Journal: Cells

Article Title: Humanized FcεRI Expressed on Mouse Eosinophils Mediates IgE-Facilitated Eosinophil Antigen Presentation

doi: 10.3390/cells14040301

Figure Lengend Snippet: IgE-hFcεRI engagement facilitates eosinophil antigen presentation in vivo to enhance pLN T cell proliferation and IL-4 release and to increase eosinophil CD40, CD80, and CD86 expression. ( A , B ) hFcεRIα eosinophils, incubated with NP-OVA or NP-OVA and chimeric anti-NP human IgE, were injected i.t. into NP-OVA-immunized mice. Controls received i.t instillation of PBS or eosinophils treated with OVA, NP-OVA, or control human IgE. ( A ) To block hFcεRIα-mediated cIgE-facilitated NP-OVA presentation, anti-FcεRIα mAb (15.1) doses were used. Three days after eosinophil instillation, pLN cells were assayed for T cell proliferation ( B ) Three days after i.t. eosinophil transfer, pLN cells were cultured and assayed for IL-4 and INF-γ release. ( C ) FcεRI engagement increases expressions of CD40, CD80, and CD86 on airway eosinophils. On BAL eosinophils from OVA-sensitized and -challenged hFcεRIα mice, hFcεRI was cross-linked with either cIgE Ab and mouse anti-human IgE (left panel) or anti-hFcεRIα mAb (15.1) and anti-mouse IgG1 (right panel). Histograms show expressions of costimulatory proteins on resting eosinophils (middle histograms) and FcεRI cross-linked eosinophils (bold line right histograms) vs isotype control Ab (left histograms). ( D ) Western blotting shows that hFcεRIα cross-linking, elicited by 15.1 mAb + anti-mIgG1 and cIgE + anti-hIgE, increases CD40, CD80, and CD86 proteins in eosinophils. Eosinophils were treated with PBS, control mIgG1 mAb, 15.1 mAb only, 15.1 mAb + anti-mIgG, or cIgE only, cIgE + mouse isotype IgG, and cIgE+ anti-hIgE.

Article Snippet: Blots were blocked overnight at 4 °C in Tris-buffered saline (TBS) containing 0.1% Tween 20 and 5% milk before incubations with goat anti-mouse CD40, CD80, or CD86 Abs (Serotec, Oxford, UK) diluted in TBS/0.1% Tween 20 for 2 h at room temperature.

Techniques: Immunopeptidomics, In Vivo, Expressing, Incubation, Injection, Control, Blocking Assay, Cell Culture, Western Blot

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: HAECs were transfected with vectors encoding the CD40 siRNA or control siRNA and then incubated with CD40L (80 ng/mL) for 24 h. (A) Representative blot showing CD40 protein expression in HAECs transfected with control or CD40 siRNA. (B) VCAM-1 protein expression and (C) THP-1 adhesion were determined as indicated. Data are shown as representative blots or are expressed as the means ± SEM by three independent assays from 4 independent experiments. * P <0.05 vs CD40L plus Control siRNA.

Article Snippet: Antibodies used in the present study include the following: mouse anti-PKCβ antibody (BD Biosciences), mouse anti–VCAM-1 (Chemicon International), rabbit anti–NF-κB p65 antibody, rabbit anti-IκBα antibody, rabbit anti–β-actin antibody (Cell Signaling Technology), and goat anti-CD40 antibody (Academy Biomedical).

Techniques: Transfection, Incubation, Expressing

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Overview of the treatment scheme. i.t: intratumorally; i.v: intravenously; ICOSL: inducible co-stimulator ligand.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques:

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Viral DNA extraction from CsCl-purified virus particles using proteinase K. The AdV-D24-ICOSL-CD40L sequencing library was made using a Nextera XT kit (Illumina). The size of the library was assessed by high-resolution agarose gel electrophoresis. The presence of the 24-bp deletion in the E1A CR2 domain, the CMV-ICOSL-IRES-CD40L expression cassette inserted in place of the E3 region and the hybrid Ad5/3 fiber were confirmed. Analysis of sequence coverage did not detect the presence of a virus sub-population with a genome characterized by a major rearrangement such as a large insertion or deletion. The figure was generated by SnapGene ® .

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: DNA Extraction, Purification, Sequencing, Agarose Gel Electrophoresis, Expressing, Generated

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of ICOSL and CD40L expression in tested melanoma cells. ( A ) ICOSL concentration was detected on supernatants collected 24–48 h after treatment using ELISA kit. ( B ) CD40L was detected from supernatants collected 24–48 h after treatment using an ELISA kit according to the manufacturer’s instructions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (ns = not significant, p > 0.05). ( C ) Transgene expression. The expression of ICOSL and CD40L from AdV-D24-ICOSL-CD40L was assessed by infecting A549 cells with the virus, harvesting proteins 48 h after the infection and detecting ICOSL and CD40L by Western blot. 1–4: protein extracts from A549 cells infected with AdV-D24-ICOSL-CD40L clones #1–4. N: protein extract from non-infected A549 cells. M: PageRuler Prestained Protein Ladder (ThermoScientific # 26616). ICOSL MW: 70 kDa (due to heavy protein glycosylation); CD40L MW: 30 kDa.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Infection, Western Blot, Clone Assay

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Evaluation of cell viability by MTS assay (cell cytotoxicity assay). ( A ) Cell viability was evaluated 72 h post-infection with AdV-24-ICOSL-CD40L and AdV-24 at the concentration of 100 VP/cell and combination with anti-PD1 (pembrolizumab). Data are expressed as the percentage of viable cells according to MTS cell viability assay protocol (CellTiter 96 ® AQueous One Solution Cell Proliferation Assay, Promega). Statistical analysis was carried out with a Mann–Whitney test to compare two groups ( p > 0.05). ( B , C ) Microscopic photographs visualizing the morphology and cytopathic effect (CPE) of the cells representing the investigated groups and assessed by MTS assay at 48 and 72 h post-infection (magnification 10×) (** = p ≤ 0.001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: MTS Assay, Cytotoxicity Assay, Infection, Concentration Assay, Viability Assay, Proliferation Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Immunogenic cell death assessment. ( A ) Evaluation of calreticulin (CRT) exposure by melanoma cell lines after treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured 48 h post-treatments with anti-calreticulin antibody staining AlexaFluor ® 488 and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( B ) Assessment of ATP release after the treatment. ATP concentration in a supernatant was evaluated 72 h after infection with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. ( C ) Evaluation of high-mobility group box 1 (HMGB-1) release after treatment with oncolytic adenoviruses and the combination with anti PD-1. HMGB-1 level was measured from the supernatant collected 72 h after infection with ELISA kit (MBL International, Woburn, MA, USA), according to manufacturer’s dispositions. Statistical analysis was carried out with a Mann–Whitney test to compare two groups (* = p ≤ 0.05; ** = p ≤ 0.001, *** = p ≤ 0.0001).

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Infection, Cell Viability Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: Combination Therapy of Novel Oncolytic Adenovirus with Anti-PD1 Resulted in Enhanced Anti-Cancer Effect in Syngeneic Immunocompetent Melanoma Mouse Model

doi: 10.3390/pharmaceutics13040547

Figure Lengend Snippet: Antitumor efficacy of oncolytic vectors and the combination therapy with anti PD-1 antibody in murine melanoma B16V (1 × 10 6 cells/flank, one mouse had 2 tumors/6 tumors per group) xenograft immunocompetent C57BL/6 model. ( A ) Tumor volume (mm 3 ) measured through the study. The treatment was performed once per day on days 1–6. The mice were treated according to the scheme with viruses (i.t.) and anti PD-1 antibody (i.v.) ( B ) At the end of the study, mice were sacrificed and tumors harvested for weight assessment. ( C , D ) Tumor volume measurement on days 6 and 20. ( E ) Body weight measurements throughout the study. ( F ) Survival profile was calculated by Kaplan–Meier test. ( H ) Evaluation of CRT exposure after the treatment with oncolytic adenoviruses AdV-24-ICOSL-CD40L and AdV-D24, and in combination with anti PD-1. CRT exposure was measured in the end of the study (after mice sacrifice) with anti-calreticulin antibody staining and subsequent flow cytometry analysis (Beckman-Coulter Cytomics FC500). ( G ) Assessment of ATP release after the treatment. ATP concentration from the tumors was evaluated in the end of the study (after mice sacrifice) with CellTiter-Glo ® Luminescent Cell Viability Assay ATP detection kit by Promega. Error bars, mean ± SEM, * = p ≤ 0.05, *** = p ≤ 0.0001.

Article Snippet: CD40L was detected using the primary antibody: goat polyclonal anti-CD40L antibody (R&D #AF617), followed by the secondary antibody: rabbit anti-goat IgG (HRP-conjugated, R&D HAF017).

Techniques: Staining, Flow Cytometry, Concentration Assay, Cell Viability Assay