Journal: Blood Advances

Article Title: In vivo HSC transduction in rhesus macaques with an HDAd5/3 + vector targeting desmoglein 2 and transiently overexpressing cxcr4

doi: 10.1182/bloodadvances.2022007975

Figure Lengend Snippet: Analysis of DSG2 expression on HSCs. (A) Flow cytometry for CD46 and DSG2 on human CD34 + cells from 2 different healthy G-CSF–mobilized donors. (B) Schematic of HDAd vector capsids and genomes. The chimeric capsids contain either affinity-enhanced Ad35 or Ad3 fiber knob domains. Both vector genomes contain an EF1α-mgmt p140k /GFP expression cassette used before. Both proteins are linked via a self-cleaving picornavirus 2A peptide. One set of vectors has an additional CMV promoter–human cxcr4 expression cassette. The 4 vectors used in this study are HDAd5/35++GFP, HDAd5/3+GFP, HDAd5/35++GFP/cxcr4, and HDAd5/3+GFP/cxcr4. (C) In vitro CD34 + cell transduction studies. Cells were infected with HDAd5/35++-GFP and HDAd5/3+-GFP virus at a multiplicity of infection of 2000 vp/cell with and without pre-incubation with recombinant soluble CD46 or DSG2 (10 μg/mL) for 1 hour. GFP expression was analyzed 24 hours' postinfection. (D) CD46 and DSG2 flow cytometry on human and rhesus peripheral RBCs stained with antibodies that recognize the receptors in both species (anti-human CD46 mAb clone M177 from Santa Cruz Biotechnology [Dallas, TX] and anti-human DSG2 polyclonal antibody AF947 from R&D Systems [Minneapolis, MN]). (E) Transduction of 293 cells with HDAd5/35++GFP and HDAd5/3+GFP in the absence and presence of human (huRBCs) and rhesus (rhRBCs) RBCs (details are provided in the Materials and methods). Shown is the percentage of GFP-positive cells measured 2 days after transduction.

Article Snippet: Recombinant human DSG-2 was from Leinco Technologies (Fenton, MO; catalog no. D340) and recombinant CD46 from Sino Biological (Beijing, China; catalog no. 12239-H08H).

Techniques: Expressing, Flow Cytometry, Plasmid Preparation, In Vitro, Transduction, Infection, Incubation, Recombinant, Staining

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: Protective genes.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Membrane

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: (A) Forward and side scatter plot of lung cells. Circles show the lymphocyte, macrophages and neutrophil populations. To the right a single color histograms showing expression of receptor CD46 from representative control, emphysema and end-stage participants. Pooled data from all participants (control, n = 4; emphysema, n = 4, end-stage n = 5) showing percent (median±SD) of total lung neutrophils, and macrophages expressing CD46; Cumulative values for lymphocytes showed a significative decrease (control, n = 6; emphysema, n = 7, end-stage n = 6, p<0.05) more patient were included with the same lung characteristics. (B) Gene expression of Casp 8, on the same patients, determined by qRT-PCR (median±SD) on emphysema patients with (n = 5) and without cancer (n = 4) shows no different expression level due to cancer away from the emphysemic region (p = 1). Mann-Whitney test was used to determine significant difference.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Expressing, Control, Gene Expression, Quantitative RT-PCR, MANN-WHITNEY

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: (A) Increased expression of CD46 in the lung tissue of no smoker mice compared to smoke exposed mice determined by IHC, arrow heads, and by western western blot on lung tissue homogenates of control mice or smoke-exposed mice (p = 0.02, n = 4, n = 4). (B) Increased deposition of C3b on lung tissue of control mice (n = 3) compared to smoke- exposed mice at 4 and 24 weeks (n = 3) determined by Western blot. Middle plot, immunoprecipitation of C3b from lung homogenate shows a significant increase of C3b deposition upon smoke exposure (p = 0.05). Elastin was stained and detected after stripping the membrane, showing significant increased co-precipitation with C3b (p = 0.01). Student t-test two tails was used to compare both groups; values in the plots represent average ± SD.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Expressing, Western Blot, Control, Immunoprecipitation, Staining, Stripping Membranes, Membrane

Journal: PLoS ONE

Article Title: CD46 Protects against Chronic Obstructive Pulmonary Disease

doi: 10.1371/journal.pone.0018785

Figure Lengend Snippet: (A) CD46 coupling with CD3 activates the transcription factor ubiquitously transcribed tetratricopeptide (UTX), which interacts with signal transducer and activator of transcription 1 (STAT1) both translocate to the nucleus to activate proliferation of CD4 + T cells. T cell receptor activates Bcl-2 that inhibits cytochrome C (Cyto C) released by chloride intracellular channel 4 (CLIC4), which positively regulates Fas Ligand receptor (CD95) mediated activation of Caspase 8 (Casp 8) and apoptosis. Homeodomain interacting protein kinase 3 (HIPK3) phophorilates CD95, and HuR stabilizes CD95 mRNA also positively regulates it. While 2 negative regulators of CD95 are methionine adenosyl transferase II, alpha (MATII) and hepatocyte growth factor receptor (HFGR). Red font signifies protective gene, black arrow apoptosis activation, black bold arrow apoptosis inhibition. Red arrow, proliferation pathway. Red proteins are kinases; blue, transcription factors; light brown, cell surface receptors. (B) Total CD46 expression on the surface of lung cells of patients (n = 14) significantly correlates with their FEV1% in a linear regression using minimum square approximation, with a coefficient r = 0.563 and a goodness of fit p = 0.036. The FasL receptor, CD95, protein expression on the same patients, also, positively correlates with FEV1% with an r = 0.711 and a goodness of fit p = 0.006. There is a significative linear association between cells surface proteins expression, CD46 + and CD95 + (r = 0.666, p = 0.012). Right top plot, total lung lymphocytes CD4 + plus CD8 + has a constant number in the lung parenchyma despite their lung FEV1% change with disease progression (r = 0.107, p = 0.6332, n = 22) although there is a significant change in the number of CD4 + and CD8 + T cells. Right middle plot, in the same patients group, CD4 + T cells (black circles) correlates directly with the FEV1% (r = 0.549, slope = 0.24, p = 0.008, n = 22), conversely CD8 + T cells (red circles) inversely correlates with FEV1% increasing its number with disease progression (r = 0.480, slope = −0.329, p = 0.024). Bottom right plot shows a direct correlation between FEV1% and depletion of CD4 + with increment of CD8 + T cells (slope = 8.4, r = 0.726, p = 0.0002). (C) There is a remarkable association among CD46 + decrement and CD4 + /CD8 + ratio of T cells, r = 0.896 p = 0.006, n = 7. (D) Increased IgG to elastin in early-onset COPD (EO-COPD) patients, relative to normal controls, tested in n = 21 and n = 28, respectively, plot represents the patients that give signal above the threshold n = 9 for both EO-COPD and control with a significant increased in the diseased group p = 0.038, determined by Mann-Whitney test, values represent median ± SD.

Article Snippet: analysis of CD46 was done using a custom (rabbit anti-mouse) antibody from Prosci (Poway, CA, USA) at 1:250 dilution.

Techniques: Activation Assay, Inhibition, Expressing, Biomarker Discovery, Control, MANN-WHITNEY

Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 3. Induction of apoptosis in HL-60 cells treated with DL-247 for 24 h. (A) Representative scattered blots of flow cytometry analysis of apoptosis by double-staining with Annexin-V and PI. The percentage of viable cells is shown in quadrant Q3. Quadrant Q4 indicates the percentage of early apoptotic cells (Annexin V-positive cells), whereas quadrant Q2 shows the percentage of late apoptotic/death cells (Annexin V-and PI-positive cells). (B) Quantitative analysis of apoptotic cell death by Annexin V and PI assay. (C) Representative histograms of flow cytometry analysis of DNA fragmentation measured by TUNEL assay. (D) Quantitative analysis of DNA fragmentation. (B,D): Data are presented as mean ± SEM of three independent experiments. Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001 vs. control.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Cytometry, Double Staining, TUNEL Assay, Comparison, Control

Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 5. Analysis of typical features of intrinsic apoptosis pathway in HL-60 cells treated with DL-247 for 24 h. (A) Percentage of phosphorylated H2AX positive cells measured by flow cytometry (Alexa Fluor 647 Mouse Anti-H2AX (pS139) antibody staining). (B) Mitochondrial membrane potential (∆Ψm) changes measured by flow cytometry (JC-1 staining). FCCP (30 µM, 30 min incubation at 37 ◦C) was used as a positive control. (C) Bax and Bcl-2 gene expression changes analyzed by real-time PCR. (D) Intracellular ROS generation measured by flow cytometry (CellROX Green Reagent staining). Menadione (200 µM, 1 h incubation at 37 ◦C) was used as a positive control. (A–D): Data are presented as mean ± SEM of three independent experiments. (A,B,D): Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test. * p < 0.05, *** p < 0.001 vs. control. C: Statistical significance was assessed using Student-t test. * p < 0.05, *** p < 0.001 vs. control.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Cytometry, Staining, Membrane, Incubation, Positive Control, Gene Expression, Real-time Polymerase Chain Reaction, Comparison, Control

Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 6. Changes in Fas concentration in HL-60 cells treated with DL-247 for 24 h analyzed by Human Fas ELISA kit. Data are presented as mean ± SEM of three independent experiments. Statistical significance was assessed using one-way ANOVA and a post-hoc multiple comparison Student–Newman–Keuls test. *** p < 0.001 vs. control.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Molecules

Article Title: A New Hybrid δ-Lactone Induces Apoptosis and Potentiates Anticancer Activity of Taxol in HL-60 Human Leukemia Cells

doi: 10.3390/molecules25071479

Figure Lengend Snippet: Figure 9. Multiparameter analysis (proliferation, DNA damage and apoptosis) of the synergistic effect of Tx (9 nM) in combination with DL-247 (1.15 µM) in HL-60 cells. Panel (A): DAPI vs. BrdU PerCP-Cy™5.5 staining profile. BrdU positive cells are in the inside frame. Panel (B): cleaved PARP (Asp214) PE vs. H2AX (pS139) Alexa profile. Squares Q1 + Q2 represent H2AX (pS139) Alexa positive cells, squares Q2 + Q4 represent Cleaved PARP (Asp214) PE positive cells, whereas square Q3 represents H2AX (pS139) Alexa and Cleaved PARP (Asp214) PE negative cells.

Article Snippet: Evaluation of the level of FAS protein was performed using Human Factor-related Apoptosis ELISA Kit (BT Lab Shanghai Korain Biotech Co, Shanghai, China) according to the manufacturer’s protocol.

Techniques: Staining

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Mechanism of Neuroinflammation: Enhanced Cytotoxicity and IL-17 Production via CD46 Binding

doi: 10.1007/s11481-010-9232-9

Figure Lengend Snippet: Comparison of proliferative responses between healthy controls and RRMS patients CD4+ T cells after 72 h of stimulation. CD4+ T cells purified by magnetic beads were stimulated with soluble IgG isotype control, anti-CD3 and anti-CD46, or anti-CD3 and anti-CD28 and pulsed with 3[H] thymidine for the last 6 h. Proliferation stimulation index=proliferation due to soluble anti-CD3 and anti-CD46 or anti-CD3 and anti-CD28/proliferation due to soluble IgG isotype alone. Statistical analysis was performed using ANOVA, and no significance was detected between groups

Article Snippet: CD46 co-stimulation enhances IL-17A production in MS The work of Kemper and colleagues showed that CD46 co-stimulation could bias T-cell lineage differentiation to favor the development of Tr1 cells in healthy donors ( Kemper et al. 2003 ).

Techniques: Comparison, Purification, Magnetic Beads, Control

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Mechanism of Neuroinflammation: Enhanced Cytotoxicity and IL-17 Production via CD46 Binding

doi: 10.1007/s11481-010-9232-9

Figure Lengend Snippet: Differential expression of IL-1b and IL-10 in healthy control and RRM patient CD4+ T cells. Purified CD4+ T cells from healthy controls or RRMS patients were stimulated with IgG isotype control, anti-CD3 and anti-CD28, or anti-CD3 and anti-CD46 antibodies for 3 days. Cytokine production in the culture supernatant was measured with the Raybio pro-inflammatory array as described in “Methods” Levels of IL-1β produced by a healthy control; c RRMS patients. IL-10 production in b healthy controls; d RRMS patients. Statistical analysis was performed with Student's t test (**p<0.05; ***p<0.005)

Article Snippet: CD46 co-stimulation enhances IL-17A production in MS The work of Kemper and colleagues showed that CD46 co-stimulation could bias T-cell lineage differentiation to favor the development of Tr1 cells in healthy donors ( Kemper et al. 2003 ).

Techniques: Quantitative Proteomics, Control, Purification, Produced

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Mechanism of Neuroinflammation: Enhanced Cytotoxicity and IL-17 Production via CD46 Binding

doi: 10.1007/s11481-010-9232-9

Figure Lengend Snippet: Intracellular flow cytometric analysis of IL-17A expression. PBMC from healthy donors (HD) and relapsing-remitting multiple sclerosis (RRMS) patients were stimulated with soluble IgG isotype control alone, anti-CD3 and IgG isotype control, anti-CD3 and anti-CD28 antibodies, or anti-CD3 and anti-CD46 for 72 h and analyzed by intracellular staining. a Representative FACS staining profile for healthy donor (top panels) and RRMS patient (bottom panels). b Quantification of percent IL-17A expressing CD4+ T cells in HD (n= 5) and RRMS patients (n=5) PBMC following the indicated in vitro stimulations. Statistical analysis was performed using 2-way ANOVA with Bonferroni correction (*p<0.001) during immune activation. Additionally, Kebir and colleagues

Article Snippet: CD46 co-stimulation enhances IL-17A production in MS The work of Kemper and colleagues showed that CD46 co-stimulation could bias T-cell lineage differentiation to favor the development of Tr1 cells in healthy donors ( Kemper et al. 2003 ).

Techniques: Expressing, Control, Staining, In Vitro, Activation Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Mechanism of Neuroinflammation: Enhanced Cytotoxicity and IL-17 Production via CD46 Binding

doi: 10.1007/s11481-010-9232-9

Figure Lengend Snippet: Structure of CD46 and known binding sites for pathogens. STP serine, threonine, and proline rich region. CYT1 cytoplasmic tail domain 1. CYT2 cytoplasmic tail domain 2. Asterisk indicates pathogens associated with autoimmune diseases

Article Snippet: CD46 co-stimulation enhances IL-17A production in MS The work of Kemper and colleagues showed that CD46 co-stimulation could bias T-cell lineage differentiation to favor the development of Tr1 cells in healthy donors ( Kemper et al. 2003 ).

Techniques: Binding Assay

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Mechanism of Neuroinflammation: Enhanced Cytotoxicity and IL-17 Production via CD46 Binding

doi: 10.1007/s11481-010-9232-9

Figure Lengend Snippet: Binding of distinct CD46 domains triggered differential IL-17A production. PBMC from healthy controls were cultured with anti-CD3 and anti-CD46 (clone J4.48) or anti-CD3 and anti-CD46 (clone MEM) for 72 h and IL-17A expression in CD4+ T cells were assessed by FACS analysis. a PBMC co-stimulated with indicated concentrations of anti-CD46 (J4.48) elicited IL-17A expression in a dose-dependent manner. b Co-stimulation of CD46 receptor with anti-CD46 antibody (MEM) did not demonstrate significant level of IL-17A

Article Snippet: CD46 co-stimulation enhances IL-17A production in MS The work of Kemper and colleagues showed that CD46 co-stimulation could bias T-cell lineage differentiation to favor the development of Tr1 cells in healthy donors ( Kemper et al. 2003 ).

Techniques: Binding Assay, Cell Culture, Expressing

Journal: Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology

Article Title: Mechanism of Neuroinflammation: Enhanced Cytotoxicity and IL-17 Production via CD46 Binding

doi: 10.1007/s11481-010-9232-9

Figure Lengend Snippet: Enhanced cytotoxicity following anti-CD3 and anti-CD46 stimulation. To evaluate the potential of cytotoxicity associated with CD46 co-stimulation, purified CD4+ T cells from healthy controls (n= 5) and RRMS patients (n=5) were co-cultured with anti-CD3 and anti-CD46 antibodies or anti-CD3 and anti-CD28 antibodies for 72 h and CD107a degranulation was assessed by flow cytometric analysis. Statistical analysis was performed with Student's t test

Article Snippet: CD46 co-stimulation enhances IL-17A production in MS The work of Kemper and colleagues showed that CD46 co-stimulation could bias T-cell lineage differentiation to favor the development of Tr1 cells in healthy donors ( Kemper et al. 2003 ).

Techniques: Purification, Cell Culture