Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: CCL3 correlates with bone resorption in vitro OCP ( n = 8 donors, four replicates/condition) differentiated into osteoclasts in medium containing anti-CCL3 or IgG1 antibody. ( A ) Anti-CCL3 exerted a concentration-dependent inhibition on osteoclastogenesis (i), reducing total tartrate-resistant acid phosphatase (TRAP)-positive cells (ii), multinucleated osteoclasts (iii) and resorption (iv). ( B ) Disks stained with TRAP and haematoxylin (day 14). M-CSF cultures lacked osteoclasts (top), M-CSF + RANKL cultures showed strong TRAP staining (bottom; scale bar = 50 µm). ( C ) Resorption pits visualized by toluidine blue (top, scale bar = 250 μm) or calcein (bottom) reduced in anti-CCL3 (8 ng/ml) cultures vs IgG1 (yellow arrows). Mean value per donor plotted. * P ≤ 0.05, ** P ≤ 0.01. OCP = osteoclast precursor cells.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: In Vitro, Concentration Assay, Inhibition, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: CCL3 inhibition had no effect on resorption pit parameters Calcein-stained ivory disks were imaged by fluorescence microscopy to quantify resorption pit parameters. ( A ) Representative topographical maps of M-CSF (i), M-CSF + RANKL + IgG1 (ii), M-CSF + RANKL + anti-CCL3 (iii) disks show their naturally undulating surface and resorption pits (spherical lacuna); scale bar = 40 μm. ( B ) Measured lacuna area (i), perimeter (ii), depth (iii) and volume (iv) for anti-CCL3 vs IgG1 were unchanged (8 ng/ml). ( C ) Levels of CCL2 (i) and sIL-6R (ii) were comparable in IgG1 and anti-CCL3 (8 ng/ml) cultures (day 14). Cells from healthy human volunteers ( n = 6) were cultured, n = ≤2 disks/condition, mean ( s . e . m .) for each donor plotted.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: Inhibition, Staining, Fluorescence, Microscopy, Cell Culture

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: Systemic inhibition of CCL3 reduced histological joint swelling Mice with CIA received IgG1 or anti-CCL3 on days 21, 23, 25, 27 and 28 (5 mg/kg, n = 6/group). ( A ) Arthritis progression monitored by clinical scores (i) and paw diameter (ii) saw no statistical differences (two-way analysis of variance). ( B ) Representative tartrate-resistant acid phosphatase (TRAP) and haematoxylin-stained elbow joints from IgG1 (i) and anti-CCL3 (ii). Assessment of inflammation (iii), erosion (iv) and arthritic index (v). ( C ) IgG1 wrist histology showed intense TRAP-staining (i), which reduced in anti-CCL3 (ii), with significant reductions in inflammation (iii), erosion (iv) and arthritic index (v). H = humerus, U = ulna, R = radius, C = carpal. * P ≤ 0.05, ** P ≤ 0.01, scale = 1 mm.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques: Inhibition, Staining

Journal: Rheumatology (Oxford, England)

Article Title: Inhibition of CCL3 abrogated precursor cell fusion and bone erosions in human osteoclast cultures and murine collagen-induced arthritis

doi: 10.1093/rheumatology/key196

Figure Lengend Snippet: Bone erosions decreased after anti-CCL3 treatment during CIA Front and hind paws from CIA mice, treated with IgG1 or anti-CCL3, were processed for histology. ( A ) tartrate-resistant acid phosphatase (TRAP)-positive cells in the elbow (i) and wrist (ii) joints significantly reduced with anti-CCL3. ( B ) Radiographs of hind paws and ( C ) front paws were acquired from mice treated with IgG1 (i) or anti-CCL3 (ii) ( n = 6 mice per group). Significant reductions in erosive radiographic score for in both hind [B (iii)] and front [C (iii)] paws were quantified in mice treated with anti-CCL3 compared with IgG1 controls. * P ≤ 0.05, ** P ≤ 0.01.

Article Snippet: Cell responses were compared using media supplemented with mouse IgG1 isotype as a control (MAB002, R&D Systems, Abingdon, UK).

Techniques:

Journal: Circulation Research

Article Title: Hemoglobin Scavenger Receptor CD163 Mediates Interleukin-10 Release and Heme Oxygenase-1 Synthesis

doi: 10.1161/01.res.0000109414.78907.f9

Figure Lengend Snippet: Figure 3. Induction of HO-1 by Hb:Hp via a CD163 receptor IL-10 autocrine mechanism. A, Representative HO-1 and HO-2 immunoblots are shown from in vitro differentiated day 7 macrophages stimulated with Hb, Hp, Hb:Hp, or Hb:Hp plus blocking anti–IL-10, IgG control, or RM3/1 antibody. Induction of the HO-1 isomer was established relative to con- stitutive expression of the HO-2 isomer by probing blots with polyclonal antibody against HO-1, followed by stripping and reprobing with anti–HO-2. B, Lysates in A were subjected to quantification of intracellular HO-1 levels by HO-1–spe- cific ELISA and normalized with respect to total cell protein. Values shown repre- sent meanSEM. Results are represen- tative of 3 similar experiments.

Article Snippet: Neutralizing anti- human IL-10 polyclonal antibody and control goat IgG antibody were purchased from R&D Systems (Abingdon, UK).

Techniques: Western Blot, In Vitro, Blocking Assay, Control, Expressing, Stripping Membranes, Enzyme-linked Immunosorbent Assay

Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD57 + GC-Th cells are more efficient than other tonsil CD4 + T cell subsets in helping B cells. (A) CD4 + T cell subsets were cultured with total tonsil CD19 + B cells for 7 days in the presence of SEB. Naïve B cells (C) or GC-B cells (D) were cultured with equal numbers of CD57 + GC-Th cells or other T cell subsets (CD57 - , CD57 - CD69 + and CD57 - CD69 - T cells) for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 5 independent experiments were combined and the averages are shown with standard errors. Relative production levels to CD57 + GC-Th cells are shown. *Significant differences from CD57 + GC-Th cells. The absolute Ig production levels (ng/ml) in panel A (GC-Th + Total B cells) were 5737 ± 1764 (IgM), 2111 ± 1185 (IgG), 577 ± 186 (IgA), and 4.8 ± 2.1 (IgE). The absolute Ig production levels (ng/ml) in panel B (GC-Th cells + naïve B cells) were 2045 ± 697 (IgM), 63 ± 21 (IgG), 40 ± 23 (IgA), and 2.9 ± 1.2 (IgE). The average levels (ng/ml) of Ig produced in the cultures of GC-Th cells and GC B cells were 750 ± 279 (IgM), 175 ± 52 (IgG), 51 ± 13 (IgA), and 1.0 ± 0.5 (IgE). (D) Isotype composition of the Ig induced by CD57 + GC-Th cells. Naïve B cells or GC-B cells were cultured with equal numbers of CD57 + GC-Th cells or CD57 - CD69 + T cells for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 4 independent experiments were combined and the averages are shown with standard errors. *Significant differences between naïve and GC-B cells, but not between the two T cell subsets, were observed.

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Produced

Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD57 + GC-Th cells have the capacities to induce AID expression and to support CSR in B cells. IgD + CD38 - naïve B cells were cultured with CD57 + GC-Th cells for indicated time periods followed by RT-PCR analysis for (A) AID expression and (B) CSR. The sizes of specific PCR products are 152 bp (IgM); 416 bp (IgG1, G2, G3), 904 bp (IgG4); 904 bp (IgA1); 891 bp (IgA2); and 179 bp (IgE). Shown are productive recombination products. (C) The expression kinetics of AID and productive IgG3 transcripts over an 8 day period are shown together in a graph. In this panel, normalized expression levels calculated after dividing the levels of AID amplification by β-actin levels are shown. The time gap to reach the peak levels of the expression between AID and productive IgG3 transcripts is shown by an arrow. Representative data from at least three independent experiments are shown (panels A and B). (D) Identification of extrachromosomal reciprocal DNA recombination products. Naïve B cells were cultured with CD57 + GC-Th cells for indicated time periods and were processed to isolate genomic DNA. Fresh GC-B cells were examined for positive controls. The switch circles were detected by a nested PCR method. Representative data out of three independent experiments are shown. (E) Detection of switch circles by a DC-PCR technique. Naive B cells, CD38 + GC-B cells and naïve B cells cultured with GC-Th cells for 5 days were examined for the presence of γ3 and α1/2 switch circles.

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Amplification, Nested PCR

Journal: BMC Immunology

Article Title: Human CD57 + germinal center-T cells are the major helpers for GC-B cells and induce class switch recombination

doi: 10.1186/1471-2172-6-3

Figure Lengend Snippet: CD40L and cytokines in regulation of the helper activity of CD57 + GC-Th cells. (A and B) Effects of endogenous CD40L and cytokines on the helper activity of CD57 + GC-Th cells were determined. In cultures of CD57 + GC-Th cells with naïve or GC-B cells, neutralizing antibodies to IL-4, IL-10, IFN-γ or CD40L or control antibodies (mouse IgG1) were added. *Significant differences from the control group (control antibody). (C and D) Effects of exogenously added cytokines on the helper activity of CD57 + GC-Th cells were determined. To cultures of CD57 + GC-Th cells with naïve or GC-B cells, IL-4, IL-10, IFN-γ and TGF-β1 were added separately. Cells were cultured for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Relative Ig secretion levels (the medium control = 1) obtained from 9 independent experiments were combined, and averages and standard errors are shown. *Significant differences from the control group (medium).

Article Snippet: Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems.

Techniques: Activity Assay, Control, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Circulating ACE2-expressing extracellular vesicles block broad strains of SARS-CoV-2

doi: 10.1038/s41467-021-27893-2

Figure Lengend Snippet: a ACE2+ EVs detected in human plasma samples of sero-negative controls (light blue), acute phase (dark green), and convalescent COVID-19 patients (green). One-tail t test (* p = 0.038, ** p = 0.0061 and ** p = 0.0016). Data are presented as mean values ± SEM. b Representative microflow vesiclometry (MFV) plots with gated ACE2+ EVs from sero-negative, acute phase and convalescent COVID-19 patients. c MFV detection of circulating ACE2 + EVs with CD63 + EVs in human plasma of convalescent COVID-19 patient samples (CSB-029 and CSB-023) (green line). Blue line is isotype IgG-negative control. d Flow profiles of ACE2 expression in HEK and HeLa parental control cells (Con, light blue line, ACE2 − ) and with ACE2 overexpression (ACE2, green line). e NanoSight NTA analysis of the sizes of HEK-derived ACE2 − (ev1Con) and ACE2 + (ev1ACE2) and HeLa-derived ACE2 − (ev2Con) and ACE2 + (ev2ACE2). f Immunoblots of HEK and HeLa (ACE2 − and ACE2 + ) EVs and cell lysates for ACE2, TSG101, CD63, CD81, GRP94 and loading control of the membrane proteins upon Ponceau staining. RIPA buffer and Bradford protein assay were used for cells/EVs lysis and protein measurement, respectively ( N = 1 experiment). g Cryo-EM images of HEK-derived EVs, ACE2 − (evCon, left) and ACE2 + (evACE2, right), stained with ACE2 (top) and CD81 (bottom). Scale bars = 100 nm. h Quantified counts of Apogee MFV-based total extracellular vesicles (EVs) and ACE2 + EVs ( N = 2 experiments with n = 6 technical replicates for total EV particles and n = 3 technical replicates for ACE2 + counts). Control EVs are in light blue and ACE2 + EVs in green. Data are presented as mean values +/− SD. i Overlay flow profiles of ACE2 positivity within CD63 + (left column) and CD81+ (right column) EVs isolated from HEK-ACE2 (top row) and HeLa-ACE2 (bottom row) cells, respectively ( n = 3 technical replicates). Light blue line for Control EVs and green line for ACE2 + EVs.

Article Snippet: Cells were blocked with mouse serum IgG (Sigma, 15381) for 10 min at room temperature and then incubated with specific antibodies; AF-647 mouse anti-human ACE2 (Clone # 535919) (R&D systems, FAB9332R), AF-488 mouse anti-human ACE2 (Clone # 171607) (R&D systems, FAB9333G) (0.4 μg/10 6 cells), AF-647 isotype control mouse IgG2b (Clone # 20102) (R&D systems, IC003R) or AF-488 isotype control mouse IgG2bAF488 (Clone # 20102) (R&D systems, IC003G) for 45 min on ice, followed by washing twice with 2% EV-free FBS/PBS.

Techniques: Clinical Proteomics, Negative Control, Expressing, Control, Over Expression, Derivative Assay, Western Blot, Membrane, Staining, Bradford Protein Assay, Lysis, Cryo-EM Sample Prep, Isolation