66b <b>(H)</b> and CD62L <b>(I)</b> for one out of five independent experiments. MFI = mean fluorescence intensity; FMO = fluorescence minus one.</p

    TC-derived soluble factors induced the expression of proinflammatory and angiogenic factors by neutrophils.

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    <p>Human neutrophils were treated with a TC-CM or the control medium for 18 hours. At the end of the incubation, the cells were harvested and lysed for RNA isolation. <i>CXCL8/IL-8</i> <b>(A)</b>, <i>VEGF-A</i> <b>(B)</b>, and <i>TNF-α</i> <b>(C)</b> mRNA levels were evaluated by real-time PCR. The results are expressed as a fold change relative to the control (mean ± SEM of six independent experiments); **p < 0.01; *p < 0.05.</p

    table_1_Secreted Phospholipases A2 in Hereditary Angioedema With C1-Inhibitor Deficiency.PDF

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    Background<p>Hereditary angioedema (HAE) caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) is a disabling, potentially fatal condition characterized by recurrent episodes of swelling. We have recently found that patients with C1-INH-HAE have increased plasma levels of vascular endothelial growth factors and angiopoietins (Angs), which have been associated with vascular permeability in several diseases. Among these and other factors, blood endothelial cells and vascular permeability can be modulated by extracellular or secreted phospholipases A<sub>2</sub> (sPLA<sub>2</sub>s).</p>Objective<p>We sought to investigate the enzymatic activity and biological functions of sPLA<sub>2</sub> in patients with C1-INH-HAE.</p>Methods<p>sPLA<sub>2</sub>s enzymatic activity was evaluated in the plasma from 109 adult patients with C1-INH-HAE and 68 healthy donors in symptom-free period and attacks. Plasma level of group IIA sPLA<sub>2</sub> (hGIIA) protein was measured in selected samples. The effect of C1-INH-HAE plasma on endothelial permeability was examined in vitro using a vascular permeability assay. The role of hGIIA was determined using highly specific sPLA<sub>2</sub> indole inhibitors. The effect of recombinant hGIIA on C1-INH activity was examined in vitro by functional assay.</p>Results<p>Plasma sPLA<sub>2</sub> activity and hGIIA levels are increased in symptom-free C1-INH-HAE patients compared with controls. sPLA<sub>2</sub> activity negatively correlates with C1-INH protein level and function. C1-INH-HAE plasma increases endothelial permeability in vitro, and this effect is partially reverted by a specific hGIIA enzymatic inhibitor. Finally, recombinant hGIIA inhibits C1-INH activity in vitro.</p>Conclusion<p>sPLA<sub>2</sub> enzymatic activity (likely attributable to hGIIA), which is increased in C1-INH-HAE patients, can promote vascular permeability and impairs C1-INH activity. Our results may pave the way for investigating the functions of sPLA<sub>2</sub>s (in particular, hGIIA) in the pathophysiology of C1-INH-HAE and may inform the development of new therapeutic targets.</p
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