Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: Human keratinocytes produce MIG/CXCL9 in response to inflammatory stimuli. A , HaCaT cells were stimulated with IFN-γ (100 units/ml). At indicated time points, RNA was isolated from the cells and MIG/CXCL9 gene expression quantified using Q-PCR. The data shown represent relative MIG/CXCL9 gene expression, where the expression at 18 h is set to a value of 1. GAPDH was used as housekeeping gene to normalize the values obtained, and each sample was analyzed in triplicate. B , MIG/CXCL9 peptide concentration in medium from HaCaT cells used for RNA isolation in A . Supernatants were collected, and the MIG/CXCL9 content was measured by ELISA. C , HaCaT cells were stimulated with IFN-γ (100 units/ml) in presence of F. magna strains 505, ALB8, and CK05 (expressing a truncated inactive form of SufA), or the S. pyogenes strain AP1 (2 × 10 7 cfu/ml). RNA was isolated from the cells, and the MIG/CXCL9 gene expression quantified using Q-PCR. Relative gene expression of MIG/CXCL9 is shown and the expression, at the individual time points, are compared with cells stimulated with IFN-γ alone, where the values was set to 1 at respective time points.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques: Isolation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: SufA-expressing strains of F. magna degrade MIG/CXCL9 produced by IFN-γ-stimulated keratinocytes. HaCaT cells were stimulated with IFN-γ alone (100 units/ml) or together with F. magna strains 505, ALB8, and CK05 (2 × 10 7 cfu). After 24 h, the cell culture media were collected, precipitated, and analyzed by Western blot using a polyclonal rabbit antibody against human MIG/CXCL9 (0.2 μg/ml). Recombinant MIG/CXCL9 (rMIG, 100 ng) was used as control.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques: Expressing, Produced, Cell Culture, Western Blot, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: Identification of the SufA cleavage sites in MIG/CXCL9. A , recombinant MIG/CXCL9 (3 μg) was incubated with buffer or SufA (100 ng) for 3 h at 37 °C. Thereafter, samples were run on a 16.5% Tris-Tricine gel. Lane 1 , intact MIG/CXCL9; lane 2 , SufA degraded MIG/CXCL9; lane 3 , SufA alone. Protein bands in lane 2 were excised and analyzed using MS and MS/MS. B , visualization of a homology model of MIG/CXCL9 using NAP-2/CXCL7 as a template. β-Strands are shown in yellow , α-helices in purple , 3 -helices in blue , and loops in turquoise . The amino acid sequences shown correspond to the flexible NH 2 -terminal and COOH-terminal ends of MIG/CXCL9 that are not included in the model. Arrowheads indicate predicted cleavage sites for SufA. C , amino acid sequence of MIG/CXCL9 holopeptide is shown at the top , with the identified bactericidal region in bold/underlined . The sequences shown below correspond to the peptide sequences present in the SufA-cleaved MIG/CXCL9, as determined by MS and MS/MS analysis. From the peptide masses matching with the MIG/CXCL9 sequence, the tools “Peptidecutter” and “Findpept” were used to search for experimentally (Asp-N + N-terminal Glu) as well as SufA-generated cleavage sites. Peptides matching a known cleavage site in one terminal and an unknown site in the other terminal were considered as relevant, if identified in three successive experiments. Results were further confirmed by MS/MS identification of sequence stretches within the different fragments. Arrowheads point at predicted cleavage sites for SufA.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques: Recombinant, Incubation, Tandem Mass Spectroscopy, Sequencing, Generated

Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: Antibacterial effect of SufA degraded MIG/CXCL9. Recombinant MIG/CXCL9 (3 μg) was incubated with SufA (100 ng) for 3 h at 37 °C. Indicated concentrations of intact and SufA-degraded MIG/CXCL9 were then incubated with 50 μl of a bacterial solution (2 × 10 6 cfu/ml) for 1 h at 37 °C. White bars , F. magna 505; black bars , S. pyogenes AP1. Bacterial killing (percent) was determined by comparing the number of bacterial colonies after exposure to MIG/CXCL9 with the number obtained after incubation in buffer alone. Data are values from three separate experiments.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques: Recombinant, Incubation

Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: Electron micrographs of S. pyogenes and F. magna incubated with MIG/CXCL9. F. magna 505 ( A and B ) and S. pyogenes AP1 ( C and D ) (2 × 10 9 cfu/ml) were incubated with MIG/CXCL9 ( A and C ), and with SufA-degraded MIG/CXCL9 ( B and D ) at a concentration of 0.36 μ m for 1 h at 37 °C. Following incubation, the bacteria were prepared for ultrathin sectioning/immunoelectron microscopy. Sections were incubated with polyclonal rabbit anti-human MIG/CXCL9 (1 μg/ml), followed by goat anti-rabbit IgG (1 μg/ml) labeled with colloidal gold (10 nm). The bar represents 100 nm.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques: Incubation, Concentration Assay, Immuno-Electron Microscopy, Labeling

Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: MIG/CXCL9 causes liposome leakage. Membrane-disruptive effects of intact MIG/CXCL9, SufA-degraded MIG/CXCL9, SufA alone, and LL-37 were analyzed by measuring the release of CF from liposomes. A , leakage induced by various peptide concentrations in 10 m m Tris-HCl buffer, pH 7.4, containing 5 m m glucose. B , kinetics of peptide induced liposome leakage. At time t = 0 s, 1 μ m peptide was added.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: SufA-degraded MIG/CXCL9 activates CXCR3. The murine pre-B cell line L1.2, stably expressing the human MIG/CXCL9 receptor CXCR3, was loaded with a calcium-sensitive fluorophore. The rise in intracellular [Ca 2+ ] was analyzed after application of the peptide. Lane 1 , MIG/CXCL9 (100 n m ); lane 2 , SufA-degraded MIG/CXCL9 (MIG/CXCL9 100 n m ; SufA 1.5 n m ); lane 3 , SufA (1.5 n m ). Data are represented as mean peak values ± S.D. values from three separate experiments.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques: Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation *

doi: 10.1074/jbc.M109.025957

Figure Lengend Snippet: SufA releases an NH 2 -terminal fragment of FAF from the ALB8 surface. A , 10 μg of recombinant full-length FAF, and the NH 2 -terminal and COOH-terminal FAF fragments, respectively, were incubated with SufA (100 ng) or buffer for 18 h at 37 °C and then separated by SDS-PAGE. The band indicated with a star was excised and subjected to NH 2 -terminal amino acid sequencing. The sequence obtained (GPLSG) corresponds to the residual amino acids of GST, in fusion with the NH 2 terminus of FAF, following Prescission proteinase removal of the GST tag. B , ALB8 bacteria were washed in 20 m m Tris-HCl, pH 7.5, and adjusted to 2 × 10 9 cfu/ml. Bacteria were incubated at 4 °C with buffer or at 37 °C with buffer, 0.5 μg SufA, or SufA+PMSF (5 m m ) for 3 h, and then vortexed and centrifuged. The resulting supernatants were separated by SDS-PAGE and blotted to an Immobilon-P membrane. FAF was detected with polyclonal rabbit anti-FAF antiserum (1:1000). C , investigation of the affinity between MIG/CXCL9 and protein FAF utilizing surface plasmon resonance. Protein FAF was immobilized and MIG/CXCL9 at concentrations ranging from 30–500 n m was injected over the surface of the chip. The affinity was determined to 4.9 n m . D , bactericidal effect of MIG/CXCL9 at various concentrations was tested against F. magna 505 (2 × 10 6 cfu/ml). Following incubation for 1 h at 37 °C, cfus were determined. Data are represented as mean ± S.D. values from three separate experiments. E , bactericidal effect of MIG/CXCL9 at a concentration of 0.28 μ m was inhibited with various concentrations of protein FAF. Data are represented as mean ± S.D. values from three separate experiments.

Article Snippet: Recombinant human IFN-γ, MIG/CXCL9, and affinity-purified polyclonal rabbit antibodies against human MIG/CXCL9 were from Peprotech.

Techniques: Recombinant, Incubation, SDS Page, Sequencing, SPR Assay, Injection, Concentration Assay

Journal: Molecular Pain

Article Title: Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury

doi: 10.1177/1744806918777401

Figure Lengend Snippet: Primer sets used in qPCR.

Article Snippet: The sections were first blocked with 5% donkey serum for 2 h at room temperature, then incubated overnight at 4°C with the following primary antibodies: CXCL9 (Rabbit, 1:500, Bio-Rad), CXCL11 (Rabbit, 1:500, Bio-Rad), glial fibrillary acidic protein (GFAP; mouse, 1:5000, Millipore), NeuN (mouse, 1:1000, Millipore), and CD11b (mouse, 1:100, Serotec).

Techniques: Sequencing

Journal: Molecular Pain

Article Title: Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury

doi: 10.1177/1744806918777401

Figure Lengend Snippet: The CXCL9 expression is increased in spinal astrocytes after SNL. (a) Time course of Cxcl9 mRNA expression in the ipsilateral dorsal horn in naive, sham-operated, and SNL mice. Cxcl9 expression was significantly increased at 3, 10, and 21 days in SNL mice. *p < 0.05, **p < 0.01, compared with sham-operated mice. Student's t test. n = 5 mice/group. (b) ELISA shows the increase of CXCL9 protein in the spinal cord 10 days after SNL. *p < 0.05, compared with sham-operated mice. Student’s t test, n = 5 mice/group. (c to d) Representative images of CXCL9 immunofluorescence in the spinal cord from naïve and SNL mice, receptively. CXCL9 was constitutively expressed in naive mice (c), but significantly increased in the ipsilateral dorsal horn 10 days after SNL mice (d). (e) CXCL9-IR was not shown after absorption with CXCL9 peptide. (f to h) Double staining shows the cellular distribution of CXCL9 in the spinal dorsal horn. CXCL9 was sparely colocalized with NeuN (f) or CD11b (g), but highly colocalized with GFAP (h) in the spinal cord 10 days after SNL.

Article Snippet: The sections were first blocked with 5% donkey serum for 2 h at room temperature, then incubated overnight at 4°C with the following primary antibodies: CXCL9 (Rabbit, 1:500, Bio-Rad), CXCL11 (Rabbit, 1:500, Bio-Rad), glial fibrillary acidic protein (GFAP; mouse, 1:5000, Millipore), NeuN (mouse, 1:1000, Millipore), and CD11b (mouse, 1:100, Serotec).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Double Staining

Journal: Molecular Pain

Article Title: Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury

doi: 10.1177/1744806918777401

Figure Lengend Snippet: Inhibition of CXCL9 or CXCL11 does not alleviate SNL-induced neuropathic pain. (a to c) The shRNA targeting Cxcl9 , Cxcl11 , or Cxcl10 reduced the mRNA expression of Cxcl9 (a), Cxcl11 (b), and Cxcl10 (c) in HEK293 cells. (d to e) Intraspinal injection of LV- Cxcl9 shRNA or LV- Cxcl11 shRNA three days after SNL did not change the paw withdrawal latency (d) or threshold (e) at days 10 and 14. p > 0.05, two-way RM ANOVA. n = 6–7 mice/group. (f to g) Intraspinal injection of LV- Cxcl10 shRNA three days after SNL significantly increased the paw withdrawal latency (f) and threshold (g) at days 10 and 14. ** p < 0.01, *** p <0.001, two-way RM ANOVA followed by Bonferroni’s test, n = 5–6 mice/group.

Article Snippet: The sections were first blocked with 5% donkey serum for 2 h at room temperature, then incubated overnight at 4°C with the following primary antibodies: CXCL9 (Rabbit, 1:500, Bio-Rad), CXCL11 (Rabbit, 1:500, Bio-Rad), glial fibrillary acidic protein (GFAP; mouse, 1:5000, Millipore), NeuN (mouse, 1:1000, Millipore), and CD11b (mouse, 1:100, Serotec).

Techniques: Inhibition, shRNA, Expressing, Injection

Journal: Molecular Pain

Article Title: Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury

doi: 10.1177/1744806918777401

Figure Lengend Snippet: CXCL9 and CXCL11 do not induce pain hypersensitivity after intrathecal injection. (a, b) Intrathecal injection of CXCL9 and CXCL11 (100 ng) did not induce heat hyperalgesia (a) or mechanical allodynia (b) in naive mice. p > 0.05, two-way RM ANOVA. n = 6 mice/group. (c, d) Intrathecal injection of CXCL10 (100 ng) induced heat hyperalgesia (C) at 1, 3, and 6 h and mechanical allodynia (d) at 1 h and 3 h in naive mice. *** p < 0.001, two-way RM ANOVA followed by Bonferroni’s test, n = 6–7 mice/group. (e) pERK expression in the spinal cord did not change 1 h after intrathecal injection of CXCL9 or CXCL11 in naive mice. p > 0.05, Student's t test, n = 4 mice/group.

Article Snippet: The sections were first blocked with 5% donkey serum for 2 h at room temperature, then incubated overnight at 4°C with the following primary antibodies: CXCL9 (Rabbit, 1:500, Bio-Rad), CXCL11 (Rabbit, 1:500, Bio-Rad), glial fibrillary acidic protein (GFAP; mouse, 1:5000, Millipore), NeuN (mouse, 1:1000, Millipore), and CD11b (mouse, 1:100, Serotec).

Techniques: Injection, Expressing

Journal: Molecular Pain

Article Title: Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury

doi: 10.1177/1744806918777401

Figure Lengend Snippet: CXCL9 and CXCL11 enhance excitatory synaptic transmission in lamina II neurons. (a) Whole-cell patch clamp recording of sEPSCs shows an increase in the frequency of sEPSCs after perfusion of CXCL9 (100 ng/ml, 2 min). However, the amplitude was not changed after perfusion of CXCL9. a1 and a2 are enlarged traces before and after CXCL9 treatment, respectively. ***p < 0.001 versus pretreatment baseline, Student’s t test, n = 5–6 mice/group. (b) sEPSCs frequency not amplitude is increased after perfusion of CXCL11 (100 ng/ml, 2 min). b1 and b2 are enlarged traces before and after CXCL11 treatment, respectively. **p < 0.01 versus pretreatment baseline, Student’s t test, n = 5–6 mice/group. (c) Miniature EPSCs (mEPSCs) were recorded in the present of TTX (500 nM). The mEPSCs frequency, not the amplitude is increased after perfusion of CXCL9 (100 ng/ml, 2 min). c1 and c2 are enlarged traces before and after CXCL9 treatment, respectively. *p < 0.05 versus pretreatment baseline, Student’s t test, n = 3 mice/group. (d) mEPSCs frequency, not the amplitude is increased after incubation of CXCL11. d1 and d2 are enlarged traces before and after CXCL11 treatment, respectively. * p < 0.05 versus pretreatment baseline, Student's t test, n = 3 mice/group.

Article Snippet: The sections were first blocked with 5% donkey serum for 2 h at room temperature, then incubated overnight at 4°C with the following primary antibodies: CXCL9 (Rabbit, 1:500, Bio-Rad), CXCL11 (Rabbit, 1:500, Bio-Rad), glial fibrillary acidic protein (GFAP; mouse, 1:5000, Millipore), NeuN (mouse, 1:1000, Millipore), and CD11b (mouse, 1:100, Serotec).

Techniques: Transmission Assay, Patch Clamp, Incubation

Journal: Molecular Pain

Article Title: Spinal CXCL9 and CXCL11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury

doi: 10.1177/1744806918777401

Figure Lengend Snippet: CXCL9 and CXCL11 enhance inhibitory synaptic transmission in lamina II neurons. (a) Patch clamp recording shows an increase in the frequency but not the amplitude of the sIPSC after perfusion of CXCL9 (100 ng/ml, 2 min). a1 and a2 are enlarged traces before and after CXCL9 treatment. *p < 0.05, Student’s t test, n = 3–4 mice/group. (b) The frequency, not the amplitude of the sIPSCs was increased during perfusion of CXCL11 (100 ng/ml, 2 min). b1 and b2 are enlarged traces before and after CXCL11 treatment. ***p < 0.001, Student’s t test, n = 3–4 mice/group. (c) Neither the frequency nor the amplitude of the sIPSCs was changed after perfusion of CXCL10 (100 ng/ml, 2 min). c1 and c2 are enlarged traces before and after CXCL10 treatment. p > 0.05, Student’s t test, n = 3–4 mice/group.

Article Snippet: The sections were first blocked with 5% donkey serum for 2 h at room temperature, then incubated overnight at 4°C with the following primary antibodies: CXCL9 (Rabbit, 1:500, Bio-Rad), CXCL11 (Rabbit, 1:500, Bio-Rad), glial fibrillary acidic protein (GFAP; mouse, 1:5000, Millipore), NeuN (mouse, 1:1000, Millipore), and CD11b (mouse, 1:100, Serotec).

Techniques: Transmission Assay, Patch Clamp

Journal: Journal of leukocyte biology

Article Title: Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.

doi: 10.1189/jlb.1003524

Figure Lengend Snippet: Fig. 1. CXCL9 and CXCL11 concentrations in synovial fluid of arthritis patients. Synovial fluids of patients suffering from osteoarthritis (OA, ), septic arthritis (SA, e), and crystal arthritis (CA, ‚) were collected, and CXCL9 and CXCL11 concentrations were determined by ELISA. The detec- tion limits for the CXCL9 (0.06 ng/ml) and CXCL11 (0.025 ng/ml) ELISA are indicated. Solid and dotted lines represent mean and median values, respec- tively. Statistical analysis was performed with the Mann-Whitney U-test, and P values are indicated on top of the figures. The scale of the y-axis is logarithmic.

Article Snippet: Microtiter plates (Maxisorp, Nunc-Immuno Plate, Invitrogen) were coated with 1.67 g/ml monoclonal mouse anti-human CXCL9 antibody (Ab; R&D Systems) or with 670 ng/ml polyclonal rabbit anti-human CXCL11 Ab (PeproTech) in phosphate-buffered saline (PBS; pH 7.4), containing 0.05% (v/v) Tween-20 (PBS/Tween) and blocked with 0.1% (w/v) casein in PBS/Tween.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of leukocyte biology

Article Title: Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.

doi: 10.1189/jlb.1003524

Figure Lengend Snippet: Fig. 2. CXCL9 and CXCL11 induction in PBMC or fibroblasts with IFN- or TLR ligands. Human PBMC (A and B) or diploid fibroblasts (C and D) were incubated with IFN- or the TLR ligands PGN, the dsRNA polyrI:rC, LPS, flagellin, or the unmethylated CpG oligonucleotide ODN2006 (ODN). Results represent the mean ( SEM) CXCL9 (A and C) or CXCL11 (B and D) concentration in the culture supernatant (five or more independent experiments). The detection limits of the CXCL9 and CXCL11 ELISA are indicated on the y-axis. Asterisks indicate significant induction of CXCL9 or CXCL11 compared with untreated (Co) cells (Mann-Whitney U-test; *, P 0.01, and **, P 0.001).

Article Snippet: Microtiter plates (Maxisorp, Nunc-Immuno Plate, Invitrogen) were coated with 1.67 g/ml monoclonal mouse anti-human CXCL9 antibody (Ab; R&D Systems) or with 670 ng/ml polyclonal rabbit anti-human CXCL11 Ab (PeproTech) in phosphate-buffered saline (PBS; pH 7.4), containing 0.05% (v/v) Tween-20 (PBS/Tween) and blocked with 0.1% (w/v) casein in PBS/Tween.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of leukocyte biology

Article Title: Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.

doi: 10.1189/jlb.1003524

Figure Lengend Snippet: Fig. 3. CXCL9 and CXCL11 induction in PMBC by IFN- in combination with TLR ligands. Human PBMC were incubated for 24 h with IFN- in combination with a TLR ligand (PGN, the dsRNA polyrI:rC, LPS, flagellin, or the unmethylated CpG oligonucleotide ODN2006). Results represent the mean ( SEM) CXCL9 and CXCL11 concentration in the culture supernatant (three or more independent experiments).

Article Snippet: Microtiter plates (Maxisorp, Nunc-Immuno Plate, Invitrogen) were coated with 1.67 g/ml monoclonal mouse anti-human CXCL9 antibody (Ab; R&D Systems) or with 670 ng/ml polyclonal rabbit anti-human CXCL11 Ab (PeproTech) in phosphate-buffered saline (PBS; pH 7.4), containing 0.05% (v/v) Tween-20 (PBS/Tween) and blocked with 0.1% (w/v) casein in PBS/Tween.

Techniques: Incubation, Concentration Assay

Journal: Journal of leukocyte biology

Article Title: Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.

doi: 10.1189/jlb.1003524

Figure Lengend Snippet: Fig. 4. CXCL9 and CXCL11 induction in fibroblasts by IFN- in combination with TLR ligands. Human diploid fibroblasts were cultured to confluency and incubated for 72 h with IFN- in combination with a TLR ligand (PGN, the dsRNA polyrI:rC, LPS, or flagellin). Results represent the mean ( SEM) CXCL9 and CXCL11 concentration in the culture supernatant (four or more independent experiments).

Article Snippet: Microtiter plates (Maxisorp, Nunc-Immuno Plate, Invitrogen) were coated with 1.67 g/ml monoclonal mouse anti-human CXCL9 antibody (Ab; R&D Systems) or with 670 ng/ml polyclonal rabbit anti-human CXCL11 Ab (PeproTech) in phosphate-buffered saline (PBS; pH 7.4), containing 0.05% (v/v) Tween-20 (PBS/Tween) and blocked with 0.1% (w/v) casein in PBS/Tween.

Techniques: Cell Culture, Incubation, Concentration Assay

Journal: Journal of leukocyte biology

Article Title: Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-gamma in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids.

doi: 10.1189/jlb.1003524

Figure Lengend Snippet: Fig. 5. CXCL9 and CXCL11 induction in fibroblasts by dsRNA in combina- tion with PGN or LPS. Human diploid fibroblasts were cultured to confluency and incubated for 72 h with the dsRNA polyrI:rC in combination with PGN or LPS. Results represent the mean ( SEM) CXCL9 and CXCL11 concentration in the culture supernatant (four or more independent experiments).

Article Snippet: Microtiter plates (Maxisorp, Nunc-Immuno Plate, Invitrogen) were coated with 1.67 g/ml monoclonal mouse anti-human CXCL9 antibody (Ab; R&D Systems) or with 670 ng/ml polyclonal rabbit anti-human CXCL11 Ab (PeproTech) in phosphate-buffered saline (PBS; pH 7.4), containing 0.05% (v/v) Tween-20 (PBS/Tween) and blocked with 0.1% (w/v) casein in PBS/Tween.

Techniques: Cell Culture, Incubation, Concentration Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1 high CD11b + cells elimination in hepatocellular carcinoma

doi: 10.1016/j.apsb.2022.08.006

Figure Lengend Snippet: Anti-PD-L1 antibody enhanced infiltration of CD8 + T cells by increasing proportion of cDC1 cells that secrete CXCL9. (A, B) Flow cytometry plots of CD3 + CD8 + T cells gated on CD45 + cells and CD39 + IFN- γ + gated on CD3 + CD8 + cells in all 6 groups. (C–E) Relative quantification of CD3 + CD8 + T cells gated on CD45 + cells and CD39 + IFN- γ + cells gated on CD3 + CD8 + T cells. (F) Immunofluorescence staining of tumor tissue sections. CD8 + T cells were stained with Alexa Fluor 594, CXCL9 was stained with Alexa Fluor 488. Scale bar = 200 μm. (G) The percentages of CD8 + T cells and CXCL9 + spots in every field of view found in all different 6 groups; three separate fields from each mice and three mice from each group were calculated. ∗ P < 0.05, ∗∗ P < 0.01,∗∗∗ P < 0.001, one-way ANOVA with Tukey's post hoc tests. All data are means ± SEM. (H,I) Representative flow cytometry analysis of the frequency of CXCL9 gated from different cell types in tumor microenvironment. (J, K) Analysis of flow cytometric quantification of CD11c + CD11b − cDC1 cells gated on tumor infiltrating CD45 + cells. (L, M) H22 cells were inoculated subcutaneously into BALB/c mice. 12 days later, cryoablation or microwave-ablation was proceeded, with anti-mouse CXCL9 neutralizing antibody (10 mg/kg) or anti-mouse PD-L1 antibody (10 mg/kg) being infused via tail injection every 3 days. Anti-mouse IgG was used as negative control. Tumor volume and frequency of infiltrated CD8 + T cells were revealed. n = 6 for each groups, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. ns, no significant difference, t- tests. All data are means ± SEM.

Article Snippet: Mouse CXCL9 was stained by Rabbit anti-Mouse FITC-CXCL9 Antibody (CUSABIO, P18340, Wuhan, China).

Techniques: Flow Cytometry, Quantitative Proteomics, Immunofluorescence, Staining, Injection, Negative Control

Journal: Frontiers in Veterinary Science

Article Title: Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis

doi: 10.3389/fvets.2023.1193332

Figure Lengend Snippet: Serial dilutions in triplicate for IFN-γ, IL-2, IL-6, CCL4, CXCL9, and CXCL10. Respective recombinant bovine cytokines were spiked in assay buffer and analyzed using UCP-LFAs. Shown are the ratios ( R ) obtained by dividing the signal at the respective test (T) lines by the signal at the flow control (FC) lines for each recombinant bovine protein. Mean values with error bars (±1 SD) are shown.

Article Snippet: The UCP conjugates for the six tested analytes were prepared to utilize the following antibodies: mouse anti-bovine IFN-γ (MT307; Mabtech, Stockholm, Sweden); mouse anti-bovine IL-2 (MT3B3; Mabtech, Stockholm, Sweden); chicken anti-bovine IL-6 (AHP2380B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL9 (AHP2369B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL10 (AHP2368B; Bio-Rad Laboratories, Hercules, CA, USA); and goat anti-bovine CCL4 (PBB0479B-050; Kingfisher Biotech, St Paul, MN, USA) at a concentration of 25 μg antibody per mg of UCP according to the methods described previously for the development of UCP-LFAs for the detection of M. leprae infection ( ).

Techniques: Recombinant, Control

Journal: Frontiers in Veterinary Science

Article Title: Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis

doi: 10.3389/fvets.2023.1193332

Figure Lengend Snippet: IFN-γ, IL-2, IL-6, CCL4, CXCL9, and CXCL10 levels in PPDb-stimulated whole blood supernatants were measured by UCP-LFAs. The ratios among groups were compared using the Kruskal–Wallis test with Dunn's multiple comparison post-test. N: Naive Animals ( n = 16, empty circles); V: BCG-vaccinated animals ( n = 10, green circles); V/C: BCG-vaccinated and Mycobacterium bovis challenged ( n = 12, blue circles); C: M. bovis challenged only ( n = 9, red circles). The bars of scatter dot plots show mean values, and error bars show ±1 SD. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The UCP conjugates for the six tested analytes were prepared to utilize the following antibodies: mouse anti-bovine IFN-γ (MT307; Mabtech, Stockholm, Sweden); mouse anti-bovine IL-2 (MT3B3; Mabtech, Stockholm, Sweden); chicken anti-bovine IL-6 (AHP2380B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL9 (AHP2369B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL10 (AHP2368B; Bio-Rad Laboratories, Hercules, CA, USA); and goat anti-bovine CCL4 (PBB0479B-050; Kingfisher Biotech, St Paul, MN, USA) at a concentration of 25 μg antibody per mg of UCP according to the methods described previously for the development of UCP-LFAs for the detection of M. leprae infection ( ).

Techniques: Comparison

Journal: Frontiers in Veterinary Science

Article Title: Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis

doi: 10.3389/fvets.2023.1193332

Figure Lengend Snippet: Receiver operator curve (ROC) analysis.

Article Snippet: The UCP conjugates for the six tested analytes were prepared to utilize the following antibodies: mouse anti-bovine IFN-γ (MT307; Mabtech, Stockholm, Sweden); mouse anti-bovine IL-2 (MT3B3; Mabtech, Stockholm, Sweden); chicken anti-bovine IL-6 (AHP2380B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL9 (AHP2369B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL10 (AHP2368B; Bio-Rad Laboratories, Hercules, CA, USA); and goat anti-bovine CCL4 (PBB0479B-050; Kingfisher Biotech, St Paul, MN, USA) at a concentration of 25 μg antibody per mg of UCP according to the methods described previously for the development of UCP-LFAs for the detection of M. leprae infection ( ).

Techniques: Significance Assay

Journal: Frontiers in Veterinary Science

Article Title: Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis

doi: 10.3389/fvets.2023.1193332

Figure Lengend Snippet: ROC curve analysis of IFN-γ (red), IL-2 (green), IL-6 (pink), CCL4 (yellow), CXCL9 (black), and CXCL10 (blue) detected in PPDb-stimulated whole blood supernatants to discriminate naïve (N) from Mycobacterium bovis -challenged group (C) (A) , BCG-vaccinated (V) from M. bovis -challenged group (C) (B) , and BCG-vaccinated group (V) from those that were M. bovis -challenged post-BCG (V/C) (C) .

Article Snippet: The UCP conjugates for the six tested analytes were prepared to utilize the following antibodies: mouse anti-bovine IFN-γ (MT307; Mabtech, Stockholm, Sweden); mouse anti-bovine IL-2 (MT3B3; Mabtech, Stockholm, Sweden); chicken anti-bovine IL-6 (AHP2380B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL9 (AHP2369B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL10 (AHP2368B; Bio-Rad Laboratories, Hercules, CA, USA); and goat anti-bovine CCL4 (PBB0479B-050; Kingfisher Biotech, St Paul, MN, USA) at a concentration of 25 μg antibody per mg of UCP according to the methods described previously for the development of UCP-LFAs for the detection of M. leprae infection ( ).

Techniques:

Journal: Frontiers in Veterinary Science

Article Title: Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis

doi: 10.3389/fvets.2023.1193332

Figure Lengend Snippet: A NUM score was calculated based on IFN-γ, IL-2, IL-6, CCL4, CXCL9, and CXCL10 ratios in PPDb-stimulated whole blood supernatants for naïve (N; n = 16), BCG-vaccinated (V; n = 10), and Mycobacterium bovis -challenged animals with (V/C; n = 12) or without prior BCG vaccination (C; n = 9). The NUM score ( y -axis) combines the results of six proteins, indicating the number of proteins with levels above a threshold based on the maximal Youden's index for each marker . Group differences were determined using the Kruskal-Wallis test; the statistical significance level used was *** p < 0.001; **** p < 0.0001 (A) . Heatmap showing Pearson correlation among the ratios of the evaluated host proteins. The color corresponds to the Pearson R value as indicated in each square (B) .

Article Snippet: The UCP conjugates for the six tested analytes were prepared to utilize the following antibodies: mouse anti-bovine IFN-γ (MT307; Mabtech, Stockholm, Sweden); mouse anti-bovine IL-2 (MT3B3; Mabtech, Stockholm, Sweden); chicken anti-bovine IL-6 (AHP2380B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL9 (AHP2369B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL10 (AHP2368B; Bio-Rad Laboratories, Hercules, CA, USA); and goat anti-bovine CCL4 (PBB0479B-050; Kingfisher Biotech, St Paul, MN, USA) at a concentration of 25 μg antibody per mg of UCP according to the methods described previously for the development of UCP-LFAs for the detection of M. leprae infection ( ).

Techniques: Marker

Journal: Frontiers in Veterinary Science

Article Title: Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis

doi: 10.3389/fvets.2023.1193332

Figure Lengend Snippet: NUM scores for biomarker signature.

Article Snippet: The UCP conjugates for the six tested analytes were prepared to utilize the following antibodies: mouse anti-bovine IFN-γ (MT307; Mabtech, Stockholm, Sweden); mouse anti-bovine IL-2 (MT3B3; Mabtech, Stockholm, Sweden); chicken anti-bovine IL-6 (AHP2380B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL9 (AHP2369B; Bio-Rad Laboratories, Hercules, CA, USA); rabbit anti-bovine CXCL10 (AHP2368B; Bio-Rad Laboratories, Hercules, CA, USA); and goat anti-bovine CCL4 (PBB0479B-050; Kingfisher Biotech, St Paul, MN, USA) at a concentration of 25 μg antibody per mg of UCP according to the methods described previously for the development of UCP-LFAs for the detection of M. leprae infection ( ).

Techniques: Biomarker Discovery