Plasma membrane binding of IFI16 to HUVEC.

<p>(A) Cells were left untreated (a, negative control) or incubated with increasing concentrations of FITC-labeled recombinant IFI16 (b, 10 nM rIFI16-FITC; c, 20 nM rIFI16-FITC; d, 30 nM rIFI16-FITC). Binding was detected by confocal microscopy using an excitation wavelength of 490 nm for FITC in one channel and trans-illuminated light in the other. Representative images of three independent experiments are shown. (B) Endogenous IFI16 released by irradiated HeLa cells binds neighboring HUVEC. UV-B irradiated HeLa cells were co-cultured with HUVEC and after 24 h, 36 h and 48 h, dead cell debris were removed and immunofluorescence was performed on the remaining live HUVEC using a home-made anti-IFI16 polyclonal as primary antibody and Alexa-488- anti-rabbit as secondary antibody. The cells were then fixed, permeabilized, nuclear-stained using propidium iodide and analyzed by confocal microscopy. Fibroblasts were employed as negative control. Representative images of three independent experiments are shown.</p

IFI16 protein levels in patients’ and controls’ sera determined using an in-house capture ELISA.

<p>Each dot represents the concentration of IFI16 protein (expressed in ng/ml on a linear scale) in each individual subject: patients suffering from Systemic Sclerosis (SSc, n = 50), Systemic Lupus Erythematosus (SLE, n = 100), Sjogren’s Syndrome (SjS, n = 49), Rheumatoid Arthritis (RA, n = 50), and non-SLE glomerulonephritis (non-SLE GN n = 46) were investigated together with healthy controls (CTRL, n = 116). The horizontal bars represent the median values. Values over the dotted line indicate the percentage of subjects with IFI16 protein levels above the cut-off value (27 ng/ml) calculated as the 95^th percentile of the control population. Statistical significance: *** p<0.001 <i>vs</i>. controls (Kruskall-Wallis test).</p

Binding inhibition of [¹²⁵I]-rIFI16 using anti-IFI16 polyclonal antibodies.

<p>Antibody inhibition curve of HUVEC using anti-rIFI16 N-terminal (1–205 aa) polyclonal antibodies (dashed) and C-term polyclonal antibodies (dot-dashed). Competitive binding of rIFI16 and [¹²⁵I]-rIFI16 to HUVEC (plain) was used as the control condition. The experiment was carried out in triplicates and data was analyzed using non-linear regression equations from GraphPad Prism with 95% confidence intervals. All the experiments have been repeated at least three times and one representative is reported.</p

Binding Kinetics of [¹²⁵I]-rIFI16 on HUVEC, HDF, and 3T3 cells.

<p>(A) Specific binding of [¹²⁵I]-rIFI16 on the plasma membrane of HUVEC, HeLa, HACAT, HDF, and 3T3 cells. (B) Competitive binding of [¹²⁵I]-rIFI16 on HUVEC, HeLa, HaCaT, HDF, and 3T3 cells. (C) The binding affinity (Kd), total bound ligand (B_max) and the estimated number of binding sites per cell for different cell lines. The experiment was carried out in triplicates and data was analyzed using non-linear regression equations from GraphPad Prism with 95% confidence intervals. All the experiments have been repeated at least three times and one representative is reported.</p

Table_3_Autoantibodies Recognizing Secondary NEcrotic Cells Promote Neutrophilic Phagocytosis and Identify Patients With Systemic Lupus Erythematosus.DOCX

<p>Deficient clearance of apoptotic cells reportedly contributes to the etiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). Based on this knowledge, we developed a highly specific and sensitive test for the detection of SLE autoantibodies (AAb) utilizing secondary NEcrotic cell (SNEC)-derived material as a substrate. The goal of the present study was to validate the use of SNEC as an appropriate antigen for the diagnosis of SLE in large cohort of patients. We confirmed the presence of apoptotically modified autoantigens on SNEC (dsDNA, high mobility group box 1 protein, apoptosis-associated chromatin modifications, e.g., histones H3-K27-me3; H2A/H4 AcK8,12,16; and H2B-AcK12). Anti-SNEC AAb were measured in the serum of 155 patients with SLE, 89 normal healthy donors (NHD), and 169 patients with other autoimmune connective tissue diseases employing SNEC-based indirect enzyme-linked immunosorbent assay (SNEC ELISA). We compared the test performance of SNEC ELISA with the routine diagnostic tests dsDNA Farr radioimmunoassay (RIA) and nucleosome-based ELISA (anti-dsDNA-NcX-ELISA). SNEC ELISA distinguished patients with SLE with a specificity of 98.9% and a sensitivity of 70.6% from NHD clearly surpassing RIA and anti-dsDNA-NcX-ELISA. In contrast to the other tests, SNEC ELISA significantly discriminated patients with SLE from patients with rheumatoid arthritis, primary anti-phospholipid syndrome, spondyloarthropathy, psoriatic arthritis, and systemic sclerosis. A positive test result in SNEC ELISA significantly correlated with serological variables and reflected the uptake of opsonized SNEC by neutrophils. This stresses the relevance of SNECs in the pathogenesis of SLE. We conclude that SNEC ELISA allows for the sensitive detection of pathologically relevant AAb, enabling its diagnostic usage. A positive SNEC test reflects the opsonization of cell remnants by AAb, the neutrophil recruitment to tissues, and the enhancement of local and systemic inflammatory responses.</p

Image_1_Autoantibodies Recognizing Secondary NEcrotic Cells Promote Neutrophilic Phagocytosis and Identify Patients With Systemic Lupus Erythematosus.PDF

<p>Deficient clearance of apoptotic cells reportedly contributes to the etiopathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). Based on this knowledge, we developed a highly specific and sensitive test for the detection of SLE autoantibodies (AAb) utilizing secondary NEcrotic cell (SNEC)-derived material as a substrate. The goal of the present study was to validate the use of SNEC as an appropriate antigen for the diagnosis of SLE in large cohort of patients. We confirmed the presence of apoptotically modified autoantigens on SNEC (dsDNA, high mobility group box 1 protein, apoptosis-associated chromatin modifications, e.g., histones H3-K27-me3; H2A/H4 AcK8,12,16; and H2B-AcK12). Anti-SNEC AAb were measured in the serum of 155 patients with SLE, 89 normal healthy donors (NHD), and 169 patients with other autoimmune connective tissue diseases employing SNEC-based indirect enzyme-linked immunosorbent assay (SNEC ELISA). We compared the test performance of SNEC ELISA with the routine diagnostic tests dsDNA Farr radioimmunoassay (RIA) and nucleosome-based ELISA (anti-dsDNA-NcX-ELISA). SNEC ELISA distinguished patients with SLE with a specificity of 98.9% and a sensitivity of 70.6% from NHD clearly surpassing RIA and anti-dsDNA-NcX-ELISA. In contrast to the other tests, SNEC ELISA significantly discriminated patients with SLE from patients with rheumatoid arthritis, primary anti-phospholipid syndrome, spondyloarthropathy, psoriatic arthritis, and systemic sclerosis. A positive test result in SNEC ELISA significantly correlated with serological variables and reflected the uptake of opsonized SNEC by neutrophils. This stresses the relevance of SNECs in the pathogenesis of SLE. We conclude that SNEC ELISA allows for the sensitive detection of pathologically relevant AAb, enabling its diagnostic usage. A positive SNEC test reflects the opsonization of cell remnants by AAb, the neutrophil recruitment to tissues, and the enhancement of local and systemic inflammatory responses.</p

Flow cytometry evaluation (dot blot) in representative subjects of two subgroups (LEAN NORMOTENSIVES AND OBESE PATIENTS).

<p>Dot plot analysis is of CD4+ gated lymphocytes. At least 20.000 events were considered.</p

Demographic and clinical characteristics of the population evaluated.

<p>Demographic and clinical characteristics of the population evaluated.</p

Lymphocyte subpopulations in the different groups.

<p>Lymphocyte subpopulations in the different groups.</p

Correlation between body mass index (BMI) and circulating CD4+ effector memory (EM) cells (% CD4+) in the whole population of 68 subjects and patients.

<p>Correlation between body mass index (BMI) and circulating CD4+ effector memory (EM) cells (% CD4+) in the whole population of 68 subjects and patients.</p