Journal: Frontiers in Immunology

Article Title: Skin repair and immunoregulatory effects of myeloid suppressor cells from human cord blood in atopic dermatitis

doi: 10.3389/fimmu.2023.1263646

Figure Lengend Snippet: Immunobiological characterization of human umbilical cord blood (hUCB) myeloid-derived suppressor cells (MDSCs) in vitro using human T cells. (A) The proliferation- and differentiation-stage CD33 + CD11b + MDSC markers during the 6-week culture period of hUCB-MDSCs. Flow cytometric analysis of hUCB-MDSCs stained with individual MDSC-positive or -negative surface marker antibodies. (CD33 + CD11b + ≥ 70%, CD14 + ≥ 90%) (B) The expression of immune suppressive molecules by hUCB-MDSCs was analyzed using FITC anti-inducible nitric oxide synthase (iNOS)2 antibody, PE anti-indoleamine 2,3-deoxygenase (IDO) antibody, and PerCP-Cy5.5 anti-arginase-1 (ARG1) antibody staining. Cells were analyzed using a FACS Lyric device after two washes in stain buffer and incubation on ice for 30 min. Black, isotype control antibody. Red, each specific antibody. (iNOS ≥ 200 MFI, IDO ≥ 450 MFI, ARG1 ≥ 400 MFI) (C) hUCB-MDSC-mediated immune suppression of T cells, stimulated by anti-CD3/CD28 beads for 6 days in human peripheral blood mononuclear cells (PBMCs), was tested at a PBMC–hUCB-MDSC ratio of 1:1. (Suppression ≥ 45%). (D) hUCB-MDSC-mediated immunoregulation of human CD4 + T-cell differentiation, stimulated using Th2 or Th17 differentiation kits for 13 or 6 days in human CD4 + T cells, was tested at a human CD4 + T cells–hUCB-MDSC ratio of 1:1. * p < 0.05, ** p < 0.01.

Article Snippet: ARG1 (Nor-NOHA 0.5 mM; Selleckchem, Houston, TX, USA) or iNOS inhibitor (1400W 0.1 mM; Medchem Express, Monmouth, NJ, USA) and hUCB-MDSCs was simultaneously treated.

Techniques: Derivative Assay, In Vitro, Staining, Marker, Expressing, Incubation, Control, Cell Differentiation

Journal: PLoS ONE

Article Title: A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses

doi: 10.1371/journal.pone.0042386

Figure Lengend Snippet: Aortic rings with endothelium intact (+EC) and denuded (−EC) were treated for 24 hours with media alone (CTRL), C12-iE-DAP 1 µg/ml (NOD1 agonist) or LPS 1 µg/ml (TLR4 agonist) (A) In some experiments, cells were treated for a further 24 hour period with replacement of media and agonists (48 hours; B). In addition, endothelium intact rings were pre-treated with the iNOS inhibitor 1400 W 1 µM (C) or the IκBβ inhibitor SC-514 30 µM (D) for 30 minutes prior to 24 hours treatment with agonists. iNOS activity was assessed by measurement of nitrite (breakdown product of nitric oxide) by the Griess assay. Results are expressed as mean ± SEM for n = 9 (A+B) or n = 4 (C+D) animals. Data was analysed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test (*P<0.05) or by two-way ANOVA followed by Bonferroni’s post tests (+P<0.05).

Article Snippet: The inhibitor of NFκB kinase subunit beta (IκBβ) inhibitor SC-514, transforming growth factor β activating kinase (TAK1) inhibitor 5Z-7-oxozeaonol and the pan-caspase inhibitor Z-VAD-fmk were from Tocris Bioscience (UK) and the inducible nitric oxide synthase (iNOS) inhibitor 1400 W from Cayman Chemical (USA).

Techniques: Activity Assay, Griess Assay

Journal: Biomedicines

Article Title: Differential Effects of D9 Tetrahydrocannabinol (THC)- and Cannabidiol (CBD)-Based Cannabinoid Treatments on Macrophage Immune Function In Vitro and on Gastrointestinal Inflammation in a Murine Model

doi: 10.3390/biomedicines10081793

Figure Lengend Snippet: The influence of pure CBD/THC and cannabis extracts on nitric oxide production of LPS-activated peritoneal macrophages. Peritoneal macrophages from C57BL/6 female ( a ) and male ( b ) mice were activated for 24 h with LPS, in the presence of cannabinoid treatments (5 µg/mL). 1400W (1400), a specific iNOS inhibitor, served as control. NO• levels in the supernatant were analyzed. ( a )— n = 10 mice, from 3 independent experiments; ( b )— n = 7 mice, from 3 independent experiments. The differences of all treatments as compare with LPS-activated control (indicated on the graphs) are highly significant. The differences of THCE and THC between CBDE and CBD are significant in ( a , b ); ( c ) NO• levels in the supernatant of activated peritoneal macrophages from young (2 months old) and aged (18 months old) C57BL/6 female and male mice. n = 7 mice per group, from 3 independent experiments. The differences between aged and young mice in each treatment, are indicated on the graph; ( d ) the expression levels of CB1 and CB2 in peritoneal macrophages from young and aged C57BL/6 female mice ( n = 3/group) were assessed by real-time PCR analysis. The results are expressed as mean + SEM. p -value *, <0.05; **, <0.01; ***, <0.001. NA—non-activated; LPS—lipopolysaccharide-activated macrophages; THC—D9 tetrahydrocannabino; CBD—cannabidiol; THCE—high THC cannabis extract; CBDE—high CBD cannabis extract; n.s.—not significant.

Article Snippet: Controls: non-activated cells, activated cells + vehicle, activated cells + 1400W dihydrochloride (NOS2 inhibitor, Enzo Life Sciences Inc., Lausen, Switzerland).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: SOCS-1 inhibition of type I interferon restrains Staphylococcus aureus skin host defense

doi: 10.1371/journal.ppat.1009387

Figure Lengend Snippet: A) Gram staining of skin biopsies collected at day 1 post-MRSA skin infection from iKIR and scrambled KIR treated mice. Gram staining to label gram-positive bacteria is shown in purple/brown. Magnifications are as shown. Black arrows indicate extracellular MRSA clusters. Images are representative of 3–5 mice per group. B) Phagocytosis of GFP tagged MRSA by BMDM’s from SOCS1 fl and SOCS1 Δmyel mice or BMDM’s from WT mice treated with the SCR KIR or iKIR peptide. C) Determination of Bacterial killing of GFP tagged MRSA by BMDMs from B as described in Phagocytosis and Killing assays—Methods. D) mRNA expression of Nos2 in the skin of infected mice treated with either SCR KIR or iKIR peptide at day 1 post-infection as determined via qPCR. E) Determination of bacterial killing of GFP-tagged MRSA as in C with BMDMs from WT mice treated with either SCR KIR, iKIR, or iKIR+ an iNOS inhibitor (1400W dihydrochloride). F) Measurement of nitric oxide in the supernatant of BMDMs from E . G) H 2 O 2 levels in the skin of mice treated with either SCR KIR or iKIR at day 1 post infection as determined by Amplex Red assay. Data represent the mean ± SEM from 3–6 mice from 2–3 independent experiments. *p < 0.05 vs. SOCS1 fl or SCR KIR treated mice. #p < .05 vs. iKIR treated mice.

Article Snippet: To determine the role of NO in iKIR-mediated microbial killing, cells were treated with 50 μM of the iNOS inhibitor 1400W dihydrochloride (Tocris).

Techniques: Staining, Infection, Bacteria, Expressing, Amplex Red Assay

Journal: PLoS ONE

Article Title: A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses

doi: 10.1371/journal.pone.0042386

Figure Lengend Snippet: Aortic rings with endothelium intact (+EC) and denuded (−EC) were treated for 24 hours with media alone (CTRL), C12-iE-DAP 1 µg/ml (NOD1 agonist) or LPS 1 µg/ml (TLR4 agonist) (A) In some experiments, cells were treated for a further 24 hour period with replacement of media and agonists (48 hours; B). In addition, endothelium intact rings were pre-treated with the iNOS inhibitor 1400 W 1 µM (C) or the IκBβ inhibitor SC-514 30 µM (D) for 30 minutes prior to 24 hours treatment with agonists. iNOS activity was assessed by measurement of nitrite (breakdown product of nitric oxide) by the Griess assay. Results are expressed as mean ± SEM for n = 9 (A+B) or n = 4 (C+D) animals. Data was analysed by one-way ANOVA followed by Bonferroni’s Multiple Comparison Test (*P<0.05) or by two-way ANOVA followed by Bonferroni’s post tests (+P<0.05).

Article Snippet: The inhibitor of NFκB kinase subunit beta (IκBβ) inhibitor SC-514, transforming growth factor β activating kinase (TAK1) inhibitor 5Z-7-oxozeaonol and the pan-caspase inhibitor Z-VAD-fmk were from Tocris Bioscience (UK) and the inducible nitric oxide synthase (iNOS) inhibitor 1400 W from Cayman Chemical (USA).

Techniques: Activity Assay, Griess Assay, Comparison

Journal: PLoS ONE

Article Title: A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses

doi: 10.1371/journal.pone.0042386

Figure Lengend Snippet: iNOS activity in aortic rings stimulated by the NOD2 agonist MDP.

Article Snippet: The inhibitor of NFκB kinase subunit beta (IκBβ) inhibitor SC-514, transforming growth factor β activating kinase (TAK1) inhibitor 5Z-7-oxozeaonol and the pan-caspase inhibitor Z-VAD-fmk were from Tocris Bioscience (UK) and the inducible nitric oxide synthase (iNOS) inhibitor 1400 W from Cayman Chemical (USA).

Techniques: Activity Assay

Journal: International journal of molecular sciences

Article Title: Treatment of Status Epilepticus after Traumatic Brain Injury Using an Antiseizure Drug Combined with a Tissue Recovery Enhancer Revealed by Systems Biology.

doi: 10.3390/ijms241814049

Figure Lengend Snippet: Figure 2. Effect of in silico-identified drugs on tissue biomarkers of neuroprotection, oxidative stress, and neuroinflammation. (A) Neuronal viability. For a comparative viability analysis, neu- ronal survival in LPS/IFNγ+-treated neuronal–microglial co-cultures was set to 0% and that in co-cultures treated with iNOS inhibitor 1400 W (positive treatment control) to 100%. Trichostatin A at a 50 nM concentration increased neuronal survival from 0% to 73% (p < 0.001 compared with LPS/IFNγ+). The neuroprotective effect of TSA was almost as good as that of the positive control 1400 W (73% vs. 100%, p < 0.001). Also, chlorpromazine at a 50-µM concentration increased neuronal survival from 0% to 57% (p < 0.001) and calpain inhibitor at a 50-µM concentration increased neu- ronal survival from 0% to 47% (p < 0.001). Geldanamycin and tranylcypromine showed no consistent neuroprotection. IL10 had no effect on neuronal viability. Note that in untreated co-cultures without neuroinflammation (LPS/IFNγ−), neuronal viability was 38% of that in untreated BV2 neuronal cultures (p < 0.001) and 48% of that in 1400 W treated inflammation-exposed co-cultures (p < 0.001). Wells with cortical neurons only (BV2−cells without LPS/IFNγ+ exposure) showed the greatest viability. (B) Nitrite levels. For a comparative viability analysis, nitrite levels in LPS/IFNγ+-treated neuronal–microglial co-culture medium were set to 100% and that in co-culture medium treated with iNOS inhibitor 1400 W (positive treatment control) was set to 0%. Trichostatin A at a 50 nM concentration reduced the LPS/IFNγ+-induced nitrite release from 100% to 28% (p < 0.001). At a 10 nM concentration, there was a slight increase in nitrite levels to 121% (p < 0.01). Calpain inhibitor at 50 µM reduced nitrite release from 100% to −21% (p < 0.001). Chlorpromazine at 50 µM reduced nitrite levels to 81% (p < 0.001). At 10 µM, tranylcypromine reduced nitrite levels from 100% to 89% (p < 0.05) and at 1 µM, to 81% (p < 0.001). Interestingly, tranylcypromine at 100 µM had no effect (p > 0.05). Geldanamycin at 100 nM concentrations resulted in low nitrite levels, likely due to the cell death that was visualized in microscopic images (data not shown). IL10 had a mild effect on the LPS/IFNγ+-induced increase in nitrite levels, reducing it to 63% (p < 0.001). Note that neuron-only (BV2−) and LPS/IFNγ−neuronal–microglial co-cultures were not exposed to neuroinflammation, and thus showed no nitrite release to the culture medium. (C) TNFα levels. For a comparative viabil- ity analysis, TNFα levels in LPS/IFNγ+-treated neuronal–microglial co-culture medium were set to 100% and that in co-culture medium treated with TNFα inhibitor, IL10 (positive treatment control) to 0%. Trichostatin A at 10 nM concentration reduced TNFα levels from 100% to −2% (p < 0.001) and at 50 nM to −139% (p < 0.001), being better than the positive control. Calpain inhibitor at 50 µM reduced TNFα levels from 100% to −226% (p < 0.001). Chlorpromazine at 50 µM reduced TNFα levels from 100% to −21% (p < 0.001). The iNOS inhibitor, at 1400 W, reduced TNFα levels from 100% to 61% (p < 0.05). Abbreviations: BV2−, cortical neuronal cultures without BV2-microglial cells; IL10+, co-cultures treated with positive control anti-inflammatory incytokine, interleukin 10; LPS/IFNγ−, co-culture wells without lipopolysaccharides and interferon γ-induced neuroinflam- mation; LPS/IFNγ+, wells with lipopolysaccharides and interferon γ-induced neuroinflammation (untreated controls); TNFα, tumor necrosis factor α; 1400 W+, co-cultures treated with positive con- trol, inducible nitric oxide synthase (iNOS) inhibitor, 1400 W hydrochloride. Statistical significance: ***, p < 0.001; **, p < 0.01; *, p < 0.05 compared with LPS + IFNγ+ (linear regression in R). The number of independent experiments in panel A was three and in panels B-C was four. Data are presented as box plots with whiskers (minimum and maximum; box: interquartile range; line: median). Each dot shows an experimental value.

Article Snippet: Anti-inflammatory cytokine, interleukin 10 (IL10, 50 μg/mL, #500-M128, Peprotech, Rocky Hill, NJ, USA; final concentration of 50 ng/mL), and the neuroprotective inducible nitric oxide synthase (iNOS) inhibitor 1400 W hydrochloride (2 mM, #1415, Tocris, Bristol, UK; final concentration of 20 μM) were used as positive controls.

Techniques: In Silico, Control, Concentration Assay, Positive Control, Co-Culture Assay