Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical platform for long-term evaluation of immuno-oncology drugs using hCD34+ humanized mouse model

doi: 10.1136/jitc-2020-001513

Figure Lengend Snippet: Scheme for the generation of the humanized mouse model. (A) 4-week female NOD-scid IL2rγnull (NSG) mice were prepared to generate humanized mice. Humanized mice were generated following myeloablation and transplantation of hCD34+ hematopoietic stem cells (HSCs). NSG mice were irradiated or injected with busulfan to suppress bone marrow function. Then, hCD34+ HSCs, isolated from human umbilical cord blood, were injected to myeloablated mice via the tail vein within 24 hours of myeloablation. 8 weeks after the injection of hCD34+ HSCs, hCD45+ cells started to appear, and differentiation of human T and B cells occurred at 11 weeks. (B) In order to optimize the humanization process in terms of efficiency and durability, irradiation versus busulfan were compared as myeloablative methods, and fresh versus frozen status of cord blood were compared as the source of human HSCs. Although the humanization success rates were higher in the irradiation group than in the busulfan group, the 45-week survival rate was higher in the busulfan group (64.3%, 9/14 humanized mice) than in the irradiation group (20%, 4/20 humanized mice). The fresh versus frozen status did not significantly affect the humanization success rate or the 45-week survival rate.

Article Snippet: Mononuclear cells (MNCs) were separated from hUCB using Ficoll-Paque Plus density gradient centrifugation. hCD34+ cells were isolated from MNCs using a magnetic activated cell sorting (MACS) human CD34 MicroBead Kit (Miltenyi Biotec, Spain) according to the manufacturer’s instructions.

Techniques: Generated, Transplantation Assay, Irradiation, Injection, Isolation

Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical platform for long-term evaluation of immuno-oncology drugs using hCD34+ humanized mouse model

doi: 10.1136/jitc-2020-001513

Figure Lengend Snippet: Feasibility of the humanized mouse model for long-term immune monitoring. (A) The proportion of cells in the peripheral blood was analyzed for the humanization marker hCD45+ by flow cytometry over 12–45 weeks from the injection of hCD34+ HSCs. Percentages of hCD45+ cells are demonstrated at three time points (starting point, highest point and final point) according to the myeloablative method or cord blood status. P values were calculated by Student’s t-test. Data are presented as mean±SD. (B) Kaplan-Meier survival curve of humanized mice. Survival of humanized mice was monitored up to 45 weeks according to the myeloablative method and cord blood status (upper). Survival curve was represented according to the myeloablative method (lower). P value was calculated by Breslow (Generalized Wilcoxon) test. (C) Gating strategy for flow cytometry analysis (left). Various immune cell markers, including hCD45, hCD3, hCD4, hCD8, hCD19 and hCD56 in the peripheral blood were analyzed by flow cytometry over 12 weeks to 45 weeks from the injection of hCD34+ HSCs by myeloablative method (right). When hCD45+ cells were taken as a whole (100%), percentages of hCD4+ or hCD8+ cells were generated from hCD45 and hCD3 double positive cells, whereas percentages of hCD19+ or hCD56+ cells were generated from hCD45+ and hCD3− cells. P values were calculated by Student’s t-test. Data are presented as mean±SD. (D) Tumorigenicity test of MDA-MB-231 cells in 45-week-old humanized mice. MDA-MB-231 cells were xenografted at 45 weeks post engraftment of hCD34+ HSCs. Mice were sacrificed 43 days after the implantation of MDA-MB-231 cells; the resultant tumors are demonstrated according to the myeloablative method and cord blood status. Scale bars, 5 mm. (E) Immunohistochemical staining of hCD8 and hCD4 in MDA-MB-231 xenograft tumors of 45-week-old humanized mice. Human tonsil was used for the positive control and non-humanized mouse tumor tissues were used for negative control. H&E images were collected at ×200 magnification and immunohistochemical images were collected at ×400 magnification. The bar graph represented the average of hCD8+ and hCD4+ cells in five random, non-overlapped fields at ×400 magnification. Data are presented as mean±SD.

Article Snippet: Mononuclear cells (MNCs) were separated from hUCB using Ficoll-Paque Plus density gradient centrifugation. hCD34+ cells were isolated from MNCs using a magnetic activated cell sorting (MACS) human CD34 MicroBead Kit (Miltenyi Biotec, Spain) according to the manufacturer’s instructions.

Techniques: Marker, Flow Cytometry, Injection, Generated, Immunohistochemical staining, Staining, Positive Control, Negative Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Preclinical platform for long-term evaluation of immuno-oncology drugs using hCD34+ humanized mouse model

doi: 10.1136/jitc-2020-001513

Figure Lengend Snippet: Long-term antitumor efficacy of the programmed cell death-1 (PD-1) inhibitor in the MDA-MB-231 cell xenograft humanized mouse model. (A) Scheme of the PD-1 inhibitor test set. Humanized mice were generated by injecting hCD34+ hematopoietic stem cells (HSCs) in mice, myeloablated with busulfan. MDA-MB-231 cells were xenografted 12 weeks after the injection of hCD34+ HSCs. When tumors reached 50–100 mm 3 , the PD-1 inhibitor was administered six times every 3 days. Tumor size was monitored up to 120 days from PD-1 inhibitor treatment, and blood and tumor samples were collected at indicated time points. (B) Schematic flow diagram of humanization experiment for PD-1 inhibitor test. Thirty-eight NOD-scid IL2rγnull (NSG) mice were myeloablated by busulfan and engrafted with CD34+ HSCs isolated from fresh cord blood. Only 32 mice that fulfilled the humanization criteria (hCD45+ cells ≥ 25%) were selected for the subsequent experiments regarding the PD-1 inhibitor efficacy. (C) Demonstration of the level of hCD45+ cells (Y-axis) in individual mouse (X-axis; 38 mice). Six arrows indicate mice which did not fulfill humanization criteria. The humanization success rate was 84.2% (32/38). (D) In vivo efficacy test of the PD-1 inhibitor in the humanized mouse model. The average tumor growth inhibition curves of all mice in each group were drawn first. Then, the tumor growth inhibition curves of individual mice were drawn as durable responders versus non-durable responders of MDA-MB-231 xenografted mice, and responders versus non-responders of patient-derived tumor xenograft (PDTX) xenografted mice. P values were calculated by Student’s t-test. (E) In vivo efficacy test of the PD-1 inhibitor in the NSG mouse model without humanization. PD-1 inhibitors showed no significant anticancer efficacy in either the MDA-MB-231 xenograft or PDTX xenograft non-humanized NSG mice. P values were calculated by Student’s t-test. (F) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using tumor sections at sacrifice. Representative images in each group are shown and bar graphs represent average numbers of apoptotic cells in each group in five random, non-overlapped fields at ×400 magnification. Data are presented as mean±SD. Apoptotic cells were increased in durable responders compared with PBS-treatment controls or non-durable responders. Non-humanized mouse tumor tissues were used for negative control. P values were calculated by analysis of variance test (post-hoc p values; LSD).

Article Snippet: Mononuclear cells (MNCs) were separated from hUCB using Ficoll-Paque Plus density gradient centrifugation. hCD34+ cells were isolated from MNCs using a magnetic activated cell sorting (MACS) human CD34 MicroBead Kit (Miltenyi Biotec, Spain) according to the manufacturer’s instructions.

Techniques: Generated, Injection, Isolation, In Vivo, Inhibition, Derivative Assay, TUNEL Assay, Negative Control

Journal: Haematologica

Article Title: The Sox4/Tcf7l1 axis promotes progression of BCR-ABL -positive acute lymphoblastic leukemia

doi: 10.3324/haematol.2014.104695

Figure Lengend Snippet: Identification of Sox4-regulated genes in ALL. (A) Genes differentially expressed in transformed Sox4fl/flSE-Cre and Sox4fl/+SE-Cre cells. Fetal liver (FL) or bone marrow (BM) pro-B cells from Sox4fl/fl or Sox4fl/+ mice were transformed with p190 BCR-ABL and transduced with SE-Cre. Gene expression microarray experiments were performed and results were compared between the two types of cells. Genes with a Sox4fl/flSE-Cre/Sox4fl/+SE-Cre signal intensity ratio of <1/3 and P<0.0001 were listed. (B) Analysis of Sox4 mRNA expression by real-time RT-PCR (left) and protein by western blotting (right) in transformed Sox4fl/+SE-Cre, Sox4fl/flSE-Cre, and Sox4fl/flSE-Cre;Sox4 pro-B cells. (C) Ratio (Sox4fl/flSE-Cre;Sox4 to Sox4fl/flSE-Cre) of mRNA expression levels of differentially expressed genes in both bone marrow (BM) and fetal liver (FL) derived transformed cells by real-time RT-PCR. The relative mRNA levels of specific genes were normalized to the level of Gapdh mRNA. Note that the ratios for most genes tested were substantially above 1, suggesting that expression of these genes was reversed upon ectopic Sox4 expression. (D) Scatter diagram demonstrating the correlation between Tcf7l1 and Sox4 expression. Tcf7l1 mRNA level is correlated with Sox4 mRNA level in leukemic cells from patients with ALL (n=11; r=0.666; P=0.0253). Expression of Gapdh was used for normalization of the RT-PCR results. (E) Enrichment of Tcf7l1 promoter sequence in Sox4-specific ChIP DNA by quantitative PCR in a bioChIP analysis. Biotin-conjugating enzyme, BirA ligase, and biotin acceptor peptide (BAP)-tagged Sox4 (BAP-Sox4) (BAP served as control) were introduced into the p190 BCR-ABL-transformed pro-B cells that had Sox4fl/fl deletion (Sox4fl/flSE-Cre). Chromatin was pulled down by magnetic beads conjugated with streptavidin (Dynabeads® MyOne™ Streptavidin T1; Invitrogen, Grand Island, NY, USA). The Sox4-specific and the control ChIP DNA was purified and subjected to real-time PCR for expression of the Tcf7l1 promoter sequence. (F) Mutational analysis of a potential Sox4 binding sequence in the Tcf7l1 promoter. The 460 bp fragment upstream of the transcription start site contains the potential Sox4 binding sequence in which mutations were introduced (-28 to -23bp: ‘ctttgt’ to ‘tgctag’) by PCR (Online Supplementary Methods). This fragment was used to construct mutant reporter pSIN-luc plasmid (MU). BCR-ABL-transformed BAP and BAP-Sox4 pro-B cells were transduced with retrovirus carrying wild-type sequence (WT), MU vector, or empty vector and luciferase activities were determined 2 days after transduction using empty vector as the background control. Data are representative of three independent experiments. Values are means ± SD (n=3). *P<0.05, **P<0.01, ***P<0.001.

Article Snippet: In the presence of biotin, BirA catalyzes conjugation of biotin to BAP-Sox4 which can then be specifically pulled down, together with bound DNA fragments (Sox4 specific ChIP DNA), by magnetic beads conjugated with streptavidin (Dynabeads ® MyOneTM Streptavidin T1; Invitrogen, Grand Island, NY, USA).

Techniques: Transformation Assay, Transduction, Gene Expression, Microarray, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Real-time Polymerase Chain Reaction, Control, Magnetic Beads, Purification, Binding Assay, Construct, Mutagenesis, Plasmid Preparation, Luciferase

Journal: Journal of Neuroinflammation

Article Title: Identification of a specific α-synuclein peptide (α-Syn 29-40) capable of eliciting microglial superoxide production to damage dopaminergic neurons

doi: 10.1186/s12974-016-0606-7

Figure Lengend Snippet: A29-V40 peptide activates Nox2 to produce superoxide via binding to gp91 phox . a Membrane translocation of p47 phox and p67 phox in rat microglia-derived HAPI cells. After stimulation by PMA or α-Syn peptides A19-A30 and A29-V40, cells were lysed in hypotonic lysis buffer. The membrane fraction and the cytosol were extracted from the lysates and were dissolved in SDS-PAGE and transferred to PVDF membranes. Samples were immunoblotted for p47 phox , p67 phox , gp91 phox , and tubulin. PM plasma membrane; Cyt cytosol. b Quantitative analyses of p47 phox and p67 phox membrane translocation according to the immunoblot data as shown in a . ANOVA was performed; n = 3–5; and * P < 0.05, ** P < 0.01 compared to the corresponding basal controls. c Superoxide production in mouse microglial cultures. WT or gp91 phox− / − microglia were seeded in 96-well plates. Upon stimulation using PMA (positive control) or α-Syn peptides A29-V40, superoxide production was measured. n = 4–6; * P < 0.05 and *** P < 0.001, compared to the corresponding basal control; ### P < 0.001, compared as indicated. d Representative images showing membrane translocation of WT mouse microglial p47 phox . After stimulation using PMA or α-Syn peptides A19-A30 and A29-V40, the cells were fixed, permeabilized, and immunostained for CD11b and p47 phox . e Representative images showing membrane translocation of gp91 phox null microglial p47 phox . After stimulation using PMA or α-Syn peptides A19-A30 and A29-V40, gp91 phox null microglia were fixed, permeabilized, and immunostained for p47 phox . f Binding of A29-V40 peptide to gp91 phox . Flag-fused A29-V40 peptide was incubated with WT or gp91 phox−/− microglial lysates, and the mixtures were further incubated with protein G-conjugated magnetic beads which were coated with control IgG or anti-gp91 phox polyclonal Ab. After washes, beads were boiled and the supernatants were dissolved in SDS-PAGE. Samples were immunoblotted for Flag, gp91 phox , or tubulin. g Binding of A29-V40 peptide or A19-A30 peptide to gp91 phox . h Representative images illustrating the cell surface binding of FITC-labeled A29-V40 peptide but not A19-A30 peptide to gp91 phox . WT or gp91 phox−/− microglia were incubated with FITC-labeled A29-V40 peptide or A19-A30 peptide on ice for 30 min and imaged by a confocal microscope. Bar = 10 μm

Article Snippet: Simultaneously, goat anti-gp91 phox polyclonal antibodies (Santa Cruz Biotech., Dallas, TX) or control IgGs were conjugated to protein G magnetic beads using a Dynabeads protein G immunoprecipitation kit (Life Technologies, Grand Island, NY) at RT for 10 min. Next, the α-Syn peptides and cell lysate mixtures were incubated with the antibody- or control IgG-conjugated protein G magnetic beads at RT for 30 min. After washing, the beads were boiled in Laemmli sample buffer and the supernatants were resolved using SDS-PAGE.

Techniques: Binding Assay, Translocation Assay, Derivative Assay, Lysis, SDS Page, Western Blot, Positive Control, Incubation, Magnetic Beads, Labeling, Microscopy

Journal: Pharmaceutics

Article Title: Enhanced Antioxidant Effects of the Anti-Inflammatory Compound Probucol When Released from Mesoporous Silica Particles

doi: 10.3390/pharmaceutics14030502

Figure Lengend Snippet: Percentage of ( a ) ROS+ and ROS- human brain endothelial cells (HBEC) after incubation with LPS at different test concentrations compared to the negative control (cells with media only) and the positive control (1000 μM hydrogen peroxide). ( b ) Cell viability assay. Production of ROS only caused a relatively small amount of cell death in comparison to the negative control. ( c ) Analysis of ROS levels using the DCFDA assay suggested an increase in hydrogen peroxide, hydroxyl and peroxyl levels, consistent with increases in mitochondrial hydroxyls, nitric oxide and peroxynitrite after incubation of 1 μg/mL LPS for 24 h in HBEC cells compared to the negative control (clear bar). Markers of inflammation: COX activity, prostaglandin E2 and TNF-α levels are also shown. Results show the mean ± SD of triplicates. Treatment with LPS resulted in significant increases in ROS and inflammatory markers ( p < 0.05) as compared to negative controls.

Article Snippet: The human TNF-α ELISA kit (ab181421, Abcam, Melbourne, Australia) was used to measure levels of the cytokine in cell culture media following the incubation of endothelial cells for 24 h with or without the PB, AMS-6PB or ascorbic acid.

Techniques: Incubation, Negative Control, Positive Control, Viability Assay, Activity Assay