Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia concentration is increased in cancer cell–conditioned medium and TIF. Ammonia concentration in the tumor-conditioned medium, collected after 48 hours of incubation, was measured using a Dimension Ammonia assay (Siemens; n = 3). Cells were cultured at different densities: lymphoma (0.5 × 10 6 , 1.0 × 10 6 , 1.5 × 10 6 /mL; A ), multiple myeloma (1 × 10 6 , 2 × 10 6 , 4 × 10 6 /mL; B ), and breast cancer cell lines (0.5 × 10 6 , 1 × 10 6 , 1.5 × 10 6 /mL; C ). In A–C , empty medium incubated for 48 hours (without the cells) at 4°C or 37°C is presented as a control. D, Schematic presentation of the TIF and SCF isolation from mice. TIF was collected from tumors not exceeding 1,500 mm 3 . SCF was isolated at the same time from the contralateral tight as a control tissue fluid. E–G, The concentration of ammonia in TIF and SCF isolated from Raji tumor–bearing NSG ( n = 5; E ), MM.1s tumor–bearing SCID mice ( n = 5; F ), and breast cancer–bearing mice (EMT6 in BALB/c mice, n = 5; E0771 in BALB/c mice, n = 5; 4T1 in BALB/c mice; n = 5; MCF7 in NSG mice, n = 4; MDA-MB-231 in NSG mice, n = 3; G ). P values were calculated using a paired t test. Data show individual values and means ± SEM. n values are the number of biological replicates in in vitro experiments or the number of mice used to obtain the data. D, Created with BioRender.com. Winiarska, M. (2025) https://BioRender.com/x56a982 .

Article Snippet: In the case of the experiments involving MCF7 cells, slow-release pellets containing 17β-estradiol (Innovative Research of America) were implanted subcutaneously 4 days before tumor cell inoculation.

Techniques: Concentration Assay, Incubation, Cell Culture, Control, Isolation, In Vitro

Journal: Cancer Research

Article Title: Ammonia Suppresses the Antitumor Activity of Natural Killer Cells and T Cells by Decreasing Mature Perforin

doi: 10.1158/0008-5472.CAN-24-0749

Figure Lengend Snippet: Ammonia inhibits natural cytotoxicity and ADCC of NK cells and CAR NK cells. A, The viability of NK cells incubated with different concentrations of NH 4 Cl for 4 hours was assessed using propidium iodide staining and flow cytometry ( n = 3). B, Natural cytotoxicity of NK cells against K562 cells in the presence of different concentrations of NH 4 Cl ( n = 3). K562 cells were stained with CFSE and incubated with NK cells in different concentrations of ammonia. C, RTX-dependent cell cytotoxicity of NK cells against Raji cells in the presence of different concentrations of NH 4 Cl ( n = 4). Raji cells were stained with CFSE and incubated with NK cells and 100 μg/mL RTX in different concentrations of ammonia. D, Daratumumab (Dara)-dependent cell cytotoxicity of NK cells against Daudi cells in the presence of different concentrations of NH 4 Cl ( n = 5). Daudi cells were stained with CFSE and incubated with NK cells and 1 μg/mL daratumumab in different concentrations of ammonia. In B–D , cytotoxicity was assessed after 4 hours using flow cytometry and plotted as the percentage of propidium iodide–positive CFSE-positive target tumor cells. E, Trastuzumab-dependent cell cytotoxicity of NK cells against MCF7 cells in the presence of different concentrations of NH 4 Cl ( n = 4). Cytotoxicity was assessed using RTCA for 12 hours. The right panel presents the normalized cell index at the 12-hour time point. F, CD19 CAR NK cell cytotoxicity against Raji cells in the presence of different concentrations of NH 4 Cl ( n = 3). Cytotoxicity was determined after 18 hours in a luciferase-based killing assay, with Raji cells stably expressing luciferase as target cells. G, PD-L1 CAR NK cell cytotoxicity against MDA-MB-231 cells in the presence of 5 mmol/L NH 4 Cl ( n = 3). Cytotoxicity was assessed using RTCA for 24 hours. P values were calculated using two-way ANOVA with Tukey post hoc test. Data show individual values and means ± SEM. n values are the numbers of biological replicates in in vitro experiments.

Article Snippet: In the case of the experiments involving MCF7 cells, slow-release pellets containing 17β-estradiol (Innovative Research of America) were implanted subcutaneously 4 days before tumor cell inoculation.

Techniques: Incubation, Staining, Flow Cytometry, Luciferase, Stable Transfection, Expressing, In Vitro

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: (2A) RAW 264.7 cells were co-incubated with NIH1 (left) and ATCC 17978 (right) isogenic wild-type strains and Δ gtr6 mutants. (2B) RAW 264.7 cells were co-incubated with ATCC 17978, the HUMC1:: gtr6 mutant strain with repaired gtr6 , or wild-type HUMC1. (2C) RAW 264.7 cells were co-incubated with ATCC 17978 wild type, Δ gtr6 , Δ gtr6 /pSC1a (the knockout mutant with a plasmid-borne functional copy) in the presence of complement-active serum, and Δ gtr6 /pSC1a in the presence of heat-inactivated serum. *p < 0.001. (2D) Gentamicin protection assay with RAW 264.7 cells and wild-type ATCC 17978 (black bars) or ATCC 17978 Δ gtr6 (white bars). Cytochalasin D was added as an inhibitor of phagocytosis. Total bacteria plated for CFUs and expressed as a proportion of initial bacterial inoculum. * = significant vs. bacteria-only group, ⸸ = significant vs. bacteria + RAW 264.7 cell group. *,⸸ = p < 0.01 (2E) RAW 264.7 cells were incubated with ATCC 17978, HUMC1, ATCC 17978 Δ gtr6 , and HUMC1:: gtr6 . Stained with Wright-Giemsa stain, total magnification is 1000x. Results are from two repeat experiments with duplicate samples in each. White arrows denote adherent or internalized bacteria.

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Incubation, Mutagenesis, Knock-Out, Plasmid Preparation, Functional Assay, Staining, Giemsa Stain

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: ( 4A ) 2.0×10 8 CFU of ATCC 1778 and HUMC1 had total capsule carbohydrate capsule extracted in parallel and total carbohydrate content measured via phenol-sulfuric acid colorimetry. ( 4B ) Incubation of macrophages and bacteria with purified capsule from gtr6 + (ATCC 17978, 15827) and gtr6 - (HUMC1, ATCC 17978 Δ gtr6 ) strains. Extract-free uptake was used as a control. * p < 0.0001 (4C) RAW 264.7 cells were pre-incubated with soluble mannan (0.5mg/mL), laminarin (0.5mg/mL), and dextran sulfate (0.1mg/mL) or an untreated control prior to co-incubation with ATCC 17978. *p < 0.0001. Two biological replicates for in vitro . Wide bars denote median, error bars denote IQR.

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Colorimetric Assay, Incubation, Purification, In Vitro

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: (5A) RAW 264.7 cells were pre-incubated with anti-Dectin-1, anti-CR3, anti-MR neutralizing monoclonal antibodies or an isotype control prior to co-incubation with ATCC 17978. *p < 0.0005, * *p < 0.0001 (5B) Knockdown of Dectin-1 and/or CR3 in RAW 264.7 cells followed by incubation with ATCC 17978. *p < 0.0001 (5C) Primary peritoneally-elicited macrophages from C57BL/6 mice followed by phagocytosis assays with ATCC 17978. *p < 0.05, * *p < 0.0001 (5D) Phagocytosis assays of ATCC 17978 with peritoneal neutrophils from wild-type mice with disruption of phagocytosis upon the addition of heat-inactivated serum (HI-S) or complement-active serum (CA-S). *p < 0.0001 (5E) Phagocytosis assays with RAW 264.7 macrophages with gtr6 + and capsule-free strains (ATCC 17978 WT, 15827, ATCC 17978 ΔitrA ), and gtr6 - strains (ATCC 17978 Δgtr6 , HUMC1), with complement active (CA-S) or heat-inactivated (HI-S) serum. *p < 0.0001. Experiments repeated once with two biological replicates. Wide bars denote median, error bars denote IQR.

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Incubation

Journal: PLoS Pathogens

Article Title: Capsule carbohydrate structure determines virulence in Acinetobacter baumannii

doi: 10.1371/journal.ppat.1009291

Figure Lengend Snippet: (6A) Incubation of RAW 264.7 cells with ATCC 17978 in the presence of 100μg/mL GlcNAc (NAG), a CR3 lectin domain inhibitor. (6B) Serial two-fold dilutions of complement-active mouse serum in a RAW 264.7 cell phagocytosis assay with ATCC 17978. *p < 0.0001 (6C) Male C57BL/6 mice aged 10 weeks were infected intravenously with 2.0×10 8 CFUs of 15827, with or without administration of 15μg cobra venom factor (CVF) 48 h prior to infection. *p < 0.001. Experiments repeated once, n = 5 per group for in vivo and two technical replicates for in vitro .

Article Snippet: Bacterial strains were grown in Tryptic Soy Broth (TSB) (VWR, Radnor, PA USA #90000–372) overnight at 37°C with shaking at 200 rpm, sub-cultured to logarithmic phase, washed three times in PBS, diluted to 2×10 8 CFUs/mL based on OD 600 measurements, and added to RAW 264.7 cells at a multiplicity of infection of 20:1 in Hanks’ Balanced Salt Solution (HBSS) (VWR, #45001–101) supplemented with 10% complement-active CD-1 mouse serum (Innovative Research Inc., Novi, MI USA).

Techniques: Incubation, Phagocytosis Assay, Infection, Combined Bisulfite Restriction Analysis Assay, In Vivo, In Vitro