Journal: Arthritis Research

Article Title: Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines

doi:

Figure Lengend Snippet: Differentiation of human TRAP-positive mononuclear cells derived from peripheral-blood monocytes into multinucleated cells after stimulation with IL-3, IL-5, IL-7, and GM-CSF

Article Snippet: Neutralizing antibodies to human IL-3, IL-5, IL-7, and GM-CSF were purchased from Genzyme (Cambridge, MA, USA).

Techniques: Derivative Assay

Journal: Arthritis Research

Article Title: Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines

doi:

Figure Lengend Snippet: Scanning electron micrographs of dentin slices, showing (a) resorption areas formed on dentin by TRAP-positive multinucleated giant cells derived from monocytes and stimulated with granulocyte-macrophage-colony-stimulating factor for 96 h and (b) a control slice incubated with the TRAP-positive mononuclear cells in the absence of cytokines. Scale lines = 50 μm.

Article Snippet: Neutralizing antibodies to human IL-3, IL-5, IL-7, and GM-CSF were purchased from Genzyme (Cambridge, MA, USA).

Techniques: Derivative Assay, Incubation

Journal: Arthritis Research

Article Title: Differentiation of monocytes into multinucleated giant bone-resorbing cells: two-step differentiation induced by nurse-like cells and cytokines

doi:

Figure Lengend Snippet: Inhibition of formation of multinucleated giant cells by neutralizing antibodies specific for IL-3, IL-5, IL-7, and GM-CSF

Article Snippet: Neutralizing antibodies to human IL-3, IL-5, IL-7, and GM-CSF were purchased from Genzyme (Cambridge, MA, USA).

Techniques: Inhibition

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

Article Title: Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation CK2 Regulates the Th17/Treg Axis

doi: 10.4049/jimmunol.1601912

Figure Lengend Snippet: (A, B) T cells were cultured in (A) Th1 and (B) Th17 conditions in the absence or presence of CX-4945 (2 μM) and at 72 h stained for IFN-γ and IL-17A. (C, D) Cells were cultured in (C) Treg and (D) Th17 conditions in the absence or presence of CX-4945 and at 72 h stained for Foxp3 and IL-17A. Data are representative of at least 3 independent experiments. (E) Mean frequencies of IL-17A+ and Foxp3+ cells in Th17 conditions +/− SEM are shown (n=6). (F–I) T cells were activated for 24 h, cultured in serum free media +/− CX-4945 (2 μM) for 4 h, then stimulated with (F) IL-6 (10 ng/ml) for 2 h, (G) IL-2 (5 ng/ml) for 30 min, or (H) TGFβ1 (5 ng/ml) for 30 min, and stained for phosphorylated Y705 STAT3, Y694 STAT5 or S463/465 Smad2 and S465/467 Smad3, respectively. NC: no cytokine. Representative histograms and MFIs normalized to isotype controls +/− SEM are shown (n=3). (I) Il17fThy1.1.Foxp3GFP T cells were polarized to the Th17 phenotype in the presence of CX-4945 (Th17+CX) or the Treg phenotype. GFP+ cells were sorted and co-cultured with CFSE-labeled effector T cells and proliferation assessed by CFSE dilution after 72 h. Representative histograms and mean frequencies of cells undergoing 3 or more divisions +/− SEM are shown (n=3). (J, K) T cells were transfected with the indicated siRNAs and polarized in Th17 conditions for 72 h. (J) A representative FACS plot and (K) mean frequencies of IL-17A+ and Foxp3+ cells +/− SEM are shown (n=3). (L, M) PBMCs were isolated from human blood and cultured in Th17 conditions for 6 days. (L) A representative FACS plot and (M) mean frequencies of IL-17A+ and Foxp3+ cells +/− SEM are shown (n=3). *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell.

Techniques: Cell Culture, Staining, Labeling, Transfection, Isolation

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

Article Title: Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation CK2 Regulates the Th17/Treg Axis

doi: 10.4049/jimmunol.1601912

Figure Lengend Snippet: (A–C) Naïve CD4+ T cells from Il17fThy1.1.Foxp3GFP reporter mice were cultured in Th17 conditions. At 72 h Thy1.1+GFP− cells were sorted and reactivated with anti-CD3 and anti-CD28 antibodies and IL-12 (10 ng/ml) in the absence or presence of CX-4945 (2 μM). (A, B) After 48 h of restimulation, cells were stained for IFN-γ. (A) Representative FACS plots and (B) mean frequencies of Thy1.1+IFN-γ+ cells +/− SEM are shown (n=3). (C) At 72 h, mRNA was extracted and gene expression analyzed by qRT-PCR. Data represent the mean relative expression to DMSO control (n=3). (D) 2D2 T cells were activated and polarized under Th17 conditions in the absence or presence of CX-4945 (2 μM) [CX-4945 (day 0)]. On day 3, cells were reactivated for 24 h. Cells that were polarized in the absence of CX-4945 were reactivated in the absence (DMSO) or presence of CX-4945 [CX-4945 (day 3)]. On day 4, 2 x 106 cells from each condition were transferred into Rag1−/− recipient mice. (E) Mice were scored daily for symptoms of classical EAE. Data are pooled form 2 separate experiments; n=5/group. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell.

Techniques: Cell Culture, Staining, Expressing, Quantitative RT-PCR

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

Article Title: Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation CK2 Regulates the Th17/Treg Axis

doi: 10.4049/jimmunol.1601912

Figure Lengend Snippet: (A) A schematic of disease induction, scoring and progression is shown with asterisks indicating time-points for CK2α detection. At days 9 and 12 post immunization (PI), cells were enriched from the draining lymph nodes (dLN) and spinal cord, respectively, and stained for intracellular CK2α. Data are representative of at least 3 independent experiments. (B–D) Mice were treated with CX-4945 (20 mg/kg/day) or sodium phosphate buffer (Vehicle) from 7 days before immunization throughout disease. Data are from 1 representative experiment of at least 2 independent experiments. (B) Clinical scores from days 0 to 23 PI are show; n=5/group. (C) On day 9, CD4+ T cells were enriched from the spleens via magnetic selection, lysed and immunoblotted for phosphorylated S129 and S473 Akt and T389 and S371 S6K p70; n=5/group. (D) On day 23, mononuclear cells were enriched from the spinal cord and Tregs identified by CD25 and Foxp3 staining; n=5/group. (E, F) Mice were treated with vehicle or CX-4945 (20 mg/kg/day) via oral gavage from days 0 to 7 PI. Data were pooled from 2 independent experiments. (E) At day 7, CD4+ T cells from the dLNs were stained for IL-17A and IFN-γ; n=8/group. (F) At day 7, CD4+ T cells from the spleens were stained for CD25 and Foxp3; n=5/group. *p<0.05, ***p<0.001.

Article Snippet: Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell.

Techniques: Staining, Selection

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

Article Title: Protein Kinase CK2 Controls the Fate Between Th17 Cell and Regulatory T Cell Differentiation CK2 Regulates the Th17/Treg Axis

doi: 10.4049/jimmunol.1601912

Figure Lengend Snippet: (A, B) Mice were treated with vehicle or CX-4945 via oral gavage for 8 days beginning at the onset of clinical symptoms. Data are from 1 representative experiment of at least 2 independent experiments. (A) Clinical scores from day 0 to 22 PI are shown; n=5/group. (B) At day 22, during disease resolution, mononuclear cells were enriched from the spinal cord and Tregs identified by CD25 and Foxp3 staining; n=5/group. (C, D) Mice were treated with vehicle or CX-4945 beginning on day 7 PI. At disease peak, effector T cells from the spinal cords were stained for (C) IL-17A and IFN-γ or (D) IL-17A and GM-CSF. Representative flow plots and frequencies of single and double positive cells +/− SEM are shown. Data were pooled from 3 independent experiments; vehicle, n=6; CX-4945, n=8. *p<0.05.

Article Snippet: Anti-mouse CD3, anti-mouse CD28 and mouse and human neutralizing antibodies to IL-4 and IFN-γ were purchased from BioXCell.

Techniques: Staining