Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: PLoS ONE

Article Title: Novel PDE4 Inhibitors Derived from Chinese Medicine Forsythia

doi: 10.1371/journal.pone.0115937

Figure Lengend Snippet: A. 5×10 5 RAW264.7 cells were seeded in 96 wells for 18 h. Cells were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. B. PBMC (0.2 ml at 1×10 5 /ml) were primed with compounds at different concentration for 3 h before treated with LPS (1 ng/ml) for additional 8 h. TNF cytokine releases were monitored by ELISA. % of TNF secretion were calculated and graphed. C. Summary of compound IC 50 . The data represent n = 3–6 experiments.

Article Snippet: IL1β, TNFα, IL6 mouse ELISA kit, human TNFα were from R&D systems.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Overexpression of TRIM38 inhibits TNFα- and IL-1β–triggered signaling. (A) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HEK293 cells. HEK293 cells (1 × 105) were transfected with the NF-κB luciferase plasmid (0.01 μg) and an HA-TRIM38 plasmid (0.2 or 0.4 μg). Twenty hours after transfection, cells were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) or left untreated for 10 h before luciferase assays were performed. Expression of transfected TRIM38 in each unstimulated sample was examined by immunoblot analysis. (B) Effects of TRIM38 on TNFα- and IL-1β–triggered NF-κB activation in HCT116 and HeLa cells. The experiments were performed as in A. (C) Effects of TRIM38 on IFNγ-induced activation of the IRF1 promoter. The experiments were performed as in A except that the IRF1 promoter reporter plasmid was used and transfected cells were treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 on TNFα- and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then total RNA was prepared for qPCR analysis. Expression of TRIM38 in the stable cell lines was examined by immunoblot analysis (Right). (E) Effects of TRIM38 on TNFα- and IL-1β–induced cytokine of TNFα, IL-6, and IL-8. HEK293 cells were transduced with either an empty vector or an HA-TRIM38 plasmid to establish stable cell lines. Cells (4 × 105) from both stable cell lines were treated with TNFα or IL-1β for the indicated times, and then the medium was collected for ELISA analysis. (F) Effects of TRIM38 on IFNγ-induced transcription of IRF1 gene. Cells (4 × 105) were left untreated or treated with IFNγ (100 ng/mL) for the indicated times, and total RNA was extracted for qPCR analysis. Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Activation Assay, Transfection, Luciferase, Plasmid Preparation, Expressing, Western Blot, Transduction, Stable Transfection, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: Knockdown or knockout of TRIM38 potentiates TNFα- and IL-1β–triggered signaling. (A) Efficiencies of TRIM38-RNAi plasmids on TRIM38 levels. (Upper) HEK293 cells (4 × 105) were transfected with expression plasmids for TRIM38-Flag and HA-β-actin (0.1 μg each) and the indicated RNAi plasmids (1 μg each). Twenty-four hours after transfection, cell lysates were analyzed by immunoblot with anti-Flag or anti-HA. (Lower) HEK293 cells (1 × 107) were transfected with control or the indicated TRIM38-RNAi plasmids (10 μg each) for 36 h. Cell lysates were analyzed by immunoblot with anti-TRIM38 or anti–β-actin. (B) Effects of TRIM38-RNAi on TNFα- and IL-1β–triggered NF-κB activation in HEK293 and HeLa cells. The cells (1 × 105) were transfected with RNAi plasmids (1 μg each) along with the NF-κB reporter plasmid (0.01 μg). Thirty-six hours after transfection, cells were left untreated or treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for 10 h before luciferase assays were performed. (C) Effects of TRIM38-RNAi on IFNγ-induced IRF1 promoter activation. Reporter assays were performed as in B except that cells were transfected with IRF1 promoter reporter plasmid and treated with IFNγ (100 ng/mL). (D) Effects of TRIM38 deficiency on TNFα and IL-1β–induced transcription of TNFA, IL-6, and IL-8 genes. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then total RNA was extracted for qPCR analysis. (E) Effects of TRIM38 deficiency on TNFα and IL-1β–induced cytokine production. The indicated cells (4 × 105) were treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for the indicated times, and then the medium was collected for ELISA analysis. (F) TRIM38 deficiency potentiates TNFα-triggered MAPK activation. The indicated cells (1 × 107) were left untreated or treated with TNFα (10 ng/mL) for the indicated times. Cells were lyzed and immunoblot analysis was performed with the indicated antibodies. (G) TRIM38 deficiency potentiates IL-1β–triggered MAPK activation. The experiments were performed as in E, except that cells were treated with IL-1β (10 ng/mL). Graphs show mean ± SD; n = 3. *P < 0.05; **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Knockdown, Knock-Out, Transfection, Expressing, Western Blot, Control, Activation Assay, Plasmid Preparation, Luciferase, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 interacts with and destabilizes TAB2 through its C-terminal PRY-SPRY domain. (A) TRIM38 interacts with TAB2 and TAB3 in mammalian overexpression system. HEK293 cells (1 × 107) were transfected with the indicated plasmids for 24 h. Coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (B) Endogenous TRIM38 interacts with TAB2/3. HEK293 cells (3 × 107) were left untreated or treated with TNFα (Left) or IL-1β (Right) for the indicated times. Endogenous coimmunoprecipitation and immunoblots were performed with the indicated antibodies. (C) TRIM38 specifically destabilizes TAB2/3. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h, and then immunoblots were performed with the indicated antibodies. (D) Effects of TRIM38 truncation mutants on destabilization of TAB2. HEK293 (4 × 105) cells were transfected with the indicated plasmids for 24 h before immunoblots were performed with the indicated antibodies. (E) Analysis of TRIM38 expression in TRIM38−/− cells stably transduced with an empty vector (II), TRIM38-Flag (III), TRIM38(63-465)-Flag (IV), or TRIM38(290-465)-Flag (V), respectively and in TRIM38+/+ cells stably transduced with an empty vector (I). Cells (1 × 107) (I, II, III, IV, V) were harvested and lysed. Immunoblot analysis was performed with the indicated antibodies. (F) Reconstitution of TRIM38 or TRIM38 mutant (63–465) into TRIM38-deficient cells leads to down-regulation of TAB2. Cells (1 × 107) (I, II, III, IV, V) were left untreated or treated with TNFα or IL-1β for the indicated times. Immunoblot analysis was performed with the indicated antibodies.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Over Expression, Transfection, Western Blot, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: TRIM38 inhibits TNFα- and IL-1β–triggered NF-κB activation by mediating lysosome-dependent degradation of TAB2/3

doi: 10.1073/pnas.1318227111

Figure Lengend Snippet: TRIM38 mediates lysosomal degradation of TAB2. (A) Effects of inhibitors on TRIM38-mediated destabilization of TAB2. HEK293 cells (4 × 105) were transfected with the indicated plasmids. Fourteen hours after transfection, the cells were treated with the indicated inhibitors for 6 h before immunoblot analysis was performed. (B) Effects of NH4Cl and MG132 on down-regulation of TAB2 triggered by TNFα and IL-1β. HEK293 (1 × 107) cells were treated with NH4Cl or MG132 for 4 h and then further treated with TNFα and IL-1β for 2 h before immunoblot analysis was performed. (C) TRIM38 promotes translocation of TAB2 to the lysosome. HEK293 cells (1 × 105) were transfected with Cherry-TAB2 and GFP-LAMP1 (Left) or CFP-TRIM38 (Right). Twenty hours after transfection, cells were fixed with 4% (wt/vol) paraformaldehyde and subjected for confocal microscopy. (D) Effect of TRIM38 deficiency on TNFα- or IL-1β–induced colocalization of TAB2 with the lysosomes. TRIM38+/+, TRIM38−/−, or TRIM38−/− cells reconstituted with the PRY-SPRY domain (1 × 105) were transfected with GFP-TAB2. Twenty hours after transfection, cells were stained with Red Lysotracker (200 nM) for 2 h and treated with TNFα (20 ng/mL) or IL-1β (20 ng/mL) for 1 h and then fixed with 4% (wt/vol) paraformaldehyde and subjected to confocal microscopy. A random 10 cells in each sample were used for calculating the colocalization dots that were normalized to the total lysosome-red dots. Graphs show mean ± SD; n = 3. **P < 0.01.

Article Snippet: Recombinant human TNFα, IL-1β, and IFNγ (R&D Systems); mouse monoclonal antibodies against Flag (Sigma), HA (Covance), and β-actin (Sigma); mouse anti-TAK1, p-TAK1, p-IKKα/β; rabbit anti-JNK, p-JNK, p38, p-p38, Erk1/2, p-Erk1/2 (CST); rabbit anti-TRAF6, RIP1, IRAK1, TRAF2 (Santa Cruz Biotechnology); rabbit anti-TAB3 (Epitomics); LysoTracker (Invitrogene); and ELISA kits for human TNFα, IL-6, and IL-8 (BOSTER) were purchased from the indicated manufacturers.

Techniques: Transfection, Western Blot, Translocation Assay, Confocal Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: High Tidal Volume Induces Mitochondria Damage and Releases Mitochondrial DNA to Aggravate the Ventilator-Induced Lung Injury

doi: 10.3389/fimmu.2018.01477

Figure Lengend Snippet: Lung injury and inflammation in animals with spontaneous breathing (CON) or mechanical ventilation at normal tidal volume (NTV) or high tidal volume (HTV). (A) Lung edema was assessed by determining the weight ratio between wet and dry lung (W/D). (B) Pathological scores were assessed by results of hematoxylin and eosin (HE) staining. (C) Total protein concentration in bronchoalveolar lavage fluid (BALF). (D) Infiltrated cell counts in BALF. (E) Levels of interleukin (IL)-1β in BALF and plasma. (F) Levels of IL-6 in BALF and plasma. (G) Levels of tumor necrosis factor (TNF)-α in BALF and plasma. (H) Levels of myeloperoxidase (MPO) in BALF and plasma. (I) Histology of lung tissue was stained with HE. Magnification, 40×. (J) Transmission electron micrographs of alveolar cells. Areas marked with arrows signify continuous membrane and triangles mark areas of disrupted cytoplasmic and nuclear structure. Areas marked with stars represent the osmilphilic multilamellar bodies, the unique structure of AT-IIs. Tissue from CON and NTV groups appeared normal, but tissue from HTV group exhibited a disrupted cytoplasmic and nuclear structure, as well as cell membrane discontinuities. In addition, the osmilphilic multilamellar bodies show characteristic vacuolation in the HTV group. Magnification, 20,000×. Both of these experiments were in triplicate. a P < 0.05 vs. CON group; b P < 0.05 vs. NTV group.

Article Snippet: Enzyme linked immunosorbent assay (ELISA) kits were used to test the levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and myeloperoxidase (MPO) (CUSABIO, Wuhan, China).

Techniques: Staining, Protein Concentration, Clinical Proteomics, Transmission Assay, Membrane

Journal: Frontiers in Immunology

Article Title: High Tidal Volume Induces Mitochondria Damage and Releases Mitochondrial DNA to Aggravate the Ventilator-Induced Lung Injury

doi: 10.3389/fimmu.2018.01477

Figure Lengend Snippet: Lung injury and inflammation in animals with spontaneous breathing (CON) or mechanical ventilation at high tidal volume (HTV) with saline, antimycin A (AmA), or cyclosporine A (CsA). (A) Lung edema was assessed by determining the weight ratio between wet and dry lung (W/D). (B) Pathological score was assessed by result of hematoxylin and eosin (HE) staining. (C) Total protein concentration in bronchoalveolar lavage fluid (BALF). (D) Infiltrated cell counts in BALF. (E) Levels of interleukin (IL)-1β in BALF and plasma. (F) Levels of IL-6 in BALF and plasma. (G) Levels of tumor necrosis factor (TNF)-α in BALF and plasma. (H) Levels of myeloperoxidase (MPO) in BALF and plasma. (I) Histology of lung tissue was stained with HE. Magnification, 100×. (J) Transmission electron micrographs of alveolar cells. Areas marked with arrows signify continuous membrane and triangles mark areas of disrupted cytoplasmic and nuclear structure. Areas marked with stars represent the osmilphilic multilamellar bodies, the unique structure of AT-IIs. Tissue from CON group appeared normal, but tissue from HTV and AmA groups exhibited a disrupted cytoplasmic and nuclear structure, as well as cell membrane discontinuities. Moreover, the osmilphilic multilamellar bodies show characteristic vacuolation in the HTV and AmA groups. But these effects were attenuated when administered in the presence of CsA. Magnification, 20,000×. Both of these experiments were in triplicate. a P < 0.05 vs. CON group; b P < 0.05 vs. HTV group; and c P < 0.05 vs. AmA group.

Article Snippet: Enzyme linked immunosorbent assay (ELISA) kits were used to test the levels of inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and myeloperoxidase (MPO) (CUSABIO, Wuhan, China).

Techniques: Saline, Staining, Protein Concentration, Clinical Proteomics, Transmission Assay, Membrane

Journal: PLoS ONE

Article Title: Elevated Membrane and Soluble CD64: A Novel Marker Reflecting Altered FcγR Function and Disease in Early Rheumatoid Arthritis That Can Be Regulated by Anti-Rheumatic Treatment

doi: 10.1371/journal.pone.0137474

Figure Lengend Snippet: Fcγ receptor expression and function reflecting severity of joint inflammation in drug naïve early RA patients ( n = 20).

Article Snippet: Briefly, microtiter plates were coated with mouse anti-human TNFα capture ab (2 μg/ml) (MAB610/clone 28401; R&D Systems, USA) overnight in a humid chamber.

Techniques: Expressing, Binding Assay

Journal: PLoS ONE

Article Title: Elevated Membrane and Soluble CD64: A Novel Marker Reflecting Altered FcγR Function and Disease in Early Rheumatoid Arthritis That Can Be Regulated by Anti-Rheumatic Treatment

doi: 10.1371/journal.pone.0137474

Figure Lengend Snippet: IgG1 and IgG3 IC-binding (A-B), ratio of IgG1-IC/IgG3-IC binding (C) and IgG1- and IgG3-IC stimulated TNFα-release (index) (D-E) in DMARD- and steroid naive early RA patients ( n = 20) and in healthy controls (HC) ( n = 33).

Article Snippet: Briefly, microtiter plates were coated with mouse anti-human TNFα capture ab (2 μg/ml) (MAB610/clone 28401; R&D Systems, USA) overnight in a humid chamber.

Techniques: Binding Assay