Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits the proliferation of human primary T cells. CD4+ T cells were isolated by negative selection (>97% purity), activated with immobilized CD3/CD28 Abs, and concurrently treated with the indicated concentrations of DIDOX, DOX, or vehicle control. After 24h of culture, 1 μCi of 3[H] thymidine was added to each well for an additional 24h of culture and proliferation measured using a thymidine incorporation assay. Data are normalized relative to values obtained in cultures treated with vehicle control. Data from three independent experiments are presented with error bars depicting S.E.

Article Snippet: Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Isolation, Selection, Control, Thymidine Incorporation Assay

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell CD40L expression. Primary human CD4+ T cells were isolated by negative immunomagnetic selection (~98% purity). Cells were activated with immobilized CD3/CD28 Abs either concurrent to treatment with GPX -150 for 6h (A & B) or following an 18h DIDOX pretreatment followed by 6h CD3/CD28 activation (C & D). After activation, cells were harvested, stained with a FITC-conjugated CD40L Ab and analyzed by flow cytometry. A) Concurrent DIDOX treatment and TCR activation (6h) inhibits T cell CD40L expression, n=4. B) A representative histogram is depicted with control cultures staining 57% positive for CD40L and 50 μM DIDOX treated cultures staining 32.6% positive, iso = isotype, NT = no treatment. C) DIDOX pretreatment (18h) prior to TCR activation (6h) inhibits CD40L expression, n=5. D) A representative histogram is depicted with control cultures staining 74.6% CD40L positive and 50 μM DIDOX treated cultures staining 12.0% positive. Error bars depict S.E and asterisks indicate statistical significance from control.

Article Snippet: Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Expressing, Isolation, Selection, Activation Assay, Staining, Flow Cytometry, Control

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell CD25 expression. Primary CD4+ T cells were isolated by negative selection (~95% purity) and pretreated with various concentrations of DIDOX or vehicle for 18h. Cells were subsequently activated with CD3/CD28 antibodies for an additional 24h, stained with a FITC-conjugated CD25 Ab, and analyzed by flow cytometry. A) Data are normalized relative to values obtained in TCR activated cultures treated with vehicle alone, n=6. Error bars depict S.E and asterisks indicate significance from baseline control. B) A representative histogram depicts CD25 staining in control cultures (NT; 73.4% CD25 positive staining) and cultures treated with 50 μM DIDOX (20.1% CD25 positive) relative to staining with an isotype control antibody (iso).

Article Snippet: Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Expressing, Isolation, Selection, Staining, Flow Cytometry, Control

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell production of TNF-α Primary CD4+ T cells were isolated by negative immunomagnetic selection (>98% purity). Cells were activated for 48h with immobilized CD3/CD28 antibodies, 40 U/ml IL-2, and treated with various concentrations of DIDOX. Cell-free conditioned media was harvested via successive centrifugations and analyzed for TNF-α by ELISA. Data from 5 representative experiments is presented. Error bars depict S.E. and asterisks indicates statistical significance from baseline control.

Article Snippet: Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay, Control

Journal:

Article Title: In Vitro and In Vivo Immunosuppressive Activity of a Novel Anthracycline, 13-deoxy, 5-iminodoxorubicin 1

doi: 10.1016/j.intimp.2007.01.010

Figure Lengend Snippet: DIDOX inhibits T cell production of IL-2. Primary CD4+ T cells were isolated by negative immunomagnetic selection (>98% purity). Cells were activated for 48h with immobilized CD3/CD28 antibodies and treated with various concentrations of DIDOX or vehicle control. Cell-free conditioned media was harvested via successive centrifugations and analyzed by ELISA. Data from 3 representative experiments is presented. Error bars depict S.E. and asterisks indicates statistical significance from baseline control.

Article Snippet: Purified CD4 + cells were cultured in RPMI/10% FCS at 1 × 10 6 cells/ml and activated with immobilized CD3 (0.3 μg/well of clone OKT3, ATCC, Rockville, MD) +/− CD28 antibodies (Abs) (0.08 μg/well of clone CD28.2, PharMingen, San Diego, CA), +/− 100 U/ml human recombinant IL-2 (Sigma) on 96 well plates in 200 μl total volume.

Techniques: Isolation, Selection, Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Antibody–Drug Conjugate Made of Zoledronic Acid and the Anti-CD30 Brentuximab–Vedotin Exert Anti-Lymphoma and Immunostimulating Effects

doi: 10.3390/cells13100862

Figure Lengend Snippet: Bre–Ved–ZA ADC reactivity. ( A ) Bre–Ved–ZA titration. Upper panels: the HL-cell lines KMH2, L428, L540, HS445, and RPMI6666 were incubated with 20–0.2 µg/mL/10 6 cells of Bre–Ved–ZA ADC (upper histograms, red lines) or Bre–Ved (lower histograms, gray lines), followed by the AlexaFluor647-labeled α-hIg antiserum and FACS analysis. Results expressed as Log far red fluorescence intensity (a.u.) vs. the number of cells. ( B ) HL/CD30 + lymphoblastoid cell lines were incubated with 20–2.0–0.2 µg/mL/10 6 cells of Bre–Ved–ZA ADC (red) or Bre–Ved (gray) as in ( B ). Results expressed as mean fluorescence intensity (MFI-AF647, a.u.).

Article Snippet: KMH2 or L428 (from pleural effusion) and L540 (from bone marrow) classical HL-cell lines (purchased and certified from DSMZ GmbH, Braunschweig, Germany), or the CD30 + lymphoblastoid RPMI6666 and HS445 cell lines (provided and certified by ATCC, Manassas, VA, USA) were incubated with serial dilutions (20–0.02 μg/mL/10 6 cells) of Bre–Ved or Bre–Ved–ZA ADC, for 1 h at 4 °C, followed by the AlexaFluor647 antihuman Ig (AF647-α-hIg) labeling.

Techniques: Titration, Incubation, Labeling, Fluorescence

Journal: Cells

Article Title: Antibody–Drug Conjugate Made of Zoledronic Acid and the Anti-CD30 Brentuximab–Vedotin Exert Anti-Lymphoma and Immunostimulating Effects

doi: 10.3390/cells13100862

Figure Lengend Snippet: HL-cell-line proliferation and apoptosis upon Bre–Ved–ZA or Bre–Ved treatment. ( A ) ATP content, determined using the CellTiter-Glo ® Luminescent Cell Viability Kit, in KMH2, L428, L540, HS445, or RPMI6666 exposed to serial dilution of Bre–Ved or Bre–Ved–ZA (20, 2.0, 0.2 μg/mL) and incubated at 37 °C for 5 days. Luminescence was detected with the VICTORX5 reader and expressed as luminescence arbitrary units (a.u). ( B ) Caspase activation in KMH2 and RPMI6666 cell lines evaluated at 72 h with the luciferase-based Caspase Glo 3/7 3D Assay, expressed as luminescence units (RLU)/5 × 10 4 cells. Results are the mean ± SD of three independent experiments from 4 replicated wells. *** p < 0.001 of Bre–Ved–Za vs. Bre–Ved. ( C ) KMH2 cultured for 72 h with 20 or 2.0 or 0.2 μg/mL Bre–Ved–ZA were labeled with FITC-annexin-V (AV) and propidium iodide (PI) and subjected to FACS analysis. CTR: staining in untreated cells. Apoptotic cells are identified as AV + PI + . ( D ) Confocal microscopy of KMH2 cells treated for 72 h with 2 μg/mL Bre–Ved–ZA upon nuclear staining with Syto16 (left images). Central images: bright field; right images: merged dark and bright fields. Magnification: 20× objective. Bar: 10 µm. ( E ) Confocal microscopy as in ( D ) at higher magnification (40× objective). Bar: 10 µm. Results are representative of three independent experiments. CTR: KMH2 cultured in medium without antibody. The white arrows in ( D , E ) indicate the apoptotic nuclei.

Article Snippet: KMH2 or L428 (from pleural effusion) and L540 (from bone marrow) classical HL-cell lines (purchased and certified from DSMZ GmbH, Braunschweig, Germany), or the CD30 + lymphoblastoid RPMI6666 and HS445 cell lines (provided and certified by ATCC, Manassas, VA, USA) were incubated with serial dilutions (20–0.02 μg/mL/10 6 cells) of Bre–Ved or Bre–Ved–ZA ADC, for 1 h at 4 °C, followed by the AlexaFluor647 antihuman Ig (AF647-α-hIg) labeling.

Techniques: Serial Dilution, Incubation, Activation Assay, Luciferase, Cell Culture, Labeling, Staining, Confocal Microscopy