Journal: Nature immunology

Article Title: IgG3 regulates tissue-like memory B cells in HIV-infected individuals

doi: 10.1038/s41590-018-0180-5

Figure Lengend Snippet: a, Flow cytometry of CD20-gated B cells from a chronically infected HIV-viremic individual with high-intensity IgG3 on IgM+ B cells, showing binding of C1q and IgG3 on naive B cells and TLM B cells (blue, CRP overlay). Numbers in blue indicate percent CRP among cells in that quadrant. Data are representative of 11 experiments. b, Calcium uptake by C1q+CRP+and C1q−CRP− populations (horizontal axis) of IgG3+IgD+ TLM B cells and naive B cells from chronically infected HIV-viremic individuals (n = 9) with moderate- to high-intensity IgG3 on IgM+ B cells (presented as in Fig. 4b). c, Mean fluorescence intensity of CD32b (evaluated by flow cytometry) on B cells isolated from HIV-viremic individuals (n = 14) and identified as naive, RM, AM or TLM B cells (horizontal axis) by their expression of CD21 and CD27. d, Flow cytometry (left half) showing the expression of IgG3 and IgM by CD20-gated B cells obtained from a chronically infected HIV-viremic individual with high-intensity IgG3 on IgM+ B cells and maintained for 20 min at 4 °C (top) or treated at 37 °C with control goat IgG (bottom left) or goat anti-CD32b (bottom right); middle and right, frequency of IgG3+IgM+ B cells (middle right) and calcium uptake (far right) among B cells obtained from chronically infected HIV-viremic individuals (n = 8 subjects (middle right) or n = 7 subjects (far right)) with moderate- to high-intensity IgG3 on IgM+ B cells and treated with antibody as at bottom left (horizontal axis); results for calcium uptake presented as in b. Each symbol (b–d) represents an individual subject (identified by color in b,c); small horizontal lines (b,c) indicate the median; diagonal lines (d) connect values for the same subject. *P < 0.05, **P < 0.01 and ****P ≤ 0.0001 (two-tailed Wilcoxon matched-pairs signed rank test, after significance was obtained by Friedman ANOVA of the full set (in b,c)).

Article Snippet: Membranes were sequentially incubated with goat anti-IgM-HRP (Southern Biotech; Cat# 2020–05) or primary antibody rabbit anti-C1q (Novus; Cat# H00000712-D01P), mouse anti-CRP (R&D Systems; clone 232026), rabbit anti-CD32 (GeneTex; Cat# GTX133371) or rabbit anti- IgG3 (Abcam; clone EPR4419), followed by the secondary antibody, goat anti-rabbit-HRP (Bio-Rad; Cat# 1662408EDU) or goat anti-mouse-HRP (Bio-Rad; Cat# 1000450), diluted in 4% milk powder in 0.1% Tween in PBS.

Techniques: Flow Cytometry, Infection, Binding Assay, Fluorescence, Isolation, Expressing, Two Tailed Test

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Relationship between sLAIR1, sLAIR2, and C1q serum levels in children with malaria. Differences between circulating sLAIR1 (ng/mL), sLAIR2 (ng/mL), and C1q (ng/mL) in children with non-SMA and SMA were determined using Student t -test and are presented as mean ± SEM. Relationships between sLAIR1, sLAIR2, C1q, and haemoglobin levels were determined by Spearman's correlation test. (a) Normalised sLAIR1 levels in children with non-SMA and SMA. Children with SMA had elevated sLAIR1 levels relative to non-SMA. (b) Normalised sLAIR1 vs. haemoglobin concentrations in children with non-SMA and SMA. Circulating sLAIR1 levels were inversely correlated with haemoglobin levels. (c) Normalised sLAIR1 levels in children with non-SMA and SMA. sLAIR2 serum levels were elevated in children with SMA relative to non-SMA. (d) Normalised sLAIR2 vs. haemoglobin concentrations in children with non-SMA and SMA. Normalised sLAIR2 vs. haemoglobin. sLAIR2 levels were inversely correlated with haemoglobin concentrations. (e) Normalised sLAIR1 vs. normalised sLAIR2 in children with non-SMA and SMA. Circulating sLAIR1 and sLAIR2 levels were positively correlated. (f) Circulating C1q levels in children with non-SMA and SMA. Circulating C1q levels were lower in children with SMA.

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques:

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Effect of intraleukocytic Pf Hz on LAIR1 and SHP-1 phosphorylation. Temporal kinetics of the signalling pathway in response to the different treatment conditions was determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). (a) Human Phospho-immunoreceptor array results showing LAIR1 and SHP-1 phosphorylation upon no treatment (baseline), C1q (25 μg/mL), Pf Hz (10 μg/mL), and Pf Hz + C1q. Phosphorylation (spots) in the different conditions with control represented by blue boxes, LAIR1 by red boxes, and SHP-1 by green boxes. (b) Densitometric analysis of human phosphor-immunoreceptor array data. Data presented as (mean ± SEM). Across group comparisons analysed using ANOVA. *indicates significant differences ( P < .05) determined by Student t-test in pLAIR1 and pSHP-1 compared to C1q treatment. Phagocytosis of Pf Hz resulted in a reduction of pLAIR1 relative to C1q (alone) treatment. Similarly, ingestion of Pf Hz also caused a marked decrease in pSHP-1 levels relative to C1q treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques: Phospho-proteomics, Control

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: Effect of haemozoin on NF-κB activation and cytokine production. Measurements ndwere determined in PBMCs from malaria-naïve donors (n = 6, measured in triplicate). Data presented as mean ± SEM. Across group and pairwise comparisons determined using Student t-test and Anova analyses, respectively. (a) Comparisons of phosphorylated NF-κB immediately after stimulation of PBMCs with no treatment (NT), C1q (25 μg/mL), Pf Hz (10 μg/mL), and Pf Hz + C1q. NF-κB phosphorylation levels were elevated in the Pf Hz and Pf Hz + C1q treatment groups relative to no treatment (denoted by * P < .05). (b) IL-1β levels (pg/mL) were elevated in culture supernatants from PBMCs treated with Pf Hz and Pf Hz + C1q. (c) IL-6 levels (pg/mL) were elevated in culture supernatants from PBMCs treated with Pf Hz and Pf Hz + C1q. (d) TNF-α levels (pg/mL) were elevated in culture supernatants from PBMCs treated with Pf Hz and Pf Hz + C1q.

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques: Activation Assay, Phospho-proteomics

Journal: EBioMedicine

Article Title: Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling

doi: 10.1016/j.ebiom.2019.06.040

Figure Lengend Snippet: LAIR1 signalling pathway in malaria. The LAIR1 pathway is activated by attachment of collagen and collagenous ligands (C1q) to LAIR1 extracellular surface. This results in phosphorylation of intracellular LAIR1 ITIM tyrosine residues by Src family kinases. Phosphorylated ITIMs serve as docking sites for recruitment of SHP-1 and SHP-2 phosphatases. SHP-1 and SHP-2 become localized to phosphorylated ITIMs through their regulatory SH2 domains, subsequently inducing their phosphatase activity. Activated SHP-1 has been shown to block activation and nuclear translocation of nuclear factor nuclear factor-kappa beta (NF-κB) through de-phosphorylation of inhibitor of kappa-beta kinase complex (IKK). SHP-1 phosphatase activity also inhibits activation and translocation of IRFs from the cytoplasm to the nucleus by preventing TANK-binding kinase 1 (TANK-1) phosphorylation of interferon regulatory factors (IRFs). These events subsequently block transcription of inflammatory mediator encoding genes with response elements for IRFs and NF-κB. SHP-2 inhibits activation of IRF 8 and blocks expression of phagocyte NADPH oxidase (gp91 PHOX ). LAIR1 inhibitory signalling is regulated by soluble LAIR1 and LAIR2 (soluble homolog of LAIR1) through competition for collagenous ligands. Children with SMA had increased circulating levels of sLAIR1 and sLAIR2 indicative of enhanced receptor shedding. C1q was reduced in children with SMA, thereby, limiting ligand availability. Phagocytosis of Pf Hz antagonizes LAIR1 signalling through down-regulation of LAIR1 ITIM and SHP-1 phosphorylation, but does not alter SHP-2 phosphorylation. Leukocytic ingestion of Pf Hz also decreases LAIR1 transcripts and protein. These events result in NF-κB activation and the consequent production of pro-inflammatory mediators that enhance the pathogenesis of SMA. Red arrows represent ligand-receptor interaction, black solid arrows represent activation, and dashed black arrows represent blockade. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: C1q was measured in serum using matched antibody pair (Cat No. H00000712-AP21) and human recombinant C1q protein (Cat No. H00000712-P01) (Novus Biologicals, 8100 Southpark Way # A8, Littleton, CO 80120).

Techniques: Phospho-proteomics, Activity Assay, Blocking Assay, Activation Assay, Translocation Assay, De-Phosphorylation Assay, Binding Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function

doi: 10.3390/ijms22052646

Figure Lengend Snippet: Effects of serum heat-inactivation on complement-bearing immune complexes, complement (co-)factors, and regulators. ( Center ) The depicted scheme represents the initiating steps and essential (co-)factors and regulators of the classical and alternative complement cascade as well as the cleavage pathway of complement factor C3. ( A – G ) Concentrations of complement-bearing immune complexes ( A ) C1q-IgG and ( B ) C3d-IgG. ( C ) The concentration and ( D ) activity of complement regulator C1 inhibitor as well as the concentration of complement (co-)factors ( E ) C3c, ( F ) C4, and ( G ) factor B were determined in native sera (blue dots) and after heat-inactivation (red dots). (median of (A) native n = 17, heat-inactivated (HI) n = 16; (B,E,F,G) n =11; (C) native n = 15, HI n = 14; (D) native n = 8, HI n = 7; Wilcoxon test; * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: In order to induce complex formation with serum contained C1q and therewith imitate the effect of heat-inactivation, we added a final concentration of 50 μg/mL anti-human anti-C1q antibody (NB100-64420, Novus Biologicals, Centennial, CO, USA) to cells cultured in native sera.

Techniques: Concentration Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function

doi: 10.3390/ijms22052646

Figure Lengend Snippet: Effects of heat-inactivation are not related to Fcγ receptors II (CD32) and III (CD16) expression but are partially reverted by C1 inhibitor and mimicked by anti-C1q antibody supplementation. ( A , B ) Histogram plots display expression of CD16, CD32, and CD64 (opaque colors) compared to isotype controls (black) and unstained cells (transparent color) in native (A) and HI cultured cells (B) after 48 h of stimulation. One representative experiment is shown. ( C , D ) ΔMFI (difference of MFI antibody and MFI isotype) of CD16 ( C ) and CD32 ( D ) is depicted in a time-dependent manner. Shown is the median ± interquartile range of n = 4 (24 h/48 h/72 h/6 d) and n = 2 (0 h). ( E – H ) C1 inhibitor was added to T cells cultured with heat inactivated serum. In the presence of C1 inhibitor IFNγ ( E ), TNF ( F ), CD28 ( G ), and CD69 ( H ) were analyzed at indicated time points. (Shown is the median of n = 6, each dot represents one donor, Friedman test, post-hoc Dunn’s, * p < 0.05, ** p < 0.01). ( I – K ) Anti-C1q antibody was added to T cells cultured with native sera. TNF secretion ( I ) was measured by ELISA and CD28 ( J ) and CD69 ( K ) expression was analyzed by flow cytometry at indicated time points. (Shown is the median of n = 13; Friedman test, post-hoc Dunn’s, * p < 0.05 ** p < 0.01; *** p < 0.001).

Article Snippet: In order to induce complex formation with serum contained C1q and therewith imitate the effect of heat-inactivation, we added a final concentration of 50 μg/mL anti-human anti-C1q antibody (NB100-64420, Novus Biologicals, Centennial, CO, USA) to cells cultured in native sera.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function

doi: 10.3390/ijms22052646

Figure Lengend Snippet: Schematic illustration of the effects of HI, HI + C1inh, and native + anti-C1q on human CD4+ T cells compared to cells in native serum. Effects are analyzed after 72 h (HI, HI + C1inh) and 48 h (native + anti-C1q), respectively. Each arrow represents a significantly increased (↑), significantly decreased (↓) effect or no significant effect (↔). Effects being also induced by the carrier solution are depicted as no significant effect.

Article Snippet: In order to induce complex formation with serum contained C1q and therewith imitate the effect of heat-inactivation, we added a final concentration of 50 μg/mL anti-human anti-C1q antibody (NB100-64420, Novus Biologicals, Centennial, CO, USA) to cells cultured in native sera.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Heat-Inactivation of Human Serum Destroys C1 Inhibitor, Pro-motes Immune Complex Formation, and Improves Human T Cell Function

doi: 10.3390/ijms22052646

Figure Lengend Snippet: Tests, equipment, and methods used for determination of heat-induced changes.

Article Snippet: In order to induce complex formation with serum contained C1q and therewith imitate the effect of heat-inactivation, we added a final concentration of 50 μg/mL anti-human anti-C1q antibody (NB100-64420, Novus Biologicals, Centennial, CO, USA) to cells cultured in native sera.

Techniques: Activity Assay, Binding Assay, Concentration Assay, Electrophoresis

Journal: Scientific reports

Article Title: Complement classical and alternative pathway activation contributes to diabetic kidney disease progression: a glomerular proteomics on kidney biopsies.

doi: 10.1038/s41598-024-84900-4

Figure Lengend Snippet: Fig. 3. Immunohistochemistry (IHC) staining for C1q, C3, C4, C5b-9, CFB, CFH, MBL, and MASP. (a–e) Representative images of C1q, C3, C4, C5b-9, and CFB staining showing a GBM pattern of immunostaining in kidney tissues from patients with DKD. (f) Immunohistochemical diagram of CFH in kidneys with DKD depicting mesangial deposition. (g, h) Representative images of MBL and MASP staining showing no deposition was found in the glomeruli of kidneys with DKD. Scale bars represent 50 μm. CFB, complement factor B; CFH, complement factor H; MBL, mannose-binding lectin; MASP, mannose-binding lectin- associated serine protease; GBM, glomerular basement membrane; DKD, diabetic kidney disease.

Article Snippet: After washing, sections were incubated with either anti-C1q antibodies (1:200 dilution, 16889-1-AP; Proteintech), anti-C3 antibodies (1:200 dilution, ab200999; Abcam), anti-C4 antibodies (1:300 dilution, 22233-1-AP; Proteintech), anti-C5b-9 antibodies (1:400 dilution, ab55811; Abcam), anti-MBL antibodies (1:200 dilution, ab23457; Abcam), anti-MASP1 antibodies (1:400 dilution, 21837-1-AP; Proteintech), anti-CFB antibodies (1:50 dilution, ab192577; Abcam), or anti-CFH antibodies (1:50 dilution, ab170036; Abcam) overnight at 4 °C in a moist chamber.

Techniques: Immunohistochemistry, Staining, Immunostaining, Immunohistochemical staining, Binding Assay, Membrane

Journal: Environmental Science and Pollution Research International

Article Title: Does ( −)-epigallocatechin-3-gallate protect the neurotoxicity induced by bisphenol A in vivo?

doi: 10.1007/s11356-021-18408-z

Figure Lengend Snippet: Effect of EGCG pre-treatment and BPA exposure on the oxidant/antioxidant balance in rats’ hippocampi. A SOD activity (µmol /min/g tissue), B circulating adiponectin (µg/ml), C NO concentration (µmol/g tissue), and D MDA concentration (nmol/g tissue). Rats were treated with corn oil (0.6 ml/kg/day, P.O.; Co), ( −)-epigallocatechin-3-gallate (10 mg EGCG/kg, P.O.; EGCG), bisphenol A (25 mg/kg, P.O.; BPA), and EGCG 2 h before BPA (EGCG + BPA) once every day for eight weeks. Data are presented as mean ± SD; One-way ANOVA followed by Tukey’s honestly significant difference (HSD) post-hoc; n = 7 per group. *, $ Different symbols indicate significant difference ( p < 0.05) vs. Co group or EGCG group, respectively

Article Snippet: Rat ELISA Kit for adiponectin (Cat No: EK1327) was obtained from Boster Biological Technology Co. (Ltd., USA).

Techniques: Activity Assay, Concentration Assay

Journal: Molecular Medicine Reports

Article Title: Proteomics analysis of lung tissue reveals protein makers for the lung injury of adjuvant arthritis rats

doi: 10.3892/mmr.2023.13051

Figure Lengend Snippet: Immunofluorescence results for C1qc, C9 and Clu (×400). (A) Immunofluorescence expression of C1qc. (B) Immunofluorescence expression of C9. (C) Immunofluorescence expression of Clu. Quantitative analysis of immunofluorescence results of (D) C1qc, (E) C9 and (F) Clu proteins. Relative expression of (G) C1qc, (H) C9 and (I) Clu proteins were analyzed by proteomic analysis. *P<0.05 or **P<0.01 vs. CN group in rats. C, complement; CN, control; Clu, clusterin.

Article Snippet: The sections were incubated with primary antibodies against C1qc (1:100; cat. no. A05666; Boster Biological Technology), C9 (1:100; cat. no. K107807P; Beijing Solarbio Science and Technology Co., Ltd.), Clu (1:100; cat. no. 12289-1-AP; Proteintech Group, Inc.) at 4°C overnight and washed with PBS 3 times (5 min each).

Techniques: Immunofluorescence, Expressing, Control