Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 1. Dinaciclib and anti-PD1 combina- tion therapy inhibits tumor growth in syn- geneic mouse tumor models. Dinaciclib was tested alone and in combination with anti- PD1 mAb in (A and C) C57/BL6J, (B) BALB/c, and (D) Rag1–/– mice implanted with (A and D) MC38, (B) CT26, or (C) MB49 tumor cells. Tumor volume is represented as the mean ± SEM. The percentage of TGI on day 20 is pre- sented for each treatment group compared with the control group. Arrows indicate the treatment time points. Data represent at least 2 independent experiments (n = 10–12 mice/group). ***P < 0.001 and *P < 0.05, by 2-way ANOVA with Bonferroni post-test.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: Control

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 2. Dinaciclib and anti-PD1 combination therapy induces immune cell infiltration and activation in tumors. Mice with established CT26 tumors were treated with dinaciclib and anti-PD1 mAb as described in Figure 1. Tumors were isolated on day 14, and immune cells were analyzed by flow cytometry (n = 5 mice/group). Shown are the numbers of tumor-infiltrating (A) CD8+ T cells, (B) CD4+ T cells, and (E) CD11b+CD11c+ DCs in the different treatment groups. Also shown is the activation status of these cell populations as measured by the percentage of CD69+ CD4+ and CD8+ T cells (C and D) and MHCII, CD80, and CD86 mean fluorescence intensity (MFI) on DCs (F). For functional analysis, TILs were isolated from dissociated tumors using density-gradient centrifugation. For the detection of intracellular cytokines, harvested TILs were stimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours. Shown are the percentages of (G) IFN-γ+, (H) TNF-α+, and (I) GzB+ CD8+ T cells. Data represent at least 2 independent experiments. ***P < 0.001, **P < 0.01, and *P < 0.05, by 1-way ANOVA with Bonferroni post-test.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: Activation Assay, Isolation, Flow Cytometry, Fluorescence, Functional Assay, Gradient Centrifugation

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 3. Dinaciclib treatment induces a type I IFN signature within tumors. (A) Fluidigm qPCR analysis of MC38, CT26, and MB49 cells treated with dinaciclib in vitro for 24 hours either continuously or by washing and replacing medium after a 2-hour pulse. Heatmap indicates type I IFN signature genes with a greater-than 2-fold change (log10 scale) over the untreated control and a P value of less than 0.05. Genes with a FC of less than 2 and a P value of greater than 0.05 are blacked out. (B and C) Mice bearing 100 mm3 MC38 tumors were treated as described in Figure 1. Twenty-four hours after the first dose, tumors were isolated, and gene expression was analyzed by RNA sequencing (n = 5/group). (B) The top upregulated functional pathways in the dinaciclib and dinaciclib plus anti-PD1 groups as determined by GO analysis and IPA. (C) Expression of type I IFN response genes is depicted by a heatmap showing the log10 FC only of genes that were significantly upregulated (>2-fold and P < 0.01) compared with the isotype control group. Genes that were upregulated by less than 2-fold and that had a P value of greater than 0.01 are blacked out (represented as FC = 0).

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: In Vitro, Control, Isolation, Gene Expression, RNA Sequencing, Functional Assay, Expressing

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 4. Dinaciclib induces immunogenic cancer cell death. (A–D) CT26 cells were treated for 24 hours in vitro with dinaciclib at the indicated concentrations. Graphical data show (A) the percentage of tumor cell apoptosis, release of (B) HMGB1 and (C) ATP into the culture supernatants, and (D) surface expression of CRT on viable cells. Data represent the mean value ± SEM of 2 to 3 replicates from 1 representative experiment. ***P < 0.001 and *P < 0.05, for comparisons between individual dinaciclib-treated groups and the untreated group (0 μM). Statisti- cal data obtained via 1-way ANOVA with Bonferroni post-test. (E) CT26 cells either treated in vitro with dinaciclib or freeze- thawed were inoculated s.c. into BALB/c mice. After 10 days, mice were rechallenged with live CT26 cells. Shown is the percentage of tumor-free mice pooled from 2 indepen- dent experiments. ***P < 0.001, by log-rank (Mantel-Cox) test.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: In Vitro, Expressing

Journal: Journal of Clinical Investigation

Article Title: Dinaciclib induces immunogenic cell death and enhances anti-PD1–mediated tumor suppression

doi: 10.1172/jci94586

Figure Lengend Snippet: Figure 6. Dinaciclib induces PD1 expression and is efficacious in PD1-KO mice. (A) CT26, MB49, and MC38 tumor-bearing mice were treated as described in Figure 1. Tumors were isolated on day 14, and PD1 expression on CD8 + T cells was analyzed by flow cytometry (n = 5 mice per group). (B) WT or PD1-KO mice with established MB49 tumors (~100 mm3) were treated with dinaciclib or vehicle as described in Figure 1. Tumor growth is represented as the mean tumor volume ± SEM. Data represent at least 2 independent experiments. Arrows indicate the treatment time points. *P < 0.05 and ***P < 0.001, by unpaired t test (A) applied to calculate 2-tailed P value to estimate statistical difference between vehicle and dinaciclib treatment groups and 2-way ANOVA with Bonferroni post-test (B) applied to assess differences in tumor growth kinetics.

Article Snippet: The CT26 cell line (BALB/c mouse colon adenocarcinoma) was obtained from ATCC.

Techniques: Expressing, Isolation, Flow Cytometry

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Polymorphonuclear leukocytes induce PDGF release from IL-1beta-treated endothelial cells: role of adhesion molecules and serine proteases.

doi: 10.1161/01.atv.18.10.1534

Figure Lengend Snippet: Figure 1. Line plots showing the effect of PMNs on mitogen release from untreated or IL-1b–treated HUVEC: kinetics. HUVEC (2.53105/well) were incubated in the absence or presence of PMNs (106/mL), for 1, 3, 6, and 15 hours. Conditioned medium was collected, sedimented by centrifugation, and tested for [3H]-TdR incorporation into BALB/c 3T3, as described in Methods. Each point represents mean6SEM of 3 experiments.

Article Snippet: To determine the level of the total mitogenic activity of endothelial cell–conditioned medium, BALB/c 3T3 mouse fibroblasts (clone A31, American type Culture Collection) were used as previously described.23 The cells were grown to near confluence in 96-well microtiter plates and then induced to quiescence by 48-hour exposure to DMEM supplemented with 5% heat inactivated plasma–derived human serum.

Techniques: Incubation, Centrifugation

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

doi: 10.1007/s00018-010-0384-9

Figure Lengend Snippet: Cell death induction by enterococci in J774A.1 macrophages. J774A.1 macrophages were infected at different MOI with E. faecium ATCC 6057 and S. aureus ATCC 29213, respectively. Extracellular bacteria were killed 2.5 h after infection by the addition of lysozyme (10 mg/ml) or lysostaphin (40 μg/ml) and incubated further for 3.5 h. Six hours after infection, cells were stained with TMRE and propidium iodide (PI) and analyzed by flow cytometry. Lysostaphin (LS)-, lysozyme (LZ)-, FCCP (100 μM)-, staurosporine (Stauro, 4 μM)-treated cells, and cells heated for 10 min at 60°C were used as controls. Data shown in a are representative of three independent experiments and data shown in b are mean ± standard deviations of three independent experiments

Article Snippet: Table 1 Strain/isolate Description/reference ATCC 6057 E. faecium isolated from cheese ATCC 29212 E. faecalis isolated from urine ATCC 14025 E. avium isolated from human feces ATCC 8043 E. hirae isolated from intestine/gut ATCC43076 E. saccharolyticus ST4014 E. faecium isolated from stool, vancomycin-susceptible (colonized patient from our clinic) S269 esp + Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), positive for the esp (extracellular surface protein)-gene [ 39 ] S261 esp − Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), negative for the esp-gene [ 39 ] ATCC 29213 S. aureus isolated from wound Open in a separate window Enterococcus and Staphylococcus strains used in this study.

Techniques: Infection, Bacteria, Incubation, Staining, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

doi: 10.1007/s00018-010-0384-9

Figure Lengend Snippet: E. faecium induces cell death in bone marrow-derived macrophages (BMDM) independently of TLR2 and TLR4 signaling. BMDM obtained from C57BL/6x129Sv, TLR2−/−, TLR4−/−, and TLR2−/− × TLR4−/− mice, respectively, were infected at MOI 20 with E. faecium ATCC 6057. At 2.5 h after infection, extracellular bacteria were killed by the addition of lysozyme (LZ) (10 mg/ml) and incubated for another 3.5 h. At 6 h after infection, cells were stained with TMRE and propidium iodide (PI) and analyzed by flow cytometry. Lysozyme (LZ)-, staurosporine (Stauro, 4 μM)-treated cells, and cells heated for 10 min at 60°C were used as controls. Cells incubated with silica beads (Beads) (1 μm, MOI 20) with or without addition of lysozyme after 2.5 h were used as an additional negative control. Data shown are mean ± standard deviations of four independent experiments. P values result from the comparison of macrophages infected with E. faecium with or without addition of lysozyme

Article Snippet: Table 1 Strain/isolate Description/reference ATCC 6057 E. faecium isolated from cheese ATCC 29212 E. faecalis isolated from urine ATCC 14025 E. avium isolated from human feces ATCC 8043 E. hirae isolated from intestine/gut ATCC43076 E. saccharolyticus ST4014 E. faecium isolated from stool, vancomycin-susceptible (colonized patient from our clinic) S269 esp + Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), positive for the esp (extracellular surface protein)-gene [ 39 ] S261 esp − Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), negative for the esp-gene [ 39 ] ATCC 29213 S. aureus isolated from wound Open in a separate window Enterococcus and Staphylococcus strains used in this study.

Techniques: Derivative Assay, Infection, Bacteria, Incubation, Staining, Flow Cytometry, Negative Control, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

doi: 10.1007/s00018-010-0384-9

Figure Lengend Snippet: Mechanisms of cell death induced by E. faecium. J774A.1 macrophages were incubated with zVAD-fmk (zVAD, 100 μM), cytochalasin D (CytD, 2 μg/ml) for 1 h and then infected at MOI 20 with E. faecium ATCC 6057 and S. aureus ATCC 29213, respectively; 2.5 h after infection, bacteria were either not killed or killed by the addition of lysozyme (LZ) (10 mg/ml), rifampicin (Rifa, 20 μg/ml) or lysostaphin (LS) (40 μg/ml, all S. aureus conditions) and incubated further for 3.5 h. Cells were stained with TMRE and propidium iodide (PI) 6 h after infection and analyzed by flow cytometry. Uninfected, lysostaphin (LS)-, lysozyme (LZ)-, zVAD-, cytochalasin D-, rifampicin (20 μg/ml) and staurosporine (Stauro, 4 μM)-treated cells, cells heated for 10 min at 60°C, and cells incubated with silica beads (1 μm, MOI 20) to provide an antigen of similar size compared to that of enterococci were used as controls. Data shown are mean ± standard deviations of three independent experiments

Article Snippet: Table 1 Strain/isolate Description/reference ATCC 6057 E. faecium isolated from cheese ATCC 29212 E. faecalis isolated from urine ATCC 14025 E. avium isolated from human feces ATCC 8043 E. hirae isolated from intestine/gut ATCC43076 E. saccharolyticus ST4014 E. faecium isolated from stool, vancomycin-susceptible (colonized patient from our clinic) S269 esp + Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), positive for the esp (extracellular surface protein)-gene [ 39 ] S261 esp − Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), negative for the esp-gene [ 39 ] ATCC 29213 S. aureus isolated from wound Open in a separate window Enterococcus and Staphylococcus strains used in this study.

Techniques: Incubation, Infection, Bacteria, Staining, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

doi: 10.1007/s00018-010-0384-9

Figure Lengend Snippet: Characteristics of necrotic cell death in E. faecium-infected macrophages. J774A.1 macrophages were infected at MOI 20 with E. faecium ATCC 6057 if not otherwise depicted. At 2.5 h after infection, extracellular bacteria were either killed or not by the addition of lysozyme (LZ) (10 mg/ml) and then incubated further. a Lack of caspase 3 activation. At different time points after infection, cells were harvested, lysed, and immunoblotting for caspase 3 was performed from cell extracts. Data shown are representative of three independent experiments. Staurosporine (Stauro, 4 μM)-treated cells were used as a positive control. As a loading control, membranes were stripped and incubated with antibodies to β-actin. b Lack of cell shrinkage. The cell size was assessed by analysis of the mean forward scatter (FSC) at different time points after infection (upper panel). Staurosporine (Stauro, 4 μM)-treated cells and cells heated for 10 min at 60°C were used as controls. In parallel, the percentages of PI-positive cells were determined by flow cytometry (lower panel). Data shown are mean ± standard deviations of three independent experiments. c LDH release. At 6 h after infection with E. faecium ATCC 6057 at two different MOI cell culture supernatants were analyzed for their LDH content. All values (% control) are referred to untreated control cells (medium without lysozyme). As a positive control, cells heated for 10 min at 60°C were used. Data shown are mean ± standard deviations of three independent experiments performed in duplicates. d Transmission electron microscopic analysis. Electron microscopic pictures were taken from cells infected for 6 h with E. faecium ATCC 6057. Staurosporine (4 μM)-treated cells and cells heated for 10 min at 60°C were used as controls for apoptosis and necrosis, respectively. Bar, 2 μm each

Article Snippet: Table 1 Strain/isolate Description/reference ATCC 6057 E. faecium isolated from cheese ATCC 29212 E. faecalis isolated from urine ATCC 14025 E. avium isolated from human feces ATCC 8043 E. hirae isolated from intestine/gut ATCC43076 E. saccharolyticus ST4014 E. faecium isolated from stool, vancomycin-susceptible (colonized patient from our clinic) S269 esp + Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), positive for the esp (extracellular surface protein)-gene [ 39 ] S261 esp − Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), negative for the esp-gene [ 39 ] ATCC 29213 S. aureus isolated from wound Open in a separate window Enterococcus and Staphylococcus strains used in this study.

Techniques: Infection, Bacteria, Incubation, Activation Assay, Western Blot, Positive Control, Control, Flow Cytometry, Cell Culture, Transmission Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

doi: 10.1007/s00018-010-0384-9

Figure Lengend Snippet: An enterococcal protein is causative for cell death induction by E. faecium. J774A.1 macrophages were infected at MOI 200 with E. faecium ATCC 6057. Prior to infection of macrophages, viable bacteria were preincubated with PBS with or without the glycosidases lysozyme (LZ, 10 mg/ml) or mutanolysin (Mut, 400 U/ml) for 1 h at 37°C. Thereafter, bacterial suspensions were centrifuged, supernatants were removed, and pellets were incubated in PBS with or without proteinase K (ProtK, 100 or 300 μg/ml) or phospholipase C (PhosphoC, 2 U/ml) for 1 h at 37°C. After centrifugation, supernatants were discarded, and pellets, after resuspension in PBS, were used for infection of macrophages for 6 h. After staining with PI, macrophages were analyzed by flow cytometry. Data shown are mean ± standard deviations of one experiment performed in triplicates representative of four independent experiments. P values result from the comparison with cell death rates of the corresponding PBS-treated controls after treatment with LZ and Mut, respectively

Article Snippet: Table 1 Strain/isolate Description/reference ATCC 6057 E. faecium isolated from cheese ATCC 29212 E. faecalis isolated from urine ATCC 14025 E. avium isolated from human feces ATCC 8043 E. hirae isolated from intestine/gut ATCC43076 E. saccharolyticus ST4014 E. faecium isolated from stool, vancomycin-susceptible (colonized patient from our clinic) S269 esp + Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), positive for the esp (extracellular surface protein)-gene [ 39 ] S261 esp − Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), negative for the esp-gene [ 39 ] ATCC 29213 S. aureus isolated from wound Open in a separate window Enterococcus and Staphylococcus strains used in this study.

Techniques: Infection, Bacteria, Incubation, Centrifugation, Staining, Flow Cytometry, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages

doi: 10.1007/s00018-010-0384-9

Figure Lengend Snippet: Cell death induction by E. faecium in peritoneal macrophages in vivo. C57BL/6x129Sv mice were injected intraperitoneally with 108 E. faecium ATCC 6057 or PBS. After 2.5 h, a second i.p. injection with lysozyme (LZ) or PBS was administered contralaterally. The mice were killed 24 and 48 h after infection, peritoneal lavages were performed, and leucocyte counts were determined using a hematocytometer (a). Additionally, 24 h after infection, peritoneal cells were triple stained with TMRE, propidium iodide (PI), and APC-conjugated antibodies to F4/80. F4/80+ macrophages were analyzed for depolarization of the inner mitochondrial transmembrane potential (ΔΨm low cells) and uptake of propidium iodide (PI) (b)

Article Snippet: Table 1 Strain/isolate Description/reference ATCC 6057 E. faecium isolated from cheese ATCC 29212 E. faecalis isolated from urine ATCC 14025 E. avium isolated from human feces ATCC 8043 E. hirae isolated from intestine/gut ATCC43076 E. saccharolyticus ST4014 E. faecium isolated from stool, vancomycin-susceptible (colonized patient from our clinic) S269 esp + Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), positive for the esp (extracellular surface protein)-gene [ 39 ] S261 esp − Vancomycin-resistant E. faecium , patient’s isolate (outbreak 2005/06 at our clinic), negative for the esp-gene [ 39 ] ATCC 29213 S. aureus isolated from wound Open in a separate window Enterococcus and Staphylococcus strains used in this study.

Techniques: In Vivo, Injection, Infection, Staining