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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Complement Is Required for Microbe-Driven Induction of Th17 and Periodontitis.

doi: 10.4049/jimmunol.2200338

Figure Lengend Snippet: FIGURE 5. C3a-induced IL-6 release in human gingival epithelial cells. (A) Human immortalized gingival keratinocytes (HIGKs) were stimulated, or not, with heat-killed P. gingivalis (MOI of 10:1), C3a, or C5a and combinations thereof (at the indicated concentrations). Culture media were collected after a 24-h incubation and assayed for IL-6 by ELISA. (B) HIGKs were stimulated for 24 h with heat-killed P. gingivalis (MOI of 10:1) or Pam3Cys lipopeptide (1 mg/ml) and C3aR expression was measured by FACS. Representative histogram (left) and bar graphs for mean fluorescence intensity (MFI) of C3aR expression (right). (C) HIGKs were stimulated, or not, with C3a (500 ng/ml), Pam3Cys (1 mg/ml), or both, and IL-6 was measured in collected culture supernatants after a 24-h incubation. (D) HIGKs were stimulated for 24 h, or not, with P. gingivalis (MOI of 10:1) alone or with C3a (500 ng/ml), in the presence or absence of 10 mg/ml anti-TLR2 neutralizing Ab or isotype control (IC), which were added 2 h prior to stimulation. IL-6 release was assayed by ELISA. (E) HIGKs were pretreated with PD98059 (10 mM; MEK/ERK inhibitor), SP600125 (50mM; JNK inhibitor), SB202190 (20mM; p38 MAPK inhibitor), or SN50 (50mM, NF-kB inhibi- tor). After 1 h, Pam3Cys (1 mg/ml) was added in the cultures and C3aR expression (MFI) was determined by FACS after a 24-h incubation. (F) HIGKs were stimulated with Pam3Cys (1 mg/ml) for the indicated time lengths. Total protein was extracted and immunoblot analysis was performed with specific Abs against phosphorylated and total ERK1/2, JNK, and p38 MAPK as well as against GAPDH (loading control). (G) HIGKs were pretreated with Pam3Cys (1 mg/ml) for 4 h and then exposed to PD98059 (10 mM; MEK/ERK inhibitor), SP600125 (50mM; JNK inhibitor), SB202190 (20mM; p38 MAPK inhibitor), or SN50 (50mM, NF-kB inhibitor). After 1 h, C3a (500 ng/ml) was added in the cultures. Culture media were collected after 24 h and assayed for IL-6 by ELISA. Data are means ± SD (n 5 6 cultures per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (A, D, E, and G, one-way ANOVA and a Tukey’s test; B and C, Dunnett’s multiple comparison tests).

Article Snippet: The cells were seeded into 96-well plates at a density of 2 × 105 cells per well for 18 h and then challenged with heat-killed (65◦C, 1 h) P. gingivalis at a multiplicity of infection (MOI) of 10:1, in the presence or absence of different concentrations of recombinant human C3a or C5a (catalog no. 3677-C3-025 or 2037- C5-025/CF, R&D Systems) or the synthetic microbial lipopeptide Pam3Cys (catalog no. tlrl-pms, InvivoGen, San Diego, CA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Control, Western Blot, Comparison