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: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) Schematic illustration of the 1D11 administration paradigm for histological analysis. (B) Representative images of H&E stained sections (left) showing whole eyes (top panels) and corneas (lower panels) from E18.5 mice and quantification graph (right) showing increased corneal stromal thickness in Col4a1+/G1344D mice treated with 1D11 compared to those that received the control IgG1 antibody. Scale bars = 200 μm (top) and 50 μm (bottom). n = 20 and 24 corneas from IgG1- and 1D11-treated Col4a1+/+ mice, and 24 and 30 corneas from IgG1- and 1D11-treated Col4a1+/G1344D mice, respectively. (C) Schematic illustration of the 1D11 administration paradigm for qPCR analyses. (D) qPCR analyses revealed that 1D11 treatment partially prevented the increased expression of TGFβ target genes in anterior segments from P0 Col4a1+/G1344D mice compared to their IgG1-treated counterparts. n = 5–6 samples per genotype. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, two-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: Timed-pregnant B6 females crossed with Col4a1 +/ G1344D males were injected intraperitoneally with the 1D11 pan-TGFβ neutralizing antibody (clone 1D11.16.8, BioXCell, West Lebanon, NH) or IgG1 isotype control antibody (clone MOPC-21, BioXCell) diluted in inVivoPure Dilution Buffer (pH 7.0, BioXCell) (20 mg/kg) every other day from E8.5 to E16.5 and animals were harvested at E18.5 or P0 for histological and molecular analyses, respectively.

Techniques: Staining, Control, Expressing, Comparison

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: PLOS Pathogens

Article Title: B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice

doi: 10.1371/journal.ppat.1011187

Figure Lengend Snippet: Mice were treated with the anti-CD20 mAb 5D2 beginning 2 days prior to a low-dose (100 CFU) aerogenic challenge with M . tuberculosis Erdman, as described in "Materials and Methods". B cell depletion was maintained throughout the duration of the experiment. The control mouse group (WT) received non-specific rat IgG. The lung tissues were examined at 5 months post-infection. The levels of granulomatous inflammation response were analyzed histologically by light microscopy on H&E-stained lung sections (A) and enumeration of total number of lung cells (B). The level of lung CD4 + T cell response was assessed by in vivo BrdU labeling to examine the proliferation capacity of this T cell subset (C), as well as by enumeration of IFN-γ-producing CD4 + T cells (D). Ex vivo evaluation of lung cells for the level of IL-10 production (E) was conducted as described in . Four to 5 mice per group were evaluated per group. Data depicted in B, C, D, and E are presented as means ± SEM. The data shown are representative of two experiments. The results demonstrated that the inflammation, Th1 response, and IL-10 phenotypes observed in the μMT mice are recapitulated in mice depleted for B cells.

Article Snippet: At day 90 post infection, 1 mg of anti-mouse IL-10 receptor (IL-10R; clone 1B1.3A; BioXcell) or isotype control Rat IgG1 antibody (BioXcell) was administered intraperitoneally (i.p.) per mouse.

Techniques: Control, Infection, Light Microscopy, Staining, In Vivo, Labeling, Ex Vivo

Journal: PLOS Pathogens

Article Title: B cells promote granulomatous inflammation during chronic Mycobacterium tuberculosis infection in mice

doi: 10.1371/journal.ppat.1011187

Figure Lengend Snippet: C57BL/6 mice were depleted for B cells via administration of 5D2 beginning 2 days prior to infection with a low dose (100 CFU) of M . tuberculosis Erdman delivered by aerosol. B cell depletion was maintained for the duration of the experiment. At 3 months after the infection, IL-10R blockade was initiated using the anti-mouse IL-10R antibody clone 1B1.3A. The control group received non-specific rat IgG. The IL-10R blockade was continued for two months. At five months post-infection (2 months after initiation of IL-10R blockade), mice were sacrificed and analyzed for the levels of inflammation in the lungs, as assessed by histological examination (A) and enumeration of total lung cells (B), CD4 + T cells proliferation via BrdU labeling (C), and Th1 response (D). Data shown are representation of two experiments. Three to four mice were analyzed per group. Data depicted in (B), (C), and (D) denote mean ± SEM.

Article Snippet: At day 90 post infection, 1 mg of anti-mouse IL-10 receptor (IL-10R; clone 1B1.3A; BioXcell) or isotype control Rat IgG1 antibody (BioXcell) was administered intraperitoneally (i.p.) per mouse.

Techniques: Infection, Aerosol, Control, Labeling