Journal: Computational and Structural Biotechnology Journal

Article Title: Evidence of quantum-entangled higher states of consciousness

doi: 10.1016/j.csbj.2025.03.001

Figure Lengend Snippet: Summary of specific biomarkers: descriptions, reference ranges, and measurement characteristics.

Article Snippet: BDNF levels were measured using the Rapid ELISA Kit (CE marked, Avantor®), requiring only a single drop of blood.

Techniques: Enzyme-linked Immunosorbent Assay, Acid Assay, Activity Assay, Activation Assay

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Expression, cytotoxic activity, and cytokine secretion of T cells expressing altered version of FcγRI. A, Illustration of the modified FcγRI structure with fused signaling domains: A2G (left), FcγRI composed of α-chain and homodimer of FcRγ chain; AG2G (middle), FcγRI α-chain with fused domain of FcRγ chain and homodimer of FcRγ; AGO2GO (right), FcγRI α-chain with fused FcRγ and OX40 domains and homodimer of FcRγ with fused OX40 domain. B, Flow cytometry analysis of A2G, AG2G, and AGO2GO expression following retroviral transduction of healthy donor T cells. C, Mean numbers of HT29 target cells expressing HER2 calculated by incuCyte software through 48-hour incubation with the three FcγRI-based receptor expressing T cells, with 1:1 E:T ratios with or without 6 μg/mL trastuzumab (data shows one of two experiments repeats, n = 4). Each sample is normalized to its cell number at time zero. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. D, Representative incuCyte images showing HER2-expressing HT29 cells, imaged and counted in live imaging system after 48 hours of incubation with different construct-expressing T cells with 1:1 E:T ratio. Scale bar is 100 μm. E, Human Luminex Discovery Assay measurement of cytokine levels in supernatants from 48 hours of culture of T cells 4:1 E:T ratio with HT29 cells expressing HER2 ( n = 4). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Activity Assay, Modification, Flow Cytometry, Retroviral, Transduction, Software, Incubation, Imaging, Construct, Luminex

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Characterization of AG2G-expressing T cells, activation, and cytotoxicity. A, ELISA measurement of cytokine levels of AG2G-expressing T cells after 48-hour incubation with HT29 cells expressing HER2. B, IFNγ and TNFα levels measured by ELISA in supernatants from 48-hour incubation of AG2G-expressing T cells and HT29 cells expressing HER2 in different E:T ratios. C, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with 0, 2.5, 5, 10 mg/mL IVIG, with or without 30 μg/mL trastuzumab ( n = 4). D, ELISA measurement of IFNγ levels of AG2G-expressing cells cocultured with HT29 cells expressing HER2 described in B . E, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-expressing T cells in combination with different concentrations of trastuzumab over 96 hours ( n = 2). F, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with different E:T ratios of AG2G-expressing T cells that were isolated for their CD4 (left) or CD8 (middle) population or with no isolation (right). G, ELISA measurement of cytokine levels from supernatants of 48-hour culture of 4:1 E:T ratios of isolated CD4, isolated CD8, or AG2G-expressing T cells with HT29 cells expressing HER2 and 30 μg/mL trastuzumab. Graphs show mean ±SD. Statistical significance was calculated using Student t test for a, two-way ANOVA with Sidak correction for multiple comparisons for B and C , two-way ANOVA with Tukey's correction for multiple comparisons for E and F . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Incubation, Software, Isolation

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: AG2G-expressing T cells differentiate between cells expressing high and low antigen levels, are more specific, and less exhausted, compared with classic CAR T cells. A, Mean numbers of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with transduced T cells in combination with antibodies (60 μg/mL each, n = 4). B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-transduced T cells incubated with HER2-expressing HT29 target cells, and with tumor-binding trastuzumab, or irrelevant antibodies cetuximab and rituximab (60 μg/mL). C, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. D, Normalized confluence of kidney epithelial cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells and 30 μg/mL trastuzumab, compared with trastuzumab-derived CAR (4:1 E:T ratio, n = 4). Each sample is normalized to its cell confluence at time zero. E, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D ( n = 4). F, ELISA measurement of IFNγ, granzyme B and TNFα levels in 24-hour supernatants of 10 5 AG2G-expressing T cells or CAR T cells incubated on immobilized trastuzumab or rHER2, in rising concentrations (mmol/mL) respectively. G, Confocal microscope imaging of internalization kinetics of PE-labeled IgG by AG2G-expressing cells (top), or PE-labeled HER2 by CAR T cells (bottom). H, PD-1, LAG-3, and TIM3 flow cytometry analysis of AG2G-expressing cells or CAR T cells after 48-hour incubation with or without 12 mmol/mL immobilized trastuzumab or HER2, respectively. Graphs show mean ± SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for A , C , D , and H . Two-way ANOVA with Sidak correction for multiple comparisons for E and F . One-way ANOVA with Dunnett correction for multiple comparisons for B . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Software, Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay, Derivative Assay, Microscopy, Imaging, Labeling, Flow Cytometry

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Human T cells expressing AG2G exert specific tumor cytotoxicity while sparing normal cells. A, Flow cytometry expression analysis of HER2 on different tumor cell lines and primary human normal cells. B, ELISA measurement of IFNγ levels in 48-hour supernatants of AG2G-expressing T cells with different tumor cells or primary normal cells in 4:1 E:T ratio and 60 μg/mL trastuzumab (results shown are from 3 different donors combined, n = 12). C, Quantification of HER2 receptor numbers using DAKO QIFIKIT (Agilent) beads by flow cytometer. Individual populations of the calibration are gated, a linear regression is calculated from the MFI and the ABC values of the five calibration bead populations using MS Excel. Using the linear regression, the ABCs of the beads coated with αHER2 is calculated from their MFI values. SDs are also retrieved from gated populations and transformed using linear regression. D, Mean counts of HT29 cells expressing HER2, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab (4:1 E:T ratio, n = 4). Each sample is normalized to its cell number at time zero. E, Normalized confluence of kidney epithelial (epithel) cells, calculated by incuCyte software, following incubation with AG2G-transduced T cells or ACTR707 and 30 μg/mL trastuzumab. Each sample is normalized to its cell confluence at time zero. Graphs are identical and described in . and but with the comparison to ACTR707 instead of CAR T. F, ELISA measurement of IFNγ levels in 48-hour supernatant obtained from transduced T cells and kidney epithel described in D and E ( n = 4). G, ELISA measurement of IFNγ levels in 24 hours supernatants of AG2G-expressing T cells or ACTR707 incubated with medium containing different concentration of IVIG ( n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Sidak correction for multiple comparisons. ****, P < 0.0001. Error bars represent standard error. Endothel, endothelial cells.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Transformation Assay, Software, Incubation, Comparison, Concentration Assay

Journal: Cancer Immunology Research

Article Title: T Cells Expressing a Modified FcγRI Exert Antibody-Dependent Cytotoxicity and Overcome the Limitations of CAR T-cell Therapy against Solid Tumors

doi: 10.1158/2326-6066.CIR-22-0423

Figure Lengend Snippet: Retroviral transduction of γδ-T cells with AG2G endows them with antitumor ADCC. A, Flow cytometry analysis of sham or AG2G-transduced αβ-T cells (two left panels, activated with IL2 and anti-CD3); sham or AG2G-transduced γδ-T cells (two right panels, activated with IL2 and zoledronic acid). B, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with AG2G-expressing γδ-T cells or AG2G-expressing αβ-T cells, with or without trastuzumab ( n = 3). C, Live cell numbers of HT29-HER2 expressing cells, calculated by incuCyte software, cocultured with sham untransduced or AG2G-expressing γδ-T cells with or without trastuzumab, in E:T ratios of 1:1, 2:1 or 4:1 ( n = 4). D, ELISA measurement of IFNγ levels in supernatants of 48-hour sham or AG2G-expressing γδ-T cells cocultured with HT29-HER2 expressing cells, with or without trastuzumab (E:T ratio 4:1, n = 3). Graphs show mean ±SD. Statistical significance was calculated using two-way ANOVA with Tukey's correction for multiple comparisons for B and C . Two-way ANOVA with Sidak correction for multiple comparisons for D . *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Error bars represent standard error.

Article Snippet: Cells were imaged by incuCyte S3 imager (Sartorius), and images were then used to calculate numbers of target cells or confluency by incuCyte software (version 2021A, basic analyzer).

Techniques: Retroviral, Transduction, Flow Cytometry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Medicine

Article Title: Relation between Serum Creatine Phosphokinase Levels and Acute Kidney Injury among ST-Segment Elevation Myocardial Infarction Patients

doi: 10.3390/jcm11041137

Figure Lengend Snippet: Baseline and in-hospital renal outcomes.

Article Snippet: NGAL levels were analyzed using NGAL rapid ELISA kits (Bioporto Diagnostics, Copenhagen, Denmark).

Techniques:

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Statin dose–response curves via Alamar Blue dye reduction assay. Human breast cancer cell lines (SK-BR-3, HCC1419, MDA-MB-231, and MDA-MB-468) were treated with increasing concentrations of ( A ) Simvastatin or ( B ) Fluvastatin in the presence (short dash) or absence (long dash) of recombinant Th1 cytokines (Tumor Necrosis Factor-alpha, TNF-α and Interferon-gamma, IFN-γ, 10 ng/mL each) for 72 h. Alamar Blue dye was added and, following color change, the optical density of the dye in the culture supernatants was determined. Optical Density (OD) values of untreated controls (black) and cytokine only treatment (gray) are represented as horizontal lines.

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Recombinant

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Combination of Th1 cytokines and statin drugs potentiates metabolic suppression in breast cancer lines. SK-BR-3, HCC1419, MDA-MB-231, and MDA-MB-468 human breast cancer cell lines were cultured with no additives (No Tx), treated with recombinant Th1 cytokines (“Cyto” TNF-α and IFN-γ, 10 ng/mL each), statin drugs (Simvastatin or Fluvastatin, 1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug (“Statin + Cyto”). After 72h incubation, Alamar Blue dye was added and, following color change, optical density of culture supernatants was determined. Results displayed are from one representative experiment of at least four trials +/− Standard Error of the Mean (SEM). Letter designations represent Tukey’s Honest Significant Difference (HSD) comparisons: treatments with the same letter designation are not statistically different; when letter designations differ between treatments, the p-value is less than 0.05.

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Cell Culture, Recombinant, Incubation

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Combination of Th1 cytokines and statin drugs maximize cell death in breast cancer lines. SK-BR-3, HCC1419, MDA-MB-231, and MDA-MB-468 human breast cancer cell lines were cultured with no additives (No Tx), treated with recombinant Th1 cytokines (TNF-α and IFN-γ, 10 ng/mL each), a statin drug ( A ) Simvastatin or ( B ) Fluvastatin (1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug ( A ) “Simva + Cyto” or ( B ) “Fluva + Cyto”. Flow cytometric results displayed in panels A and B are from one representative experiment. ( C ) Graphical interpretation of gated flow cytometric results comparing the percentage of stained events between groups: no additives (No Tx), treated with recombinant Th1 cytokines (TNFα and IFNγ, 10 ng/mL each), a statin drug (Simvastatin or Fluvastatin, 1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug (“Statin + Cyto”). Results displayed are from at least three trials +/− SEM. Letter designations represent Tukey’s HSD comparisons: treatments with the same letter designation are not statistically different; when letter designations differ between treatments, the p -value is less than 0.05.

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Cell Culture, Recombinant, Staining

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Statin drugs plus Th1 cytokines maximize mitochondrial membrane depolarization as assessed by tetramethylrhodamine ethyl ester (TMRE) staining. SK-BR-3, HCC1419, MDA-MB-231, and MDA-MB-468 human breast cancer cell lines were cultured with no additives (No Tx), treated with recombinant Th1 cytokines (TNF-α and IFN-γ, 10 ng/mL each), a statin drug ( A ) Simvastatin or ( B ) Fluvastatin (1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug ( A ) “Simva + Cyto” or ( B ) “Fluva + Cyto” for approximately 48 h. Flow cytometric results displayed in panels A and B are from one representative experiment. ( C ) Statistical analysis of flow cytometry results comparing the mean channel fluorescent intensity between groups: no additives (No Tx), treated with recombinant Th1 cytokines (TNF-α and IFN-γ, 10 ng/mL each), a statin drug (Simvastatin or Fluvastatin, 1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug (“Statin + Cyto”). Results displayed are from at least five trials +/− SEM. Letter designations represent Tukey’s HSD comparisons: treatments with the same letter designation are not statistically different; when letter designations differ between treatments, the p -value is less than 0.05.

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Membrane, Staining, Cell Culture, Recombinant, Flow Cytometry

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Statin drugs plus Th1 cytokines maximize apoptotic markers in breast cancer lines. SK-BR-3, HCC1419, MDA-MB-231, and MDA-MB-468 human breast cancer cell lines were cultured with no additives (No Tx), treated with recombinant Th1 cytokines (TNF-α and IFN-γ, 10 ng/mL each), a statin drug ( A ) Simvastatin or ( B ) Fluvastatin (1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug ( A ) “Simva + Cyto” or ( B ) “Fluva + Cyto”. Flow cytometric results displayed in panels A and B are of one representative experiment from 3 separate trials with each drug. ( C ) Statistical analysis of composite results for proportions of double-staining cell events between groups: no additives (No Tx), treated with recombinant Th1 cytokines (TNF-α and IFN-γ, 10 ng/mL each), a statin drug (Simvastatin or Fluvastatin, 1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug (“Statin + Cyto”). Results displayed are from three trials +/− SEM. Letter designations represent Tukey’s HSD comparisons: treatments with the same letter designation are not statistically different; when letter designations differ between treatments, the p -value is less than 0.05.

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Cell Culture, Recombinant, Double Staining

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Cells treated with a statin drug and Th1 cytokines demonstrate enhanced activation of caspase 3/7. SK-BR-3, HCC1419, MDA-MB-231, and MDA-MB-468 human breast cancer cell lines were cultured with no additives (No Tx), treated with recombinant Th1 cytokines (TNF-α and IFN-γ, 10 ng/mL each), a statin drug ( A ) Simvastatin or ( B ) Fluvastatin (1 µM MDA-MB-231; 10 µM remaining cell lines), or the combination of Th1 cytokines and a statin drug ( A ) “Simva + Cyto” or ( B ) “Fluva + Cyto” and stained with CellEvent™ fluorescent caspase substrate. The experiment was repeated at least three times; the displayed flow cytometric results are from one representative experiment. The percentage of stained events, as well as the geometric mean channel fluorescence (in parentheses), is annotated on each histogram.

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Activation Assay, Cell Culture, Recombinant, Staining, Fluorescence

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Combined treatment with Th1 cytokines and statin drugs potentiates K-Ras delocalization from membrane. MDA-MB-231 human breast cancer cells were cultured with no additives (No Tx), treated with recombinant Th1 cytokines (TNF-α and IFN-γ, 10 ng/mL each), a statin drug (Simvastatin or Fluvastatin, 1 µM), or the combination of Th1 cytokines and a statin drug (“Simva + Cyto” or “Fluva + Cyto”) for 24 h. Membrane proteins were separated from cytosolic proteins and the distribution of the K-Ras protein was analyzed by Western blot. ( A ) Representative Western blot images and ( B ) corresponding semi-quantitative densitometry analysis of K-Ras normalized to β-actin loading control. Representative Western blot images of whole-cell K-Ras, AKT, ERK ½, and phospho-ERK ½ after ( C ) 24 and ( D ) 72 h of treatment. Semi-quantitative densitometry analysis of the ( E ) GAPDH-normalized total AKT, ( F ) GAPDH-normalized total ERK-1/2, and ( G ) GAPDH-normalized phosphorylated ERK-1/2. All Western blot experiments were repeated at least three times. Displayed graphical results in panels ( E ), ( F ), and ( G ) are from at least three trials +/− SEM. Letter designations in panel ( E ) represent Tukey’s HSD comparisons: treatments with the same letter designation are not statistically different; when letter designations differ between treatments, the p -value is less than 0.05.

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Membrane, Cell Culture, Recombinant, Western Blot

Journal: Vaccines

Article Title: Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis and Ras Delocalization in Human Breast Cancer Lines and Combine with Dendritic Cell-Based Immunotherapy to Suppress Tumor Growth in a Mouse Model of HER-2 pos Disease

doi: 10.3390/vaccines8010072

Figure Lengend Snippet: Simvastatin does not interfere with T cell function and enhances effects of immunotherapy in a mouse model of HER-2 pos breast cancer. ( A ) Example IFN-γ EliSpot wells from individual healthy donor Peripheral Blood Mononuclear Cells (PBMCs) stimulated with a mixture of common viral peptide recall antigens (left panel) or tetanus toxoid (right panel) in the presence or absence of simvastatin (“Simva”; 1–10 µM). ( B ) Composite analysis of each of the 3 donors for viral recall peptides (left panel) and 3 donors for tetanus toxoid (right panel). Statistical analysis by one-way ANOVA indicated no significant difference between simvastatin-treated and untreated groups. ( C ) IFN-γ ELISA analysis of 72 h culture supernatants from allogeneic Mixed Leukocyte Reactions (MLRs) where activated dendritic cells (DC) and lymphocyte-rich elutriation fractions were co-cultured at 1:40 stimulator:responder ratios in the presence or absence of 1–10 µM simvastatin. Data displayed represents the mean IFN-γ production from seven unique allogeneic DC:lymphocyte pairings. Statistical analysis by one-way ANOVA indicated no significant difference between simvastatin-treated and untreated groups (B.D. = below detection). ( D ) TUBO-bearing Balb/c mice, 7 days after implantation, were either left untreated (“No Rx”) or treated with Simvastatin (“Simva”), peptide-pulsed DC-based therapy (“DC”), or given combined treatment with simvastatin plus peptide-pulsed DCs (“Both”). Tumor size was measured periodically with calipers during treatment. Tumor size denoted as average area +/− SEM. Statistical significance is indicated by the asterisk (*). ( E ) TUBO cells were cultured in vitro with IFN-γ plus TNF-α, IFN-γ only, or no treatment in the presence of increasing concentrations of Simvastatin for 72 h, then assessed for metabolic activity by the Alamar Blue dye assay. ( F ) TUBO-bearing Balb/c mice, 7 days after implantation, were either left untreated (“No Rx”) or treated with Simvastatin (“Simva”), Interferon-γ (“IFN-γ”) or given combined treatment with simvastatin plus IFN-γ (“Both”). Tumor size was measured periodically with calipers during treatment. Tumor size denoted as average area +/− SEM. Statistical significance is indicated by the asterisk (*).

Article Snippet: For EliSpot assays, human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from CTL Corporation (Shaker Heights, OH, USA) and stimulated in vitro with viral peptide ‘plus’ peptide pool (10 µL/well; CTL corp) or Tetanus Toxoid (2 µg/mL; Sigma) in the presence or absence of 1–10 µM simvastatin in 96-well IFN-γ EliSpot assay plates (CTL corp).

Techniques: Cell Function Assay, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Cell Culture, In Vitro, Activity Assay