Journal: Frontiers in Immunology

Article Title: Complement activation and cellular inflammation in Fabry disease patients despite enzyme replacement therapy

doi: 10.3389/fimmu.2024.1307558

Figure Lengend Snippet: Serum concentrations of C3a and C5a in FD patients. (A) C3a and (B) C5a concentrations (ng/ml) in serum samples from healthy controls (n=18) and treatment-naiüve (n=24) FD patients. (C) C3a and (D) C5a concentrations (ng/ml) in serum samples from healthy controls (n=18), treatment-naiüve FD patients and from the same patients under ERT (n=17). Data were tested for normal distribution by Kolmogorov-Smirnov test. Differences between groups were determined by Mann-Whitney test (A, B) when data were not normally distributed or Ordinary one-way ANOVA with Tukey’s posthoc test (C, D) in case of normal distribution. Data in A, B are shown as median and range with 25th and 75th percentile. Data in (C, D) are shown as mean + SEM (C, D) . *p<0.05, **p<0.01; ***p<0.001; ****p<0.0001. (E, F) Percentage of serum samples from healthy controls (white bar, n=18), treatment-naiüve FD patients (black bar, n=17) and from the same patients under ERT (grey bar, n=17) with levels of (E) ≤5,000 ng/ml or >5,000 ng/ml of C3a or (F) ≤20 ng/ml, 21-35 ng/ml or >35 ng/ml C5a in serum. (G, H) Linear regression analysis of C5a and C3a serum levels (ng/ml) in FD patients before (G) and after (H) ERT (n=17). R 2 is the coefficient of determination of linear regression and ranges from 0 (no linear regression) to 1 (perfect linear regression).

Article Snippet: For quantification of complement cleavage products C3a/C3a-desArg (C3a) as well as C5a/C5a-desArg (C5a) in serum, commercial C3a (HK354, Hycult Biotech, Uden, Netherlands) and C5a (HK349, Hycult Biotech, Uden, Netherlands) human enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer’s instructions.

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Complement activation and cellular inflammation in Fabry disease patients despite enzyme replacement therapy

doi: 10.3389/fimmu.2024.1307558

Figure Lengend Snippet: C3a, C5a and lyso-Gb3 concentrations in the circulation of FD patients with missense/nonsense mutations. (A–D) C3a, (E–H) C5a and (I–L) lyso-Gb3 concentrations (ng/ml) in samples from (A, E, I) treatment-naiüve (n=17), (B, F, J) FD patients under ERT (n=17) with missense (n=9) or nonsense (n=8) GLA mutations and from FD patients with (C, G, K) missense (n=9) or (D, H, L) nonsense (n=8) GLA mutations before (V0) or under ERT (V1). Data were tested for normal distribution by Kolmogorov-Smirnov test. Differences between groups were determined by unpaired t-test (A, B, E, F, I, J ) or paired t- test (C, D, G, H, K, L) . Data in (A, B, E, F, I, J) are shown as mean + SEM. *p<0.05; ***p<0.001.

Article Snippet: For quantification of complement cleavage products C3a/C3a-desArg (C3a) as well as C5a/C5a-desArg (C5a) in serum, commercial C3a (HK354, Hycult Biotech, Uden, Netherlands) and C5a (HK349, Hycult Biotech, Uden, Netherlands) human enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in Immunology

Article Title: Complement activation and cellular inflammation in Fabry disease patients despite enzyme replacement therapy

doi: 10.3389/fimmu.2024.1307558

Figure Lengend Snippet: C3a, C5a and lyso-Gb3 concentrations in the circulation of FD patients with ADA-negative/positive status. (A–D) C3a, (E–H) C5a and (I–L) lyso-Gb3 concentrations (ng/ml) in serum from ADA-negative (n=6) or ADA-positive (n=11) (A, E, I) patients, who were either treatment-naiüve (n=17) or (B, F, J) received ERT (n=17). (C, G, K) ADA-negative (n=6) or (D, H, L) ADA-positive (n=11) FD patients before (V0) or under ERT (V1). Red lines denote patients with missense mutations. Data were tested for normal distribution by Kolmogorov-Smirnov test. Differences between groups were determined by unpaired t-test (A, B, E, F, I, J) or paired t-test (C, D, G, H, K, L) . Data in (A, B, E, F, I, J) are shown as mean + SEM. *p<0.05; ***p<0.001.

Article Snippet: For quantification of complement cleavage products C3a/C3a-desArg (C3a) as well as C5a/C5a-desArg (C5a) in serum, commercial C3a (HK354, Hycult Biotech, Uden, Netherlands) and C5a (HK349, Hycult Biotech, Uden, Netherlands) human enzyme-linked immunosorbent assay (ELISA) kits were used according to the manufacturer’s instructions.

Techniques:

Journal: Cells

Article Title: House Dust Mite Nebulization Drives Alarmin and Complement Activation in a Murine Tracheal Air–Liquid Interface Culture System

doi: 10.3390/cells14201598

Figure Lengend Snippet: Time-dependent complement activation and anaphylatoxin receptor expression in ALI cultures exposed to HDM. ( a ) C3 and C3a concentrations in cell lysates and the SN of unstimulated (steady-state) ALI cultures and 24, 48 and 72 h after HDM exposure (steady-state, 24, 48 and 72 h: n = 6). * p < 0.05; ** p < 0.01; *** p < 0.001. ( b ) HDM and HI-HDM mediated cleavage of hC3 into hC3a ( n = 3 independent experiments). Data are shown as mean ± SEM. Data were analyzed using an unpaired T -test. *** p < 0.001. ( c ) C5 and C5a concentrations in cell lysates and the SN of steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). ( d ) Comparison of C3 and C5 ( left panel ), C3a and C5a ( right panel ) concentrations in cell lysates from steady-state ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM (steady-state, 24, 48 and 72 h: n = 6). Data were analyzed using two-way ANOVA with Šidák’s multiple comparisons test. **** p < 0.0001. ( e ) Immunofluorescence analysis of C3aR and C5aR1 expression in unstimulated ALI cultures and 24, 48 and 72 h after HDM exposure. Data are shown as mean ± SEM ( n = 3). Data were analyzed using one-way ANOVA with Holm–Šidák’s multiple comparisons test. * p < 0.05; ** p < 0.01. The number of experiments shown in ( a , c – e ) refers to biological replicates derived from 6 independent preparations of trachea, each representing a separate cell isolation and culture.

Article Snippet: Briefly, we used neo-epitope-specific anti-hC3a mAb (Hycult Biotech #HM2074—Uden, The Netherlands) as capture and biotinylated anti-hC3/C3a mAb (Hycult Biotech #HM2073—Uden, The Netherlands) as detection antibody.

Techniques: Activation Assay, Expressing, Comparison, Immunofluorescence, Derivative Assay, Cell Isolation

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: hTLQP-2 and mTLQP-21 activate ERK signalling in CHO-K1 and CHO-C3aR cells. hTLQP-21, mTLQP-21 and plasma-derived human C3a were tested in (A) non-transfected CHO-K1 or (B) CHO cells stably expressing human C3aR. CHO cells were serum-starved overnight and then stimulated with various ligands for 10 min before being lysed. The phospho-ERK1/2 content in the lysate was measured and expressed as fold-baseline before being combined. The maximum relative pERK1/2 activity induced by each ligand is shown in (C) . Data represent mean ± S.E.M. of triplicate measurements from 3-4 independent experiments (n = 3-4). Two-way ANOVA with Dunnett’s post hoc analysis. * P < 0.05, *** P < 0.001, **** P < 0.0001. Ligand treated versus medium treated cells for each cell line.

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Derivative Assay, Transfection, Stable Transfection, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: Summary of potencies and activities of TLQP-21 tested on CHO-C3aR, HMDM and BMDM.

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: TLQP-21 triggers ERK signalling through C3aR in murine bone marrow-derive macrophages. BMDMs (90,000/well) from wildtype mice (A) or with C3aR knockout mice (B) , were serum-starved overnight and then stimulated with respective ligands at the indicated concentrations for 10 min. The phospho-ERK1/2 content in the cell lysate was measured and normalised to the medium only-treated levels before being combined. Data represent mean ± S.E.M. of triplicate measurements using cells from 3 mice (n = 3).

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Knock-Out

Journal: Experimental and Therapeutic Medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Complement C3a and C5a aggravate inflammation in HRPE cells via the NF-κB signaling pathway. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) or C5a (1 µg/ml) with or without NF-κB inhibitor PDTC (10 µM), and the release of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) PGE2 and (E) IL-10 was measured by ELISA. *** P<0.001 relative to control; ### P<0.001 relative to C3a or C5a treatment. PGE2, prostaglandin E2; HRPE, human retinal pigment epithelium.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufacturers' protocols: Tumor Necrosis Factor-α Assay Kit (cat. no. H052-1), Interleukin-1β Assay Kit (cat. no. H002), Interleukin-6 Assay Kit (cat. no. H007-1-1) and Interleukin-10 Assay Kit (cat. no. H009-1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E-EL-0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB-E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB-E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Complement C3aR and C5aR antagonist inhibit inflammation and NF-κB signaling in HRPE cells challenged with complement C3a and C5a. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM), and the release of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) PGE2 and (E) IL-10 was determined by ELISA. HRPE cells were treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM), and the release of (F) TNF-α, (G) IL-1β, (H) IL-6, (I) PGE2 and (J) IL-10 was determined by ELISA. (K) The phosphorylation of NF-κB and expression of NF-κB in HRPE cells treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM) were determined by western blot. (L) The phosphorylation of NF-κB and expression of NF-κB in HRPE cells treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM) were determined by western blot. *** P<0.001 relative to control; ### P<0.001 relative to C3a or C5a treatment. p-NF-κB, phosphorylated NF-κB; C5aR, C5a receptor; HRPE, human retinal pigment epithelium; CCX, CCX168; SB, SB290157.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufacturers' protocols: Tumor Necrosis Factor-α Assay Kit (cat. no. H052-1), Interleukin-1β Assay Kit (cat. no. H002), Interleukin-6 Assay Kit (cat. no. H007-1-1) and Interleukin-10 Assay Kit (cat. no. H009-1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E-EL-0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB-E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB-E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Expressing, Western Blot, Control

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: C3A viability and LC 50 values of the 40 and 80 nm a AuNP and b PC AuNP. Data represent mean ± S.D. ( n = 3). PC human plasma protein corona, ND not determined, BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, LC 50 median lethal concentration

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time-dependent cellular uptake of the 40 nm a BPEI-AuNP, b LA-AuNP, and c PEG-AuNP, and the 80 nm d BPEI-AuNP, e LA-AuNP, and f PEG-AuNP in the absence and presence of PC in C3A cells up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona, MSD minimum significant difference. * p < 0.05; ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time- and concentration-dependent ROS/RNS production in C3A cells exposed to a the 40 nm BPEI-AuNP and b the 40 nm PEG-AuNP up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, CTRL control, MSD a minimum significant difference, PCN pyocyanin (ROS inducer). ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Concentration Assay, Control

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: An inhibitory effect of AuNP on CYP3A4 activity in C3A cells exposed to the 40 and 80 nm BPEI-, LA-, and PEG-AuNP in the absence and presence of PC for 24 h. Values represent mean ± S.D. ( n = 3). BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Activity Assay, Clinical Proteomics

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 2: Te concentrations of complement pyrolysis products were found higher in pleural efusion than in plasma from TPE patients. (a) Complement pyrolysis products, including C3a, C3b, C3d, C5a, and opsonin receptors (CR1 and CR3) were detected in human tuberculosis pleural biopsy samples by immunohistochemistry. (Original magnifcation, ×200) (n 4). (b) Higher levels of complement pyrolysis products were found in pleural efusion than in plasma in TPE patients (n 20). Te concentrations of complement pyrolysis products in pleural fuid and plasma from TPE patients were measured by ELISA (n 20).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

Techniques: Clinical Proteomics, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 3: Monocyte migration was inhibited by antibodies that blocked CXCL12. (a) CXCL12 and CXCR4 staining by immunohisto- chemistry in human pleural biopsy (original magnifcation, ×200) (n 4). (b) Te concentration of CXCL12 in pleural fuid and plasma from TPE patients was measured by ELISA (n 20). (c) CXCL12 produced by PMCs was measured by PCR and ELISA after Mpt64 and anaphylatoxin activation. PMCs were incubated for 24 hours in control media or in media with Mpt64 (20 μg/ml) and with or without human C3a (100 nM) or C3aRA (100 nM) (n 4). (d) Coexpression of CXCL12-CXCR4 in PMCs and monocytes from TPE was detected by immunofuorescence (original magnifcation, ×400) (n 4). (e) Monocytes were seeded into the top chamber of a transwell system, and the supernatant from PMCs cultured with anti-CXCL12 antibody or PBS were placed in the bottom chamber. Te migratory index was calculated by dividing the number of monocytes that migrated in response to the supernatants from cultured PMCs by the number of monocytes that migrated in response to the control. ∗vs the MO-PBS group, ∗∗P < 0.01. #vs the MO-PMC group, ##P < 0.01 (n 4).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

Techniques: Migration, Staining, Immunohistochemistry, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Produced, Activation Assay, Incubation, Control, Cell Culture