Journal: bioRxiv

Article Title: A GALLIUM-68-LABELED PEPTIDE RADIOTRACER FOR CD38-TARGETED IMAGING IN MULTIPLE MYELOMA WITH PET

doi: 10.1101/2023.05.09.540036

Figure Lengend Snippet: A) Structure of a novel bicyclic peptide AJ206 having NOTA as bifunctional chelator for 68 Ga labeling B) Surface plasmon resonance (SPR) analysis showing affinity of AJ206 for CD38 using recombinant human CD38 protein; data is represented as mean±SEM (n=2)

Article Snippet: Blots were probed with rabbit monoclonal anti-human CD38 antibody (Clone RM388, Thermo Scientific, cat# MA5-36061) after blocking with 5% BSA.

Techniques: Labeling, SPR Assay, Recombinant

Journal: bioRxiv

Article Title: A GALLIUM-68-LABELED PEPTIDE RADIOTRACER FOR CD38-TARGETED IMAGING IN MULTIPLE MYELOMA WITH PET

doi: 10.1101/2023.05.09.540036

Figure Lengend Snippet: A) [ 68 Ga]Ga-AJ206 binding (%IA) to different MM cells. Cells were incubated with [ 68 Ga]Ga-AJ206 at 4 °C for 1 hour. [ 68 Ga]Ga-AJ206 uptake is CD38 expression dependent, and co-incubation with 2 μM of unlabeled AJ206 (blocking dose) significantly reduced radiotracer uptake confirming CD38 specificity. B) Flow cytometry analysis of CD38 surface expression in MM cells. C) Cytometric-quantibrite assay to quantify CD38 receptor density in various MM cells. D) Representative western blot of total CD38 protein expression (bottom panel). Densiometric analysis of western blot preformed using Image J software and band intensities represented as a ratio of CD38 protein to GAPDH control (top panel), E) Correlation of [ 68 Ga]Ga-AJ206 uptake with CD38 receptor numbers; data in panels A, C and E are represented as mean±SD (n=3-4). ns, p ≥ 0.05; ****, p ≤ 0.0001 is by unpaired Student’s t-test. Simple linear regression and Pearson coefficient were used in E.

Article Snippet: Blots were probed with rabbit monoclonal anti-human CD38 antibody (Clone RM388, Thermo Scientific, cat# MA5-36061) after blocking with 5% BSA.

Techniques: Binding Assay, Incubation, Expressing, Blocking Assay, Flow Cytometry, Western Blot, Software

Journal: bioRxiv

Article Title: A GALLIUM-68-LABELED PEPTIDE RADIOTRACER FOR CD38-TARGETED IMAGING IN MULTIPLE MYELOMA WITH PET

doi: 10.1101/2023.05.09.540036

Figure Lengend Snippet: A) Whole-body PET/CT images of different human MM xenografts at 60 min after the injection of the radiotracer. Mice were injected with ∼ 7.4 MBq (∼200 μCi) [ 68 Ga]Ga-AJ206. B) IHC staining for CD38 expression in MM xenografts. C) [ 68 Ga]Ga-AJ206 uptake quantification in different MM xenografts by ex vivo biodistribution at 60 min after injection. D) PET/CT images of MM1S tumor xenograft-bearing mice with [ 68 Ga]Ga-AJ206, with and without pre-administration of a blocking dose (50 μg of AJ206). E) [ 68 Ga]Ga-AJ206 quantification in tumors by ex vivo biodistribution in mice treated with and without a blocking dose; data in figure C , and E are shown as box and whisker plots showing all data points (n=4-5). Ordinary one-way ANOVA using multiple comparison test in C and multiple unpaired t-test in E. ns, p ≥ 0.05; *, p ≤ 0.05; ** p ≤ 0.01; ***, p ≤ 0.001.

Article Snippet: Blots were probed with rabbit monoclonal anti-human CD38 antibody (Clone RM388, Thermo Scientific, cat# MA5-36061) after blocking with 5% BSA.

Techniques: Positron Emission Tomography-Computed Tomography, Injection, Immunohistochemistry, Expressing, Ex Vivo, Blocking Assay, Whisker Assay

Journal: bioRxiv

Article Title: A GALLIUM-68-LABELED PEPTIDE RADIOTRACER FOR CD38-TARGETED IMAGING IN MULTIPLE MYELOMA WITH PET

doi: 10.1101/2023.05.09.540036

Figure Lengend Snippet: A) IVIS-bioluminescence image of luciferase expressing MM1S disseminated tumor model (i.v) showing luciferase expression in lower limbs, unit of radiance is watt per steradian per square metre (W·sr −1 ·m −2 ) B) In vivo uptake of [ 68 Ga]Ga-AJ206 in lungs and bones C) Flow cytometry analysis of CD38 expression in lungs harvested from of MM1S-Luc cells injected mice. Lungs from PBS treated mice were used as controls (Top). IHC staining of bones to validate CD38 expression (bottom). D) In vivo and ex vivo [ 68 Ga]Ga-AJ206-PET images of lower limbs of MOLP8 injected (i.v) animals. E) IHC images of MOLP8 bone marrow tumor and PBS treated animals confirming CD38 expression F) Flow cytometry analysis of CD38 expression in cells extracted from bone marrows of PBS, MM1S-Luc and MOLP8 cell injected mice. G) Quantification of PET signals in the bone marrow of PBS, MM1S and MOLP8 injected animals H) Tumor/muscle ratios of PET measures in the bone marrows of PBS, MM1S and MOLP8 injected animals; data in figure G and H are shown as box and whisker plots showing all data points (n=7 in PBS, n=5 in MM1S and n=8 in MOLP8). Ordinary one-way ANOVA using multiple comparison test. ** p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

Article Snippet: Blots were probed with rabbit monoclonal anti-human CD38 antibody (Clone RM388, Thermo Scientific, cat# MA5-36061) after blocking with 5% BSA.

Techniques: Luciferase, Expressing, In Vivo, Flow Cytometry, Injection, Immunohistochemistry, Ex Vivo, Whisker Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Characterization of anti-CD38 chitosan NPs. A) Crosslinking reaction showing ionotropic gelation of chitosan with TPP anions (ChemDraw Professional 15.1 was used for the chemical drawings). B) Scheme representing anti-CD38 chitosan NPs loaded with bortezomib by conjugation of polymeric chitosan NPs with streptavidin and further coupling with biotinylated anti-CD38 monoclonal antibody. C) Effect of TPP crosslinker (0.25 – 1 mg/ml) on i) size and ii) stability of anti-CD38 chitosan NPs. D) TEM image of chitosan NPs showing 50μm size. E) In vitro BTZ release from anti-CD38 chitosan measured by HPLC after 72 h (n=3).

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Conjugation Assay, In Vitro

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: In vitro drug release from anti-CD38 chitosan NPs to different environments. A) i) pH of non-conditioned media and conditioned media from PB MNCs and MM cells after 72h in culture (n=3). ii) Effect of conditioned media pH on drug release from anti-CD38 chitosan NPs. B) i) Corrected pH of conditioned media from PB MNCs and MM cells after 72h in culture (n=3). ii) Effect of conditioned media with corrected pH on drug release from anti-CD38 chitosan NPs. C) Swelling of anti-CD38 chitosan NPs in non-conditioned media and MM-conditioned media over 24 h.

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: In Vitro

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Kinetics of binding/uptake of anti-CD38 chitosan NPs to MM cells. A) Flow cytometry representative histograms of CD38 expression measured as fold of MFI of anti-CD38 to isotype controls in MM cells. B) Correlation of anti-CD38 NPs binding/uptake at 2 h with CD38 expression in MM cells compared to the non-correlation of non-targeted NPs with CD38 expression. C) Kinetics of binding/uptake of anti-CD38 chitosan NPs over a period of time of 24 h to MM cells. D) Fluorescent imaging of AF633 anti-CD38 chitosan NPs after 2 h of binding/uptake to MM1s cells (Bright field (BF); AF633, Red). Scale bar= 10 μm. E) Kinetics of dissociation of anti-CD38 chitosan NPs over a period of time of 24 h to MM cells after 2 h of binding/uptake.

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Binding Assay, Flow Cytometry, Expressing, Imaging

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Specificity of binding/uptake of anti-CD38 chitosan NPs to MM cells. A) Comparison of specificity of binging of anti-CD38 chitosan NPs versus non-targeted chitosan NPs to MM cell lines (Av, n=5), CD138+ cells isolated from MM patients (Av, n=5), normal mononuclear cells isolated from the peripheral blood of normal subjects (PB MNCs, Av, n=5) and normal plasma cells isolated from the BM of normal subjects (BM MNCs, Av, n=5) analyzed by flow cytometry, *p<0.05. B) Mechanism of binding/uptake of the anti-CD38 targeted and non-targeted NPs by blocking or not with free anti-CD38 antibody, *p<0.05. C) Comparison of the biodistribution of anti-CD38 chitosan NPs, non-targeted chitosan NPs and free deactivated AF633 to MM cells (GFP+) in different organs: femurs, blood, heart, kidney, liver, lung and spleen, analyzed by fluorescent signal (fold of MFI of AF633) by flow cytometry, *p<0.05. (n=5).

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Binding Assay, Isolation, Flow Cytometry, Blocking Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Effect of bortezomib-loaded anti-CD38 chitosan NPs on proliferation, cell cycle, and apoptosis. The effect of vehicle (control), BTZ as a free drug (5 nM), empty chitosan NPs, non-targeted chitosan NPs loaded with BTZ equivalent amounts to 5 nM, and anti-CD38 chitosan NPs loaded with BTZ equivalent amounts to 5 nM for 48 h on: A) proliferation of MM cells and PB MNCs analyzed by MTT, *p<0.05; B) % Sub-G1 population of H929 cells measured by Propidium Iodide stained of DNA, *p<0.05; C) Apoptosis of MM cells by FITC-Annexin-V and Propidium Iodide, *p<0.05. D) Survival–associated molecules (pERK and pAKT) after 6h of treatment, cell cycle–associated molecule (pRB), as well as, apoptotic–associated molecules (cleaved PARP and cleaved caspase 3) after for 12h of treatment, in H929 cells were measured by immunoblotting.

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Staining, Western Blot

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Enhanced bortezomib uptake and proteasome inhibition by anti-CD38 chitosan NPs. A) The effect of no treatment (control), BTZ as a free drug (5 nM), empty chitosan NPs, non-targeted chitosan NPs loaded with BTZ equivalent amounts to 5 nM, and anti-CD38 chitosan NPs loaded with BTZ equivalent amounts to 5 nM for 2 h on proteasome activity, *p<0.05. B) Cell pellet boron concentration analyzed by ICP-OES after no treatment (control), BTZ as a free drug (100 nM), non-targeted chitosan NPs loaded with BTZ equivalent amounts to 100 nM, and anti-CD38 chitosan NPs loaded with BTZ equivalent amounts to 100 nM for 1.5 h, *p<0.05.

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Inhibition, Activity Assay, Concentration Assay

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Enhanced proteasome activity inhibition by anti-CD38 chitosan NPs endocytic internalization. A) Confocal micrographs of the MM cells showing intracellular location of AF633 anti-CD38 in red, early endosomes expressing GFP (rab5+), and merged images showing co-localization of the red NPs in the green early endosomes at 2 and 24 hours; Scale bar= 10 μm. B) Anti-CD38 chitosan NPs uptake in the presence of macropinocitosys and endocytosis inhibitors. H929 cells were pre-incubated with the following inhibitors cytochalasin D 1μM, chlorpromazine 2μM, nystatin 0.5μg/ml, and EGA 2.5μM for 30 min. Two control samples were used with no inhibitors (No inhibition) at either 37°C or 4°C, *p<0.05. C) The effect of anti-CD38 chitosan NPs loaded with BTZ equivalent amounts to 5 nM uptake in the presence of macropinocitosys and endocytosis inhibitors (cytochalasin D 1μM, chlorpromazine 2μM, nystatin 0.5μg/ml, and EGA 2.5μM for 30 min) on proteasome activity, *p<0.05. D) The effect of BTZ as a free drug (5 nM) in the presence of macropinocitosys and endocytosis inhibitors (cytochalasin D 1μM, chlorpromazine 2μM, nystatin 0.5μg/ml, and EGA 2.5μM for 30 min) on proteasome activity, *p<0.05.

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Activity Assay, Inhibition, Expressing, Incubation

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Effect of bortezomib-loaded anti-CD38 chitosan NPs on drug resistance. A) The effect of vehicle (control), BTZ as a free drug (5 nM), empty chitosan NPs, non-targeted chitosan NPs loaded with BTZ equivalent amounts to 5 nM, and anti-CD38 chitosan NPs loaded with BTZ equivalent amounts to 5 nM for 48 h on: A) cell adhesion-mediated drug resistance in co-culture with MSP-1 (myeloma-derived stroma); and B) hypoxia-mediated drug resistance, *p<0.05. C) The effect of vehicle (control), BTZ as a free drug (10 nM), empty chitosan NPs, non-targeted chitosan NPs loaded with BTZ equivalent amounts to 10 nM, and anti-CD38 chitosan NPs loaded with BTZ equivalent amounts to 10 nM for 48 h on survival of MM cells cultured in 3DTEBM, *p<0.05. D) Confocal microscopy images of MM cells cultured (green) in 3DTEBM after 24h treatment with AF633 anti-CD38 chitosan NPs (red), shown by a Z-Stack rotated view, and images at different depths of the z-stack to show the co-localization (white arrows) of NPs and MM cells in yellow; Scale bar= 50 μm.

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Co-Culture Assay, Derivative Assay, Cell Culture, Confocal Microscopy

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Inhibition of tumor progression and reduction of the side effects of bortezomib-loaded anti-CD38 chitosan NPs in vivo. The effect of vehicle, BTZ as a free drug (1 mg/kg once a week), non-targeted chitosan NPs loaded with BTZ equivalent amounts to 1 mg/kg (once a week), and anti-CD38 chitosan NPs loaded with BTZ equivalent amounts to 1 mg/kg (once a week) on: A) tumor progression shown by quantification of BLI*p<0.05; B) number of MM cells on circulation, *p<0.05; C) survival shown by Kaplan-Meier survival curves (p value targeted compared to other treatments); D) % weight loss, and E) hair loss (representative images of one mice from each group).

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Inhibition, In Vivo

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Enhancing proteasome-inhibitory activity and specificity of bortezomib by CD38 targeted nanoparticles in multiple myeloma

doi: 10.1016/j.jconrel.2017.11.045

Figure Lengend Snippet: Schematic representation of BTZ cellular uptake by normal and MM cells. (Left) Free BTZ penetrates into all cells by passive diffusion and preferentially inhibits the overexpressed proteasome in MM cells, even at a low drug concentration. (Right) BTZ-loaded anti-CD38 chitosan NPs bind to CD38+ MM cells, and BTZ can be released directly from the NPs attached to the surface or anti-CD38 NPs entered CD38+ MM cells via the endocytic pathway (inhibited by 4°C incubation). Clathrin- (inhibited by chlorpromazine, chlorp.), caveolae-mediated endocytosis (inhibited by nystatin) incorporated anti-CD38 chitosan NPs into early endosomes, which will be transformed into late endosomes (inhibited by EGA). Through both mechanisms BTZ is presented at a higher drug concentration to the proteasome based on higher availability of BTZ inside the cells through either diffusion from the anti-CD38 NPs that bind to the surface or the endocytosed anti-CD38 NPs.

Article Snippet: The in vitro cellular studies with BTZ-loaded anti-CD38 chitosan NPs demonstrated enhanced cytotoxicity and apoptosis on MM cells compared to similar concentration of BTZ as free drug; demonstrated by induction of more profound cell cycle arrest, and apoptosis leading to cell death through inactivation of proliferative cell signaling (MAPK, PI3K pathways, cell cycle) and activation of pro-apoptotic pathways (PARP and Caspase-3) ( ).

Techniques: Diffusion-based Assay, Concentration Assay, Incubation, Transformation Assay

Journal: Molecular neurobiology

Article Title: CD38 Coordinates with NF-κB to Promote Cochlear Inflammation in Noise-Induced Hearing Loss: the Protective Effect of Apigenin.

doi: 10.1007/s12035-024-04675-7

Figure Lengend Snippet: Fig. 9 CD38 coordinates with NF-κB to promote cochlear inflammation in NIHL. As the closest immune cell to organ of Corti, activated macrophages can express CD38 and consume NAD in organ of Corti, causing NAD + metabolism dysfunction and ultimately leads to cochlear inflammation. In this process, inflammatory factors such as IL-1, IL-6, and TNF-α pro- moted the activation of NF-κB signaling pathway, forming a positive feedback cycle

Article Snippet: Immunofluorescence The cochlear basilar membrane was prepared with the surface preparation method, antibodies used in this experiment were rabbit anti-NF-κB p65 polyclonal antibody (Abcam, ab32536), rat anti-F4/80 polyclonal antibody (Abcam, ab6640), and mouse anti-CD38 monoclonal antibody (Proteintech, 60,006–1-lg).

Techniques: Activation Assay

Journal: Microbiology Spectrum

Article Title: Primary Murine Macrophages as a Tool for Virulence Factor Discovery in Coxiella burnetii

doi: 10.1128/spectrum.02484-21

Figure Lengend Snippet: Phenotype of BMDM infected with C. burnetii NMII. (A) Flow cytometric analysis of untreated primary BMDM in parallel with BMDM treated with either IFN-γ plus 100 ng/mL LPS or IL-4 or infected with dotA ::Tn or wild-type NMII C. burnetii for 48 h. Cells were stained for two markers of classical activation (CD38 and CD11c) and two markers of alternative activation (EGR2 and CD206). A histogram (representative of two independent experiments with greater than 10,000 events) shows the percentage of positive events in the population for each marker and condition, where a horizonal line denotes the threshold for a positive signal. (B) Graph displaying the relative arginase activity of mock-infected BMDM or BMDM treated with either IFN-γ plus 100 ng/mL LPS or IL-4 (positive control, 100%) or infected with dotA ::Tn or wild-type NMII C. burnetii at 24 hpi. (C) Graph displaying the absolute nitrate production of the same BMDM populations described in the legend for panel B, where the population treated with IFN-γ plus 100 ng/mL LPS serves as the positive control for the assay. For both panels B and C, the data shown are the mean of results of three independent experiments, with error bars representing the standard deviations. The statistical significance relative to positive controls was calculated by one-way ANOVA, with Dunnett’s test for multiple comparisons: *** * , P < 0.0001. (D) Western blots of whole-cell lysates from mock-infected BMDM, BMDM treated with either IFN-γ plus 100 ng/mL LPS or IL-4, or BMDM infected with wild-type NMII or dotA ::Tn C. burnetii at 24 hpi. Expression of inducible nitric acid synthase (α-NOS2) and arginase (α-ARG1) is shown, with β-actin (α-actin) serving as a loading control.

Article Snippet: The following antibodies were used for primary probing: rabbit anti-NOS2, rabbit anti-CD38, rabbit anti-ARG1 (all from AbClonal).

Techniques: Infection, Staining, Activation Assay, Marker, Activity Assay, Positive Control, Western Blot, Expressing

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance.

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: Fig. 5 | Depletion of PD-1+CD38hi CD8+ T cells results in a strong antitumor response. a, Tumor growth and survival of variously treated B16-bearing Rag1−/− mice following transfer of either total or PD-1+CD38+-depleted, in vitro-activated CD8+ T cells (with the indicated number of mice per group given in parentheses); data are the average of two independent experiments. The error bars indicate the s.e.m. Left panel: for comparison purposes, an unpaired, one-tailed Student’s t-test was used; *P = 0.017 (day 14), *P = 0.0127 (day 16), **P = 0.0074 (day 20). Right panel: survival in various groups was compared using the log-rank (Mantel–Cox) test; **P = 0.0054. b, Experimental outline for Pmel-1 CD8+ T cell treatment. c, MFI and protein expression of CD38 in PD- 1+CD8+ T cells (shown in the small red box on the left). The protein expression of CD38 in flow-sorted PD-1+CD38+ T cells transfected with CD38 siRNA or scrambled RNA (scRNA) was determined by immunoblot. The expression of β-actin was used as a loading control (the uncropped full scan of the blot is shown in Supplementary Fig. 5b). d, Frequency of Ki-67+, CD40L+ and IFN-γ+ in the PD-1+CD38+ CD8+ T cell population. Data are representative of two independent experiments with at least 3–5 technical replicates per group. The error bars indicate the s.e.m. For comparison purposes, an unpaired, one- tailed Student’s t-test was used. ***P = 0.0003 (c); ***P = 0.0007 (left panel); ****P ≤ 0.0001 (middle panel); **P = 0.0012 (right panel) (d). *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: Anti-CD38 antibody for the immunoblot was obtained from Proteintech (1:100, clone 3C6G4, catalog no. 60006-1-Ig) while anti-β-actin (anti-mouse) antibody was purchased from Sigma (1:1,000, clone AC-15, catalog no. A5441).

Techniques: In Vitro, Comparison, One-tailed Test, Expressing, Transfection, Western Blot, Control

Journal: Nature immunology

Article Title: PD-1 blockade in subprimed CD8 cells induces dysfunctional PD-1 + CD38 hi cells and anti-PD-1 resistance.

doi: 10.1038/s41590-019-0441-y

Figure Lengend Snippet: Fig. 8 | The frequency of PD-1+CD38+ CD8+ T cells is a pharmacodynamic and predictive biomarker of anti-PD-1 therapy. a, The posttreatment average frequency of PD-1+CD38+ CD8+ T cells in 21 non-responding and 8 responding tumor lesions, as determined by single-cell RNA sequencing analysis, is shown on the left and the individual frequencies are shown on the right. The red line depicts the cutoff limit where at least 4% or more CD8+ T cells were PD-1+CD38+ in the tumors. b, The pretreatment average frequency of PD-1+CD38+ CD8+ T cells in 10 non-responding and 9 responding tumors lesions is shown on left and the individual frequencies are shown on the right. The red line depicts the cutoff limit where at least 10% or more CD8+ T cells were PD-1+CD38+ in the tumors. The error bars indicate the s.e.m. Left panels: an unpaired, one-tailed Student’s t-test was used. **P = 0.0045 (a); **P = 0.0048 (b). The post- and pretreatment cutoffs that best predicted responders from non-responders were determined a priori and were further confirmed using ROC analysis (right panels). c, Absolute numbers of CD8+ (top) and PD-1+CD38+ CD8+ (bottom) T cells in total viable cells in the TME in the pretreatment (non-responders: n = 10; responders: n = 9), posttreatment (non-responders: n = 21; responders: n = 8) or total (non-responders: n = 31; responders: n = 17) number of responding and non-responding tumor lesions. Each dot corresponds to one tumor lesion. The error bars indicate the s.e.m. For comparison purposes, a one-tailed Student’s t-test was used. Top panels: NS; bottom panels: *P = 0.019 (pretreatment); *P = 0.034 (posttreatment); **P = 0.0034 (total). d, Flow cytometry measurements of CD38+ cells in PD-1+CD8+ T cells in PBMCs from advanced melanoma patients at 3 and 9 weeks after anti-PD-1 treatment. For two non-responding patients, data are shown at 6 weeks since samples were not available at week 9.

Article Snippet: Anti-CD38 antibody for the immunoblot was obtained from Proteintech (1:100, clone 3C6G4, catalog no. 60006-1-Ig) while anti-β-actin (anti-mouse) antibody was purchased from Sigma (1:1,000, clone AC-15, catalog no. A5441).

Techniques: Biomarker Discovery, RNA Sequencing, One-tailed Test, Comparison, Flow Cytometry