Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Probiomimetics-Novel Lactobacillus-Mimicking Microparticles Show Anti-Inflammatory and Barrier-Protecting Effects in Gastrointestinal Models.

doi: 10.1002/smll.202003158

Figure Lengend Snippet: Figure 6. Cytokine production in inflamed macrophage-like dTHP-1 cells as well as in enterocyte-like Caco-2 cells. Cells were stimulated with lipopoly- saccharides (10 µg mL-1) and cotreated with probiomimetics or MVs. Supernatants were harvested after 6 or 24 h, and the protein content was analyzed using ELISA. To exclude the effects of different MV concentrations during the assembly of the probiomimetics, all results were normalized to the protein content. a) ELISA measurement of proinflammatory TNF-α released in dTHP-1 cells after 6 h. b) Release of anti-inflammatory IL-10 after 6 h. c) ELISA measurement of proinflammatory TNF-α released in dTHP-1 cells after 24 h. d) Release of anti-inflammatory IL-10 after 24 h. e) Release of proinflam- matory IL-8 in enterocyte-like Caco-2 cells after 24 h. Values represent the mean of 3–9 biological replicates with standard deviations.

Article Snippet: The supernatants were thawed and the concentrations of IL-10 and TNF-α were analyzed using Human IL-10 ELISA Set (Diaclone, Besançon, France), Human TNF-α ELISA Set (Diaclone, Besançon, France), and Human IL-8 ELISA Set (Diaclone, Besançon, France), respectively, according to the supplier’s protocols.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell reports. Medicine

Article Title: Targeting neoadjuvant chemotherapy-induced metabolic reprogramming in pancreatic cancer promotes anti-tumor immunity and chemo-response.

doi: 10.1016/j.xcrm.2023.101234

Figure Lengend Snippet: Figure 6. Targeting CD36 synergistically promoted AG-mediated killing of PDAC in preclinical models (A) Visual presentation of subcutaneous xenograft murine PDAC tumor models (C57 mice) for each group. (B) Measurement of tumor volumes showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models. (C) Measurement of tumor weights showed CD36 blockage synergistically promoted AG-mediated killing of PDAC in subcutaneous xenograft murine PDAC tumor models (n = 5). (D) Representative IHC staining showed Ki67 expression in subcutaneous xenografts treated with different regimens. (E) t-Distributed stochastic neighbor embedding (TSNE) analyses showed the clustering for CD36+ CD8+ T cells and GZMB+ CD8+ T cells. (F) Flow cytometry revealed that more CD8+ T cells infiltrated PDAC with NAC, while the percentage of CD36+ CD8+ T cells also increased (n = 5) (mean with standard deviation). (G) ELISA results showed the combination of AG and CD36 blockade significantly improved IFN-g and tumor necrosis factor a (TNF-a) levels intratumorally (n = 5). (H) Representative image of orthotopic murine models of PDAC. (I) Kaplan-Meier curve revealed the combination of CD36 blockade and AG significantly prolonged the survival interval of mice that received orthotopic PDAC cell transplantation (n = 10). Circle or square referred to a happened event (death or censored). Censored event means the mice is still alive at the time point that we ended follow-up. (J) CD36 blockade synergistically with AG regimens optimally narrowed the PDAC tumor size in a humanized PDX model (n = 10). (K) Representative IHC staining image of CD36-high and -low PDAC. (L) Kaplan-Meier curve showed increased CD36 expression predicted worse prognosis of PDAC patients with adjuvant AG chemotherapy. The statistical sig- nificance shown in this figure was detected using t test.

Article Snippet: The ELISA kits used in the present study were as follows: Human IFN-gamma ELISA Kit (absin, abs510007); Human IL-2 ELISA Kit (Boster, EK0397); Mouse TNF alpha ELISA Kit (absin, abs520010) and Mouse IFN- gamma ELISA Kit (absin, abs520007).

Techniques: Immunohistochemistry, Expressing, Flow Cytometry, Standard Deviation, Enzyme-linked Immunosorbent Assay, Transplantation Assay, Adjuvant

Journal: Frontiers in Immunology

Article Title: Effective cancer immunotherapy combining mRNA-encoded bispecific antibodies that induce polyclonal T cell engagement and PD-L1-dependent 4-1BB costimulation

doi: 10.3389/fimmu.2024.1494206

Figure Lengend Snippet: Functional characterization and mechanisms of action of LiTE and Albu-LiTCo antibodies. Schematic representation of EGFR-targeted LiTE and Albu-LiTCo antibodies (A) . LiTE-mediated EGFR-dependent activation of tumor cells (B) . CT26 and CT26 EGFR were cocultured with mouse splenocytes (E:T ratio 5:1) in the presence of LiTE or anti-CD3 IgG at 3.34 nM equimolar concentration. Mouse CD69 expression was measured by flow cytometry after 24 hours (B) . LiTE-mediated EGFR-dependent cytotoxicity of tumor cells (C) . Luciferase-expressing CT26 and CT26 EGFR cells were cocultured with splenocytes (E:T ratio 5:1) and different concentrations of LiTE. Specific lysis was measured by bioluminescence at 48 hours. Percent specific lysis was calculated relative to control. Data are mean ± SD (n = 3). Significance was calculated by unpaired Student’s t test. Competition ELISA for PD-L1/PD-1 interaction inhibition by anti-PD-L1 IgG or Albu-LiTCo (D) . Data are mean ± SD (n = 3). One representative experiment from two independent experiments is shown. LogIC50 is shown. Blocking activity of Albu-LiTCo in a cell-based bioassay (E) . JurkatPD-1 cells were cocultured with CHO PD-L1 cells and increasing concentrations of anti-PD-L1 IgG or Albu-LiTCo. Luminescence was measured after 6 hours. Data are expressed as fold induction relative to unstimulated JurkatPD-1 cells. Representative dose-concentration curves are shown as mean ± SD (n = 3). IC50 is shown. Agonistic activity of Albu-LiTCo (F) . Wild type CHO cells or CHO PD-L1/EGFR were cocultured with LiTE-activated mouse CD8a + T cells (3.34 nM) in the presence of Albu-LiTCo, anti-4-1BB IgG or anti-PD-L1 IgG at 6.67 nM equimolar dose. IFNγ secretion was determined after 72 h Negative controls (–) consisted of CD8 + T cells cultured with either CHO or CHO PD-L1/EGFR , and in the absence (- control in the left) or presence (- control in the right) of soluble LiTE. Data are mean ± SD (n = 3). One representative experiment of three independent experiments is shown. Significance was calculated by unpaired Student’s t test.

Article Snippet: In all cases, co-cultures were incubated for 72 hours, supernatants were collected and assayed for IFNγ secretion by ELISA (Diaclone, cat# 861.050.005) following manufacturer’s protocol.

Techniques: Functional Assay, Activation Assay, Concentration Assay, Expressing, Flow Cytometry, Luciferase, Lysis, Control, Enzyme-linked Immunosorbent Assay, Inhibition, Blocking Assay, Activity Assay, Bioassay, Cell Culture

Journal: Frontiers in Immunology

Article Title: Effective cancer immunotherapy combining mRNA-encoded bispecific antibodies that induce polyclonal T cell engagement and PD-L1-dependent 4-1BB costimulation

doi: 10.3389/fimmu.2024.1494206

Figure Lengend Snippet: Functional characterization of LiTE RNA and Albu-LiTCo RNA . Western blot detection using a HRP-conjugated anti-V HH mAb in the conditioned media of LiTE RNA and Albu-LiTCo RNA transfected HEK293 cells. Molecular mass (kDa) is indicated (A) . Specific binding of LiTE RNA (B) and Albu-LiTCo RNA (C, D) to plastic immobilized specific antigens (hEGFR and m4-1BB, mPD-L1, respectively) demonstrated by ELISA using an HRP-conjugated anti-V HH mAb cocktail (B, C) or HRP-conjugated anti-human serum albumin (HRP-HSA) mAb (D) . Data are represented as a mean ± SD (n = 3). BSA, bovine serum albumin; NFDM, non-fat dry milk. Binding of LiTE RNA and Albu-LiTCo RNA to cell surface expressed antigens mCD3 (E) , m4-1BB (F) , hEGFR (G) and hPDL-1 (H) by flow cytometry. The y-axis shows the relative cell number, and the x-axis represents the R-phycoerythrin (PE)-fluorescence, expressed on a linear scale. The anti-CD3 IgG, anti-4-1BB IgG, anti-EGFR IgG and anti-PD-L1 IgG were used as controls. One representative experiment out of two independent experiments is shown. The number indicates the percentage of positive cells (%).

Article Snippet: In all cases, co-cultures were incubated for 72 hours, supernatants were collected and assayed for IFNγ secretion by ELISA (Diaclone, cat# 861.050.005) following manufacturer’s protocol.

Techniques: Functional Assay, Western Blot, Transfection, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Fluorescence

Journal: Frontiers in Immunology

Article Title: Effective cancer immunotherapy combining mRNA-encoded bispecific antibodies that induce polyclonal T cell engagement and PD-L1-dependent 4-1BB costimulation

doi: 10.3389/fimmu.2024.1494206

Figure Lengend Snippet: Effects of LiTE RNA and Albu-LiTCo RNA on tumor cell cytotoxicity, agonistic costimulatory activity and tumor growth inhibition. EGFR-dependent cytotoxicity by LiTE RNA (A) . Luciferase-expressing CT26 and CT26 EGFR cells were co-cultured with mouse splenocytes at an E:T ratio of 5:1. Specific tumor cell lysis was measured by bioluminescence. The percentage of specific lysis was calculated relative to the same number of tumor cells cultured with splenocytes. Data are expressed as mean ± SD (n = 3). Significance was calculated by unpaired Student’s t test. CHO or CHO PD-L1/EGFR cells were plated with mouse CD8a + T cells activated with LiTE RNA in the presence of Albu-LiTCo (B) or with LiTE in the presence of Albu-LiTCo RNA (C) . IFNγ secretion was determined after 72 hours. Data are expressed as mean ± SD (n = 3). Significance was calculated by unpaired Student’s t test. Anti-4-1BB IgG and anti-PD-L1 IgG (B) or EGFP RNA (C) were used as controls. Pharmacokinetic profile of LiTE RNA (solid blue line) and Albu-LiTCo RNA (solid red line) expressed as ng/mL (left Y-axis) after a single intravenous (i.v.) administration of mRNA-LNP in BALB/c mice (D) . Ex vivo specific tumor lysis of CT26 EGFR cells mediated by LiTE-containing mouse serum (dashed blue line) and Albu-LiTCo-containing mouse serum (dashed red line) expressed as % of Lysis (right Y-axis) (D) . Target cells modified for the expression of luciferase were co-cultured with splenocytes at the effector/target (E/T) ratio of 5:1 in the presence of mouse serum obtained at 4, 96 and 168 hours post-mRNA-LNP administration. After 48 hours, the percentage of specific tumor lysis was measured by bioluminescence (right Y-axis). Data are presented as mean ± SD (n = 3). BALB/c mice were subcutaneously (s.c.) inoculated with CT26 EGFR cells, randomized into n=5/group with similar mean tumor sizes and SDs and treated with 3 i.v. injections of LiTE RNA , Albu-LiTCo RNA (10 µg/mouse) as monotherapy or combined (Combo RNA ) (E) . Average tumor volume growth of mice in each group is shown. Data are presented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s correction test for multiple comparisons. Quantitative analysis of intratumoral CD8 + T cells in mouse tumor tissue (n = 4/group) (F) . Data were calculated as percentage of CD8 + versus total cell number and presented as mean ± SD. Significance was determined by one-way ANOVA with Tukey’s test correction for multiple comparisons.

Article Snippet: In all cases, co-cultures were incubated for 72 hours, supernatants were collected and assayed for IFNγ secretion by ELISA (Diaclone, cat# 861.050.005) following manufacturer’s protocol.

Techniques: Activity Assay, Inhibition, Luciferase, Expressing, Cell Culture, Lysis, Ex Vivo, Modification

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: IL-1β (human) ELISA set , Diaclone , 851 610 020.

Techniques: Sequencing, Control, Magnetic Beads, Recombinant, Colorimetric Assay, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Cytometry, Mass Spectrometry, Imaging, Spectrophotometry