Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: VPA enhances the sensitivity of pancreatic cancer cells to NK cell-mediated lysis via a NKG2D-dependent mechanism. (A) VPA sensitized pancreatic cancer cells to NK cell-mediated lysis; * P < 0.05; ** P < 0.01. (B) Blockade of NKG2D attenuated the ability of VPA to sensitize pancreatic cancer cells to NK cell-mediated lysis; * P < 0.05 for VPA + IgG vs. VPA + NKG2D; ** P < 0.01 for VPA + IgG vs. VPA + NKG2D. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. E:T, effector/ target ratio.

Article Snippet: The human pancreatic adenocarcinoma cell lines PANC-1, MIA PaCa-2, and BxPC-3, and the human natural killer cell line NK-92 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Lysis

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: PI3K/Akt signaling is required for VPA-induced upregulation of MICA and MICB in pancreatic cancer cells. (A, B) Quantitative real-time RT-PCR analysis. The VPA-induced upregulation of MICA and MICB mRNA expression were inhibited by the HER2/HER3 inhibitor lapatinib and the PI3K inhibitor LY294002, but not by the ATM/ATR inhibitor caffeine. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. * P < 0.05; ** P < 0.01; ns P > 0.05. (C) Flow cytometry analysis. The VPA-induced upregulation of MICA and MICB protein expression on the cell surface were inhibited by the PI3K inhibitor LY294002. MFI, mean fluorescence intensity; * P < 0.05. (D) Western blotting analysis. Transfection of the PI3KCA siRNA inhibited PI3KCA protein expression at 48 h post-transfection. NC, negative control; siR1-3, PI3KCA_siR sequence 1-3; ** P < 0.01. (E) Quantitative real-time RT-PCR analysis. VPA-induced upregulation of MICA and MICB mRNA expression were attenuated in PI3KCA-knockdown cells; Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01. (F) Flow cytometric analysis. VPA-induced upregulation of MICA and MICB protein expression on the cell surface were attenuated in PI3KCA-knockdown cells. MFI, mean fluorescence intensity; * P < 0.05; ** P < 0.01. (G) LDH release assay. The VPA-induced susceptibility of cancer cells to NK cell-mediated cell lysis was reduced in PI3KCA-knockdown cells. Data are mean ± SD of a single experiment performed in triplicate, all results were reproducible in three independent experiments. ** P < 0.01 and * P < 0.05 for NC vs. siR2; ▲▲ P < 0.01 and ▲ P < 0.05 for NC vs. siR3. siR2, PI3KCA siR sequence 2; siR3, PI3KCA siR sequence 3.

Article Snippet: The human pancreatic adenocarcinoma cell lines PANC-1, MIA PaCa-2, and BxPC-3, and the human natural killer cell line NK-92 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Expressing, Flow Cytometry, Fluorescence, Western Blot, Transfection, Negative Control, Sequencing, Lactate Dehydrogenase Assay, Lysis

Journal: BMC Cancer

Article Title: Valproic acid sensitizes pancreatic cancer cells to natural killer cell-mediated lysis by upregulating MICA and MICB via the PI3K/Akt signaling pathway

doi: 10.1186/1471-2407-14-370

Figure Lengend Snippet: VPA sensitizes pancreatic cancer cells to NK cell-mediated lysis in vivo by upregulating the expression of MICA and MICB. (A, B) Excised xenograft tumors and growth curves for the xenografts; VPA enhanced the growth inhibitory effect of NK cells in vivo ; this effect was attenuated by the PI3K inhibitor LY294002. Data are mean ± SD of five mice per group; * P < 0.05 for NK-92 vs. NK-92 + VPA; Δ P < 0.05 for NK-92 + VPA + LY294002 vs. NK-92 + VPA. (C) Immunohistochemical staining and quantification of MICA and MICB expression in the xenograft tumors; VPA significantly upregulated the expression of MICA and MICB in the pancreatic cancer xenograft tumors in vivo , this effect was attenuated by the PI3K inhibitor LY294002.

Article Snippet: The human pancreatic adenocarcinoma cell lines PANC-1, MIA PaCa-2, and BxPC-3, and the human natural killer cell line NK-92 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Lysis, In Vivo, Expressing, Immunohistochemical staining, Staining

Journal: Theranostics

Article Title: Inhibition of tumor progression and M2 microglial polarization by extracellular vesicle-mediated microRNA-124 in a 3D microfluidic glioblastoma microenvironment

doi: 10.7150/thno.60851

Figure Lengend Snippet: Intratumoral infiltration of NK cells, induced by miR-124 EVs in a tri-culture system. (A) U373MG and microglia were embedded in a collagen gel at a ratio of 2:1, 2 days before seeding of NK cells. Representative images of NK cells on ( B ) day 2 and ( C ) day 4 in the microfluidic device treated with miR-NC EVs or miR-124 EVs. Live NK cells were immunostained with PE-conjugated CD 45 (red). (D) Maximum infiltration distance, ( E ) average infiltration distance, and ( F ) infiltration area of NK cells were investigated. N indicates the total number of ROI. Box-and-whisker plots show all individual points on the box.** p < 0.01, *** p < 0.001. Scale bar represents 200 µm.

Article Snippet: The human NK cell line NK-92 was purchased from American Type Culture Collection (ATCC, USA).

Techniques: Whisker Assay

Journal: medRxiv

Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

doi: 10.1101/2022.08.12.22278726

Figure Lengend Snippet: Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: CD16.NK-92 (ATCC PTA-6967) were purchased from ATCC (ATCC PTA-6967), grown at 37C, 5% CO2 and maintained in in MEM-α supplemented with 12.5% FBS, 12.5% horse serum, 1.5g/L sodium bicarbonate, 0.02mM folic acid, 0.2mM inositol, 0.1 mM 2-β-mercaptoethanol, 100U/mL recombinant IL-2.

Techniques: Neutralization, Binding Assay, Activation Assay, Expressing

Journal: medRxiv

Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

doi: 10.1101/2022.08.12.22278726

Figure Lengend Snippet: Vaccine specific IgG induction of FcγRIIIa/CD16 effector functions correlate with neutralization of wildtype and SARS-CoV-2 clinical variants. Relationships between live SARS-CoV-2 WA1 neutralization (FRNT50) and receptor binding domain (RBD) specific relative binding to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) FcγRIIb/CD32b, RBD specific antibody dependent natural killer cell activation (ADNKA) determined by (D) CD107a expression, (E) IFNγ production and (F) TNFα secretion, and (G) RBD specific antibody dependent neutrophil phagocytosis (ADNP) are shown. (H) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions ADNKA, antibody dependent cellular phagocytosis (ADCP), ADNP and antibody dependent complement deposition (ADCD). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) in duplicate for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10 4 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences).

Techniques: Neutralization, Binding Assay, Activation Assay, Expressing

Journal: medRxiv

Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

doi: 10.1101/2022.08.12.22278726

Figure Lengend Snippet: Differential fucose and sialic acid on vaccine specific IgG link to FcγRIIIa/CD16a effector functions. Stacked column graphs depict the relative abundance of individual glycoforms ( – ) with respect to (A) total bulk non-antigen specific and (B) receptor binding domain (RBD) specific IgG. Each column represents one individual study participant. Dot plots summarize differences between bulk non-antigen specific and RBD specific IgG in the collective relative abundance of all individual glycoforms ( and ) containing (C) fucose, (D) total sialic acid, (E) di-sialic acid, (F) mono-sialic acid and (G) di-galactose with statistical significance calculated by Wilcoxon matched-pairs signed rank test. Data for (H) asialylated fucosylated, (I) total sialic and (J) total di-sialic acid, the three RBD specific glycoforms that have a statistically significant relationship across all markers of ADNKA activation, are plotted with CD107a expression per RBD specific IgG, as well as IFNγ and TNFα . For comparison, data for (K) total mono-sialic acid is plotted.

Article Snippet: Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) in duplicate for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10 4 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences).

Techniques: Binding Assay, Activation Assay, Expressing

Journal: medRxiv

Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

doi: 10.1101/2022.08.12.22278726

Figure Lengend Snippet: Age influences some but not all vaccine specific antibody FcγR functions. The relationships between relative binding of receptor binding domain (RBD) specific IgG to (A) FcγRIIIa/CD16a, (B) FcγRIIa/CD32a and (C) inhibitory FcγRIIb/CD32b and age are shown. (D) Heatmap of the coefficient of determination (r 2 ) summarizes the goodness of fit across RBD and control respiratory syncytial virus (RSV), influenza (Flu) and anthrax antigens in FcγR binding and age. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; N/A not available given absence of significant detectable levels. The relationship between age and RBD antibody dependent natural killer cell activation (ADNKA) as measured by (E) CD107a and (F) TNFα, and (G) RBD antibody dependent cellular phagocytosis (ADCP) and (H) RBD antibody dependent neutrophil phagocytosis (ADNP) are shown. Linear regression with p values adjusted for sex are reported. (I) Radar plots depict vaccine specific IgG glycoforms calculated from the Z scored data for each individual RBD specific IgG glycoforms relative to bulk non-antigen specific IgG glycoforms with lines representing the median for each age group.

Article Snippet: Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) in duplicate for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10 4 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences).

Techniques: Binding Assay, Activation Assay

Journal: medRxiv

Article Title: BNT162b2 induced neutralizing and non-neutralizing antibody functions against SARSCoV-2 diminish with age

doi: 10.1101/2022.08.12.22278726

Figure Lengend Snippet: Enhanced BNT162b2 induced polyfunctional antibody breadth and magnitude against SARS-CoV-2 in younger compared to older adults. For each individual, the neutralization breadth across variants and cumulative vaccine specific Fc functional magnitude from the sum of the Z scores for each of the individual effector functions were calculated. (A) Grouped by neutralization breadth (top), each column shows the cumulative Fc functional score for one individual. Median, minimum and maximum ages characterizing each neutralization breadth group are shown (bottom). Polyfunctional antibody breadth was calculated for each individual and used to categorize individuals into high (90–100%), medium (80–90%) or low (<80%) responders. The proportions of high, median and low responders are grouped by (B) age and (C) sex. (D) Radar plots depict vaccine specific polyfunctional antibody magnitude calculated from the Z scored data for each antibody function with lines representing the median for each age group. (E) Heatmap summarizes Spearman correlations between neutralization of SARS-CoV-2 wildtype WA1 and variants with RBD specific IgG/M/A, IgG and IgA levels, relative binding of RBD specific IgG to activating (FcγRIIIa/CD16a and FcγRIIa/CD32a), inhibitory (FcγRIIb/CD32b) and ratios of activating:inhibitory FcγR (FcγRIIIa/CD16a:FcγRIIb/CD32b and FcγRIIa/CD32a:FcγRIIb/CD32b) binding and Fc effector functions antibody dependent natural killer cell activation (ADNKA), antibody dependent cellular phagocytosis (ADCP), antibody dependent neutrophil phagocytosis (ADNP) and antibody dependent complement deposition (ADCD) for each age group. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Article Snippet: Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) in duplicate for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 10 4 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences).

Techniques: Neutralization, Functional Assay, Binding Assay, Activation Assay