Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression in Breast Cancer PDXs That Have Acquired Resistance to PI3Kα Inhibition (A) Relapse of an HCI-011 PDX after treatment with GDC-0032 (HCI-011R). (B) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts receiving drug vehicle (n = 5) or GDC-0032 (n = 8). p values were defined using two-sided Wald t tests. (C) Immunoblots of lysates from (B) and from an HCI-011 tumor. (D) Mean tumor volumes (cm 3 ) ± SEM of HCI011R xenografts treated with drug vehicle (n = 2), drug vehicle plus tamoxifen (n = 3), and tamoxifen plus GDC-0032 (n = 5). p values were defined using two-sided Wald t tests. (E) Immunoblots of lysates from (D). Mean ± standard deviation (n = 2 to 4). p values were calculated using two-sided Welch's t tests. (F) Gene expression (Log2) enrichment (heatmap: red, positive; white, neutral; blue, negative) of FOXM1 gene targets in HCI-011 (n = 4) and in HCI-011R xenografts (n = 8). The top row shows false discovery rate (FDR)-adjusted p values. (G) Gene expression (Log2) enrichment of genes mediating resistance to BYL-719 in HCI-011 (n = 4 for vehicle and drug) and in HCI-011R (n = 5 vehicle, n = 8 GDC-0032). FDR-adjusted p values of drug-resistant versus drug-sensitive differential expression are shown in the top row. (H) Mean tumor volume (cm 3 ) ± SEM of drug-resistant T47DR tumors after long-term treatment with GDC-0032. p values were defined using two-sided Wald t tests. (I) Immunoblot of tumors lysates from (H). See also Figure S5 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Inhibition, Western Blot, Standard Deviation, Gene Expression, Quantitative Proteomics

Journal: Cancer Cell

Article Title: Metabolic Imaging Detects Resistance to PI3Kα Inhibition Mediated by Persistent FOXM1 Expression in ER + Breast Cancer

doi: 10.1016/j.ccell.2020.08.016

Figure Lengend Snippet: Persistent FOXM1 Expression Contributes to Drug Resistance and Can Be Explained by Cytoplasmic Localization of FOXO3a (A) Representative confocal microscopy images of T47D PTEN wt (Ctrl) and PTEN KO cells drug-treated for 72 h. FOXO-3a staining (green), DAPI staining (blue). (B) Relative number of red fluorescent MCF7 and T47D cells (mStrawberry-positive cells, co-expressing FOXM1C) in mixtures with parental controls following treatment for 120 h. p values were calculated using two-sided Welch's t tests. (C) Viability of HCI-011R PDTCs treated with the indicated drugs for 120 h. Left: viability of HCI-011R PDTCs treated for 120 h. Mean ± standard deviation (five technical replicates, n = 3 experiments). Right: mean ± standard deviation (five technical replicates, n = 4 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (D) Viability of cells dissociated from a T47DR tumor treated with the indicated drugs for 120 h. Left: mean ± standard deviation (five technical replicates, n = 4 experiments). Right: mean ± standard deviation (five technical replicates, n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (E) Viability of T47D PTEN KO cells expressing FOXM1 targeting sequences (shFOXM1 [sequence 1 or 2]) or a non-targeting control sequence (shCtrl) treated for 120 h. Mean ± standard deviation (n = 5, five technical replicates). p values were calculated using two-sided Welch's t tests on the averages of replicates. (F) Viability of HCI-011R PDTCs expressing doxycycline-inducible shRNA sequences targeting FOXM1 (sequence 1) or a control shRNA and treated for 120 h. Mean ± standard deviation (three technical replicates). p values were calculated using two-sided Welch's t tests on the averages of the replicates. (G) LDH activity in the cells used in (E) and treated for 96 h. Figure shows the mean ± standard deviation (n = 3 experiments). p values were calculated using two-sided Welch's t tests on the averages of replicates. (H) Immunoblots of lysates from cells used in (G). (I) Mean tumor volume (cm 3 ) ± SEM of T47D PTEN KO xenografts expressing doxycycline-inducible shFOXM1 (sequence 1) or shCtrl, following prolonged GDC-0032 treatment. One cohort received standard food (shCtrl n = 3 and shFOXM1 n = 3) and the other cohort food plus doxycycline (shCtrl n = 3 and shFOXM1 n = 4; 0.2 g/kg food pellet Harlan D.98186). p values were defined using two-sided Wald t tests. (J) Immunoblot of lysates from tumors in (I). p values were calculated using two-sided Welch's t tests. See also Figure S6 .

Article Snippet: PDTCs from HCI-001, HCI-011, HCI-011R tumors and cells from T47DR tumors were obtained by dissociating them using a kit (human 130-095-929, Miltenyi Biotec), as described previously ( ).

Techniques: Expressing, Confocal Microscopy, Staining, Standard Deviation, Sequencing, Control, shRNA, Activity Assay, Western Blot

Journal: Communications chemistry

Article Title: A platinum(IV) prodrug strategy to overcome glutathione-based oxaliplatin resistance.

doi: 10.1038/s42004-022-00661-z

Figure Lengend Snippet: Fig. 4 Impact of BSO-OxOAc as compared to oxaliplatin and cisplatin on the viability of the indicated cancer cell lines and their sublines with the respective acquired platinum drug resistance. a Impact of a 72 h continuous drug exposure on the viability of human colon cancer cells HCT116 and a oxaliplatin-resistant subline HCT116/OxR, as well as the human ovarian cancer cell model A2780 and the cisplatin-resistant subline A2780/Cis. One representative experiment out of at least three performed in triplicate is shown. Data points are depicted as mean ± SD. b IC50 values were derived from the dose-response curves as shown under (a) using the four-parameter logistic nonlinear regression model. Resistance factors were calculated by dividing the IC50 values for the resistant subline by those of the respective sensitive parental cell model.

Article Snippet: The human and murine colorectal carcinoma cell lines HCT116 (Expasy accession: CVCL_0291) and CT26 (CVCL_7254), respectively, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA), and the human ovarian cancer cell line A2780 (CVCL_0134, product number 93112519) together with its cisplatin-resistant subline A2780/Cis (93112517) from Sigma Aldrich.

Techniques: Derivative Assay

Journal: Communications chemistry

Article Title: A platinum(IV) prodrug strategy to overcome glutathione-based oxaliplatin resistance.

doi: 10.1038/s42004-022-00661-z

Figure Lengend Snippet: Fig. 5 Cellular uptake of BSO-OxOAc as compared to free oxaliplatin and impact of acquired oxaliplatin resistance. a Parental HCT116 and oxaliplatin- resistant HCT116/OxR cells were exposed to the indicated concentrations of BSO-OxOAc and oxaliplatin for 3 h and cellular uptake quantified by ICP-MS. Data are depicted as mean ± SD. b Log-fold difference in the platinum accumulation between parental HCT116 and HCT116/OxR calculated from the data shown in (a). Statistical significance was tested using one-way ANOVA. *p < 0.05; ***p < 0.001.

Article Snippet: The human and murine colorectal carcinoma cell lines HCT116 (Expasy accession: CVCL_0291) and CT26 (CVCL_7254), respectively, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA), and the human ovarian cancer cell line A2780 (CVCL_0134, product number 93112519) together with its cisplatin-resistant subline A2780/Cis (93112517) from Sigma Aldrich.

Techniques:

Journal: Communications chemistry

Article Title: A platinum(IV) prodrug strategy to overcome glutathione-based oxaliplatin resistance.

doi: 10.1038/s42004-022-00661-z

Figure Lengend Snippet: Fig. 6 Impact of BSO-OxOAc as single agent and in combination with the reducing agents NAC (100 µM) and AA (50 µM) on the clonogenic potential of HCT116 cells and the subline with acquired oxaliplatin resistance (HCT116/OxR). Sparsely seeded cells (1 × 103/24-well plate well) were exposed for 10 days to the indicated compounds and derived cell clones stained with crystal violet, photographed, and results evaluated by ImageJ software as described in the Methods section. One respective experiment out of three performed in duplicate is shown under (a) and the respective evaluation under (b), depicted as mean ± SD. Statistical significance was tested using two-way ANOVA. In all cases: *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: The human and murine colorectal carcinoma cell lines HCT116 (Expasy accession: CVCL_0291) and CT26 (CVCL_7254), respectively, were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA), and the human ovarian cancer cell line A2780 (CVCL_0134, product number 93112519) together with its cisplatin-resistant subline A2780/Cis (93112517) from Sigma Aldrich.

Techniques: Derivative Assay, Clone Assay, Staining, Software

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Primers for quantitative real-time RT-PCR analyses of gene transcript expression

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Basal mRNA expression of mesenchymal markers was increased in DHBE cells. Basal mRNA expression of EMT-related markers in non-COPD (NHBE, open bar, n = 3) and COPD (DHBE, closed bar, n = 3) human bronchial epithelial cells were analyzed by real-time RT-PCR. Results were normalized to NHBE and expressed as mean (SE) of three independent experiments. Data were analysed by Student t test, * p < 0.05, compared with NHBE. ( a ) E-cadherin ( b ) N-cadherin ( c ) vimentin ( d ) Ratio of E-/N-cadherin. ns: not significant

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Snail1 and E-cadherin protein expression in non-COPD and COPD lungs. Immunohistochemical localization of EMT markers in the airway epithelium of non-COPD ( a , c , e ) and COPD ( b , d , f ) lungs ( n = 3). Immunostaining for E-cadherin ( a , b ) and Snail1 transcription factor ( c , d ) and N-cadherin ( e , f ). The dark brown color is indicative of strong expression (denoted by red arrows) of E-cadherin in non-COPD ( a ), Snail1 in COPD ( d ) and N-cadherin in COPD ( f ) lung. Negative control was stained with isotype-matched control antibody (data not shown). Non-COPD subjects are non smokers. aw: airway lumen

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Immunohistochemical staining, Immunostaining, Negative Control, Staining

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: mRNA expression of EMT markers in HBE cells after 3-day treatment with CM from HLF. HBE cells from non-COPD (N) or COPD patients (D) were stimulated with 100 % CM (black bars), or without CM (white bars) collected from human lung fibroblasts (HLF) derived from non-smokers (N) or COPD subjects (D) for 3 days and total RNA was extracted. The mRNA expression of EMT markers were determined by real-time RT-PCR and normalized to GAPDH mRNA expression in the same origin of HBE cells. ( a ) E-cadherin, ( b ) N-cadherin, ( c ) vimentin, and ( d ) the ratio of E-/N-cadherin were calculated. Data were analysed and expressed as mean (SE) of from six to nine independent experiments by one-way ANOVA with Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, compared between the indicated groups

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients

doi: 10.1186/s12931-015-0232-4

Figure Lengend Snippet: Effects of CM from HLF on protein expression of EMT markers in HBE cells. HBE cells from normal subjects (N) or COPD patients (D) were lysed after stimulation with or without 100 % CM collected from human lung fibroblasts of N or D for 5 days. Total protein was prepared analysed by western blotting, using 40 μg of protein per lane. The membranes were probed with anti-E-cadherin, anti-N-cadherin, anti-vimentin, or anti-GAPDH antibodies. The intensities of the bands for target protein were normalized to the corresponding GAPDH bands for the each treated cells. ( a ) E-cadherin, ( b ) N-cadherin, ( c ) vimentin, and ( d ) the ratio of E-/N-cadherin were calculated. Data were analysed and expressed as mean (SE) of from seven to ten independent experiments by one-way ANOVA with Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01, compared between the indicated groups

Article Snippet: Paraffin-embedded lung sections were stained with specific antibodies for E-cadherin (a mouse anti-human E-cadherin antibody, 1:300 dilution, ab1416, Abcam, Cambridge, MA) or snail1 (1:250 dilution, a rabbit anti-Snail + Slug, ab85936, Abcam, Cambridge, MA) or N-cadherin (a rabbit polyclonal anti-human N-cadherin antibody, 1:300 dilution, ab18203, Abcam, Cambridge, MA).

Techniques: Expressing, Western Blot

Journal: Cancer Science

Article Title: Down‐regulation of pescadillo inhibits proliferation and tumorigenicity of breast cancer cells

doi: 10.1111/j.1349-7006.2009.01325.x

Figure Lengend Snippet: Overexpression of pescadillo in human primary breast cancer. (A) Western blotting analysis of pescadillo protein in normal breast epithelial cells (NBEC) and indicated breast cancer cell lines. α‐Tubulin was probed for loading control. (B) Expression of pescadillo mRNA in the NBEC and cultured breast cancer cell lines. Expression levels were normalized for GAPDH. (C) Western blotting analysis of pescadillo protein from human primary breast cancer (T) and paired tumor‐adjacent non‐cancerous breast tissues (n), with each pair taken from a same patient. (D) Real time RT‐PCR analysis of pescadillo mRNA from the same four pairs of breast cancer and adjacent non‐cancerous tissues. Error bars represent SDs calculated from three parallel experiments.

Article Snippet: IHC analysis was performed to study altered protein expression in five normal breast tissues and 92 human breast cancer tissues using rabbit anti‐pescadillo antibody (1:250; Bethyl Laboratories).

Techniques: Over Expression, Western Blot, Control, Expressing, Cell Culture, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Down‐regulation of pescadillo inhibits proliferation and tumorigenicity of breast cancer cells

doi: 10.1111/j.1349-7006.2009.01325.x

Figure Lengend Snippet: Increased expression of pescadillo protein in histopathological sections of breast cancer as shown by immunohistochemical analysis. (A) Representative examples of IHC staining for pescadillo expression in normal breast tissues and breast cancer of American Joint Committee on Cancer (AJCC) stages I–IV. (a,b) Normal breast tissue (c,d) clinical stage I; (e,f) clinical stage II; (g,h) clinical stage III; and (i,j) clinical stage IV. Magnifications: ×200 (a, c, e, g, and i); ×400 (b, d, f, h, and j). (B) Average mean optical densities (MODs) of pescadillo staining in all stages of breast cancers (randomly chosen 12 cases per stage) were statistically higher than that in normal breast tissues (five cases). *Statistical significance (P < 0.05).

Article Snippet: IHC analysis was performed to study altered protein expression in five normal breast tissues and 92 human breast cancer tissues using rabbit anti‐pescadillo antibody (1:250; Bethyl Laboratories).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Naive CD4+CD25−CD62LhiCD44lo T cells from spleens of C57BL/6 mice were sorted by flow cytometry and activated with anti-CD3 and anti-CD28 in the presence of TGF-β plus IL-6, anti-IFN-γ and anti-IL-4 for 72 h (first stimulation). Cells were rested 2 days in the presence of IL-2 and then reactivated with anti-CD3 and anti-CD28 (second stimulation) during 72 h in the presence of TGF-β plus IL-6, TGF-β, IL-6 and IL-23, IL-23 or without added cytokines. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug for the final 4 h, stained and analyzed by flow cytometry. CD4+ cells are shown. (b) Percentage of GM-CSF+ cells among CD4+IL-17A+ cells after the second stimulation. (c) GM-CSF, IL-10 and IL-17A concentrations in cell culture supernatants after the second stimulation measured by ELISA. (d) TH17 cells restimulated in the presence of TGF-β and IL-6 for 72 h were treated with anti-IL-10 or isotype control (goat IgG) and analyzed by flow cytometry. (e) GM-CSF concentrations in culture supernatants of cells stimulated as in (d) measured by ELISA. **p< 0.001. Data are representative of three independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Flow Cytometry, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Naive CD4+CD25−CD62LhiCD44lo T cells from spleens of C57BL/6 mice were FACS sorted and differentiated into TH17 cells during the first stimulation. Cells were then reactivated with anti-CD3 and anti-CD28 for 72 h in the presence of either IL-23 or TGF-β plus IL-6, in the presence of IL-1β (10 ng/ml) and/or TNF (10 ng/ml). CD4+ cells are shown. (b) GM-CSF, IL-17A, IL-21 and IL-22 concentrations in cell culture supernatants after the second stimulation measured by ELISA. (c) Naive CD4+ T cells from spleens of C57BL/6 and RORγt-deficient mice were activated during 72 h with anti-CD3 and anti-CD28 in the presence of either TGF-β and IL-6 or IL-1β, IL-6 and IL-23. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug, stained and analyzed by flow cytometry. *p<0.01, **p < 0.001. Data are representative of two independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: (a) Splenocytes from MOG35-55-immuzed C57BL/6 mice were activated with MOG35-55 (20 μg/ml) in the presence of TGF-β plus IL-6, IL-23 or without added cytokines. Cells were then stimulated with PMA and ionomycin in the presence of GolgiPlug, stained and analyzed by flow cytometry. CD4+ cells are shown. (b) Percentage of GM-CSF+ cells among CD4+IL-17A+ cells after the second stimulation. (c) GM-CSF, IL-10, and IL-17A concentrations in cell culture supernatants measured by ELISA. (d) GM-CSF and IL-17A concentrations in supernatants of cell cultures stimulated as in (a) in the presence of TGF-β plus IL-6 and treated with anti-IL-10 or goat IgG. (e) GM-CSF and IL-17A concentrations in supernatants of cell cultures stimulated as in (a) without added cytokines and treated with anti-IL-23p19 or goat IgG (f) Clinical scores of irradiated wild-type recipient mice that received 5×106 CD4+ cells enriched from EAE splenocytes activated in the presence of IL-23. Mice were treated with either anti-GM-CSF or rat IgG from day 2 to day 35 post cell transfer. *p< 0.01; **p < 0.001. Data are representative of two (d, e and f) or four (a, b, and c) independent experiments (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Staining, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation

Journal: Nature immunology

Article Title: The encephalitogenicity of T H 17 cells is dependent on IL-1- and IL-23-induced production of the cytokine GM-CSF

doi: 10.1038/ni.2031

Figure Lengend Snippet: Splenocytes from wild-type- or Csf2−/−-MBPAc1-11 transgenic mice were activated with MBPAc1-11 peptide in the presence of IL-12 (TH1 conditions) or TGF-β plus IL-6, anti-IFN-γ and anti-IL-4 during 72 h (TH17 conditions). Cells were rested 2 days in the presence of IL-2 and then reactivated with MBPAc1-11 peptide in the presence of IL-12 (TH1 conditions) or IL-23 (TH17 conditions). After 72 h, cells were stimulated with PMA and ionomycin in the presence of GolgiPlug. (a) Flow cytometric analysis of IL-17A and IFN-γ expression in wild-type and Csf2−/− TH1 and TH17 cells after the second stimulation. CD4+ cells are shown. (b) IL-17A, IFN-γ and GM-CSF concentrations were measured by ELISA in the supernatants after the second stimulation. (c) RORγt and T-bet expression on gated CD4+IFN-γ+ (TH1) or CD4+IL-17A+ cells (TH17) analyzed by flow cytometry after the second stimulation. Filled histograms represent isotype controls. (d) Real time PCR analysis of Ahr, CCL20, CCR6, IL-23R and RORα expression in CD4+ T cells enriched by magnetic beads after the second stimulation from wild-type or Csf2−/− MBPAc1-11-splenocytes cultured in TH17 conditions. (e) Clinical scores of recipient mice that received 5×106 of either MBPAc1-11-specific wild-type or Csf2−/− TH1 or TH17 cells enriched by magnetic beads after the second stimulation. Pertussis toxin was injected i.p. on days 0 and 2 post transfer. *p< 0.01; **p < 0.001. Data are representative of two independent experiments. (error bars, s.e.m).

Article Snippet: Anti-GM-CSF monoclonal antibody (clone 22E9.11; ATCC) was kindly provided by KaloBios Pharmaceuticals, Inc. (South San Francisco).

Techniques: Transgenic Assay, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Real-time Polymerase Chain Reaction, Magnetic Beads, Cell Culture, Injection