Journal: Central-European journal of immunology

Article Title: Targeting the serum marker interleukin 9 improves the underlying characterization and immune homeostasis in rheumatoid arthritis.

doi: 10.5114/ceji.2024.141695

Figure Lengend Snippet: Fig. 3. Interleukin 9 affected the severity of arthritis in CIA rats. A) ELISA for the detection of IL-9 levels. B) Rat arthritis score. C) Analysis of cartilage damage by saffron O-solid green staining. The descriptive statistics are pre- sented as mean ±SD. *p < 0.05 vs. the CIA group, n = 3

Article Snippet: We measured the levels of serum markers using ELISA with a specialized commercial human IL-9 ELISA Kit (CSB-E04642h, Cusabio, Wuhan, China), human Gal1 ELISA Kit (CSB-EL012882h, Cusabio), human anti-CII ELISA Kit (HB3371-Hu, Shanghai Hengyuan Biotechnology Co., Ltd, Shanghai, China), human sSR-A ELISA Kit (HB3370-Hu, Shanghai Hengyuan Biotechnology Co., Ltd), human anti-Sa ELISA Kit (ml382511V), human RF ELISA Kit (ml060678V), human ACPA ELISA Kit (ml233305V), rat IL-9 ELISA Kit (CSB-E07294r, Cusabio), rat Gal1 ELISA Kit (JL25762, Shanghai Jianglai Biotechnology Co., Ltd, Shanghai, China), rat anti-CII ELISA Kit (HB1364-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat sSR-A ELISA Kit (HB1363-Ra, Shanghai Hengyuan Biotechnology Co., Ltd), rat anti-Sa ELISA Kit (ml202241V), rat RF ELISA Kit (ml059541V), and rat ACPA ELISA Kit (ml201458V).

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Frontiers in immunology

Article Title: Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

doi: 10.3389/fimmu.2023.1207631

Figure Lengend Snippet: FIGURE 1 Interaction analysis of platelet-derived microparticles and CLL cells. (A) Flow cytometry was conducted for the characterization of CLL cell lines labeled with anti-human CD19 antibody either untreated (control) or co-incubated with platelet-derived microparticles (PMPs) labeled with MitoTracker Deep Red (MTDR). Co-incubations of CII (upper panels) or MEC-1 cells (lower panels) with PMPs were performed at 1:10 and 1:100 (cell/PMP) ratios for 48 hours. (B) CLL co-incubations (48 hours) with PMPs (1:10 and 1:100) were also photographed by fluorescence microscopy. CLL cell lines were labeled with DAPI and MitoTracker™Green FM (MTG), whereas PMPs were labeled with MTDR. (C) Relative mitochondrial DNA copy number was obtained from cell/PMP co-cultures for 24 and 48 hours using Real-time PCR on leucine tRNA (mtDNA-tRNA-Leu) normalized with nuclear DNA copy number. Each value is the mean ± SEM of six separate experiments. The asterisk (*) indicates significant differences where **p < 0.01, and ***p < 0.001.

Article Snippet: Human leukemic cell lines CII (#ACC 773), MEC-1 (ACC 497), and the megakaryocytic Set2 cell line (#ACC 608) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ Braunschweig, Germany).

Techniques: Derivative Assay, Flow Cytometry, Labeling, Control, Incubation, Microscopy, Real-time Polymerase Chain Reaction

Journal: Frontiers in immunology

Article Title: Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

doi: 10.3389/fimmu.2023.1207631

Figure Lengend Snippet: FIGURE 3 Impact of PMPs on CLL metabolic functions. CLL cell lines (CII and MEC-1) were evaluated for metabolic functions at varying co-culture ratios (1:10 and 1:100) and incubation periods (24 and 48 hours) with either PMPs or purified mitochondria isolated from the Set2 megakaryocytic cell line. (A) Oxygen consumption rate (OCR) was determined using an Oroboros-O2k for: ROUTINE respiration as the basal state; the non-phosphorylating resting state (LEAK respiration); and the electron transport system (ETS) capacity. (B) Total ATP and glycolytic ATP levels were evaluated using a firefly luciferase-based assay. Relative ATP concentrations related to specific pathways (oxidative phosphorylation/OXPHOS versus glycolysis) were determined by cell treatments to inhibit mitochondrial complexes I and III using Rotenone (ROT) and Antimycin A (AmA), respectively. (C, D) Cells were treated with AmA, ROT, and phorbol 12-myristate 13-acetate (PMA) as ROS inducers. Thereafter, samples were mixed with (C) CellROX green reagent and SYTOX Red Dead Cell Stain prior analysis by flow cytometry at Ex/Em: 508/525 nm (CellROX green) and 640/658 nm (SYTOX Red Dead Cell Stain) to determine intracellular total ROS content; or with (D) MitoSOX™reagent prior analysis by flow cytometry at Ex/Em: 510/580 nm to determine mitochondrial superoxide content. N-acetylcysteine (NAC) and Tert-butyl hydroperoxide (TBHP) were used as negative and positive controls, respectively. Results are expressed as the mean ± SEM of six biological experiments. One-way ANOVA followed by Tukey’s multiple comparisons test show significant differences in the values presenting different superscript letters (p < 0.05).

Article Snippet: Human leukemic cell lines CII (#ACC 773), MEC-1 (ACC 497), and the megakaryocytic Set2 cell line (#ACC 608) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ Braunschweig, Germany).

Techniques: Co-Culture Assay, Incubation, Isolation, Luciferase, Phospho-proteomics, Staining, Cytometry

Journal: Frontiers in immunology

Article Title: Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

doi: 10.3389/fimmu.2023.1207631

Figure Lengend Snippet: FIGURE 5 Impact of PMPs on CLL chemoresistance and metabolic gene expression. (A) CII and (B) MEC-1 cell lines were co-incubated with PMPs (1:100, 48 h) and then treated with increasing amounts of either Cytarabine, Venetoclax, and Plumbagin (5-200 nM) for 24 hours to establish the lethal doses for 50% mortality (LD50). Control groups were treated with DMSO as dilution buffer. Light microscopy of cells treated at their respective LD50 are also presented for morphological analysis. (C, D) Real-time PCR analysis was performed to evaluate differentially expressed metabolic-related genes (DEGs) in CLL groups in the presence of chemo-drugs where each column represents the PMP-treated/non-treated cell ratio. Control groups were treated with DMSO as dilution buffer. Results are expressed as the mean ± SEM of six biological experiments. One-way ANOVA followed by Tukey’s multiple comparisons test show significant differences in the values presenting different superscript letters (p < 0.05).

Article Snippet: Human leukemic cell lines CII (#ACC 773), MEC-1 (ACC 497), and the megakaryocytic Set2 cell line (#ACC 608) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ Braunschweig, Germany).

Techniques: Gene Expression, Incubation, Control, Light Microscopy, Real-time Polymerase Chain Reaction

Journal: Frontiers in immunology

Article Title: Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

doi: 10.3389/fimmu.2023.1207631

Figure Lengend Snippet: FIGURE 6 Effects of PMPs on CLL survival and apoptosis. CLL cells were co-incubated with PMPs (1:100, 48 h) prior to treatments with the cells’ respective LD50 of Cytarabine, Venetoclax, and Plumbagin for 24 hours for the evaluation of drug cytotoxic events. Flow cytometry analysis in (A) CII and (B) MEC-1 cells stained with Propidium Iodide (PI, X‐axis) and Annexin‐FITC (X‐axis) sorted cell populations according to distinctive quarter zones (Q), including the large nuclear fragments/debris or Q1 (Anx-/PI+), necrosis cells or Q2 (Anx+/PI+), apoptotic cells or Q3 (Anx+/PI-), and the living cells or Q4 (Anx-/PI-). Values for each quadrant were then plotted in relation to their respective control groups (DMSO). Each value is the mean ± SEM of six separate experiments. The asterisk (*) indicates significantly different (*p < 0.05, ***p < 0.001).

Article Snippet: Human leukemic cell lines CII (#ACC 773), MEC-1 (ACC 497), and the megakaryocytic Set2 cell line (#ACC 608) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ Braunschweig, Germany).

Techniques: Incubation, Flow Cytometry, Staining, Control

Journal: Frontiers in immunology

Article Title: Platelet-derived microparticles provoke chronic lymphocytic leukemia malignancy through metabolic reprogramming.

doi: 10.3389/fimmu.2023.1207631

Figure Lengend Snippet: FIGURE 7 Impact of PMPs on CLL cycle progression. CLL cells were co-incubated with PMPs (1:100, 48 h) prior to treatments with the cells’ respective LD50 of Cytarabine, Venetoclax, and Plumbagin for 24 hours then evaluated for cycle analysis using flow cytometry. Cell accumulation in each cell cycle phase (i.e., G2/M) for (A) CII and (B) MEC-1 cells stained with Propidium Iodide and RNase A was then plotted in relation to control groups treated with DMSO. Each value is the mean ± SEM of six separate experiments. The asterisk (*) indicates significantly different (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Human leukemic cell lines CII (#ACC 773), MEC-1 (ACC 497), and the megakaryocytic Set2 cell line (#ACC 608) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture (DSMZ Braunschweig, Germany).

Techniques: Incubation, Cytometry, Staining, Control

Journal: Frontiers in Immunology

Article Title: Cartilage Oligomeric Matrix Protein Induced Arthritis—A New Model for Rheumatoid Arthritis in the C57BL/6 Mouse

doi: 10.3389/fimmu.2021.631249

Figure Lengend Snippet: Native COMP induces antibody responses and specific B cell epitopes are characterized in B6N mice. (A, B) Native COMP induced anti-COMP antibodies but no anti-CII antibodies in B6N mice. (A) Both native COMP+CFA group (n=7) and native COMP+IFA+0.5 mg/ml M.T. group (n=6) have anti-COMP IgG, gG1 and IgG2b production throughout the experiment (* p < 0.05). There are no differences of anti-COMP IgG, IgG1, and IgG2b levels between the two groups ( p >0.05). (B) No response to rat CII could be detected in COMP immunized mice ( p >0.05) (A B6N mouse immunized with rat CII was chosen as positive control). (C) Mapping of human COMP linear B-cell epitopes using overlapping peptide library of COMP protein (n=6) (D) According to the results of experiment A (C) , We choose six cyclic COMP peptides (C14, C15, C16, C22, C23 and C24) in a cyclic COMP library, which are similar to the sequences of the candidate peptides (peptide 7, 29, 30, 37, 39). We precoated the plate with these cyclic peptides and added two dilutions of serum of B6N mice immunized with COMP with 2 dilute (diluted 100 times and 1000 times) to test the OD value by ELISA. Positive binding was defined as 3SD from the mean of naïve mice (n=4). Antibody binding was measured by ELISA. The results presented as absorbance at nm 405.

Article Snippet: Detection of serum levels of antibodies against COMP and CII: Serum antibody levels against COMP were analyzed in 96-well plate (Nunc, Thermo Fisher Scientific, Denmark) by ELISA using recombinant human COMP.

Techniques: Positive Control, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Arthritis Research & Therapy

Article Title: Anti-type II collagen immune complex-induced granulocyte reactivity is associated with joint erosions in RA patients with anti-collagen antibodies

doi: 10.1186/s13075-015-0523-7

Figure Lengend Snippet: Functional reactivity against IgG as part of surface-bound collagen type II autoantibody (anti-CII) immune complex and polyclonal IgG. Amounts and functional reactivity against IgG as part of surface-bound collagen type II autoantibody (anti-CII) immune complex and polyclonal IgG directly coated to identical enzyme-linked immunosorbent assay (ELISA) plates. (a) ELISA reactivity of IgG against type II collagen (CII) aligned to known concentrations of directly coated IgG; these anti-CII levels (μg/ml) were thereafter used in the comparisons below. (b) Peripheral blood mononuclear cell-derived tumor necrosis factor alpha (ΤΝFα). (c) , (d) Polymorphonuclear granulocyte expression of CD66b and of CD16 respectively were compared for different concentrations of anti-CII and polyclonal IgG. Panels depict one representative result out of two to four experiments performed for each comparison. MFI, mean fluorescence intensity; OD, optical density.

Article Snippet: ELISA plates were coated with native human CII (10 μg/ml) (ELISA grade; Chondrex Inc.) at 4°C overnight.

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, Fluorescence