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: Gut Microbes

Article Title: Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus

doi: 10.1080/19490976.2024.2390680

Figure Lengend Snippet: The expression of AMPs in biopsy samples from patients with chronic gastritis. RNA from 16 biopsy samples from H.pylori positive chronic gastritis (GC) cases and 8 from normal-looking stomach (N) was retrotranscribed into cDNA for the detection of AMP genes and of genes encoding for pro-inflammatory cytokines. (a)–(g). The Log10 relative expression of LTF , LCN2 , C3 , CXCL9 , TNFA , IL1B and IFNG is reported. The significance of the difference between the groups was calculated using an unpaired t-test. * p =<0.05, ** p =<0.005, *** p =<0.0005, **** p =<0.00005. (h). The Log10 fold induction value of each gene detected in the chronic gastritis biopsies was used to compute a pearson R linear correlation heatmap. 1 = highest correlation, 0 = no correlation, −1=highest negative correlation.

Article Snippet: CXCL9 , Rabbit , 1:100 , 1:1000 , Cell signaling , 30327S.

Techniques: Expressing

Journal: Gut Microbes

Article Title: Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus

doi: 10.1080/19490976.2024.2390680

Figure Lengend Snippet: Antibodies.

Article Snippet: CXCL9 , Rabbit , 1:100 , 1:1000 , Cell signaling , 30327S.

Techniques:

Journal: Gut Microbes

Article Title: Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus

doi: 10.1080/19490976.2024.2390680

Figure Lengend Snippet: Primers.

Article Snippet: CXCL9 , Rabbit , 1:100 , 1:1000 , Cell signaling , 30327S.

Techniques: Sequencing

Journal: Gut Microbes

Article Title: Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus

doi: 10.1080/19490976.2024.2390680

Figure Lengend Snippet: The bactericidal activity of antimicrobial peptides found in the mucus. ((a) A suspension of 10 5 CFU of kanamycin resistant H.pylori was incubated for 1 h and 2 h in the presence of different concentrations (5, 10, or 30 mg/mL) of AMPs. The suspension was plated in kanamycin plates and the number of colonies was counted relative to PBS to assess the bactericidal range of each antimicrobial. (b). Bactericidal activity of Lactotransferrin using the bioactive peptide lactoferricin (LFcin), the antimicrobial peptide derived from the hydrolysis of lactotransferrin. (c). Bactericidal activity of LCN2. (d). Bactericidal activity of C3a, the peptide derived from complement component 3. (e). Bactericidal activity of CXCL9. Multiple comparison test was performed after a two way ANOVA to assess if the difference in bactericidal activity, for each concentration of AMP, between different time points, is statistically significant. p values comparing t = 2 h and t = 0 are reported. ns= non significant. * p = <0.05, ** p = <0.005, *** p = <0.0005, **** p = <0.00005. (f). Mucosoids were treated with TNFα (5 ng/mL), IL1β (2.5 ng/mL), and IFNγ (10 ng/mL) for 5 days, with a fresh supply of cytokines added on the third day. Mucus samples from mucosoids derived from 3 different patients was incubated with 10 5 CFU of a kanamycin resistant isogenic P12 strain for 2 hours before plating and assessing the bactericidal activity by counting the percentage of colonies relative to PBS. Paired samples t-test was used to assess if the difference between the bactericidal activity of the two groups was significant. ***= p < 0.0005. (g) Mucosoids were treated with a combination of pro-inflammatory cytokines as before and infected on the second day with H.pylori at MOI 100 for 3 days. Mucosoids were washed with PBS to remove the dead cells, they were stained with DAPI and imaged using confocal microscopy to count the remaining cells after infection. Each dot represents the density of cells per area of imaging (0.18 mm 2 ) and paired samples t-test was used to assess if the difference between the groups is significant * p =<0.05.

Article Snippet: CXCL9 , Rabbit , 1:100 , 1:1000 , Cell signaling , 30327S.

Techniques: Activity Assay, Suspension, Incubation, Derivative Assay, Comparison, Concentration Assay, Infection, Staining, Confocal Microscopy, Imaging

Journal: Gut Microbes

Article Title: Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus

doi: 10.1080/19490976.2024.2390680

Figure Lengend Snippet: H. pylori downregulates the expression of antimicrobials of inflamed epithelial cells (a). Three different mucosoid lines were treated by four different protocols, according to the schematic. Upper: non-treated (NT); second row: H. pylori infection carried out at day 2 at MOI 100; third row: treatment with pro-inflammatory cytokines (TNFα, 5 ng/mL; IL1β, 2.5 ng/mL; IFNγ 10 ng/mL) from day 0; lower: treatment with pro-inflammatory cytokines at the same concentrations as previously from day 0, following H. pylori infection at MOI 100 at day 2. At day 5, RNA was extracted and the abundance of transcripts was measured relative to non-treated controls. Due to the large differences between the expression of genes under different treatments, results are expressed in Log10-fold change. (b). The expression of CXCL8 does not change when H. pylori infection follows the treatment with pro-inflammatory cytokines. (c)(d)(e) The expression of LTF, C3 and LCN2 is downregulated when H. pylori infection is carried out in addition to the treatment with pro-inflammatory cytokines. (f) CXCL9 expression does not significantly change when H. pylori infection is in addition to to the treatment with pro-inflammatory cytokines.

Article Snippet: CXCL9 , Rabbit , 1:100 , 1:1000 , Cell signaling , 30327S.

Techniques: Expressing, Infection

Journal: Gut Microbes

Article Title: Inflammation promotes stomach epithelial defense by stimulating the secretion of antimicrobial peptides in the mucus

doi: 10.1080/19490976.2024.2390680

Figure Lengend Snippet: Inflammation induce the secretion of AMPs in the mucus. (a). Mucus was removed from three different mucosoid lines after treatment with TNFα (5 ng/mL), IL1β (2.5 ng/mL), and IFNγ (10 ng/mL) combined or individually (as indicated) for 5 days. The total mucus secreted and accumulated was analyzed by mass spectrometry. (b). Heatmap of the abundance of the proteins in the mucus determined by label free quantification (LFQ). Only proteins with an altered expression of at least 10-fold in one of the conditions are represented. Outlined in black are those proteins with reported bactericidal activity. The other proteins are categorized by location or function. (c)(d)(e)(f) The Log 10 LFQ values are plotted for LTF (lactotransferrin), LCN2 (lipocalin 2), C3 (complement component C3), CXCL9 (C-X-C motif chemokine ligand 9) the horizontal bar represent the median of tree individual measurements from the mucus produced by three different mucosoid lines (GAT23, 27, 29). Multiple comparison test was performed after one way ANOVA to assess if the differences between treated and untreated samples are significant: * p = <0.05, ** p = <0.005, *** p = <0.0005, **** p = <0.00005. The relative abundance of other antimicrobials CXCL1 and CXCL3 did not pass the statistical test for any of the multiple comparisons. (g)(h)(i)(j) validation of expression of the antimicrobial was confirmed by Western blot using a further two samples of mucosoids (GAT28, GAT31) treated with the combination of pro-inflammatory cytokines as before. As housekeeping proteins are not yet available for the mucus, Red Ponceau was used as a loading control. (k)(l)(m)(n) Mucosoids were treated with a combination of pro-inflammatory cytokines as before, and immunofluorescence experiments carried out to localize the expression of intracellular LTF, LCN2, C3 and CXCL9. Stained whole mount mucosoids were imaged using confocal microscopy. Images were taken across the whole thickness of the monolayer (z-stacks) and the panels shows a top and a lateral projection. Scale bar = 5 µm.

Article Snippet: CXCL9 , Rabbit , 1:100 , 1:1000 , Cell signaling , 30327S.

Techniques: Mass Spectrometry, Quantitative Proteomics, Expressing, Activity Assay, Produced, Comparison, Biomarker Discovery, Western Blot, Control, Immunofluorescence, Staining, Confocal Microscopy

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