Journal:

Article Title: Systemic protein delivery by muscle-gene transfer is limited by a local immune response

doi: 10.1182/blood-2004-03-0848

Figure Lengend Snippet: OVA expression and antibody formation to OVA. Systemic OVA expression and antibody formation to OVA after intramuscular injection of AAV-CMV-OVA into wild-type BALB/c mice (A-B) or BALB/c mice transgenic for DO11.10 TCR (C-D). (A-B) Mice received doses of 1 × 1011 vg/animal (♦) or 1 × 1012 vg/animal (▪). (C-D) Mice received doses of 1 × 1012 vg/animal (▪) or were naive controls (•). All data points represent average of 5 animals ± SD. Panels A and C show plasma levels of OVA as a function of time after vector administration; panels B and D indicate levels of IgG1 anti-OVA as a function of time after vector administration.

Article Snippet: Purified CD4 + T cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinmidyl ester (CFSE flow kit; Renovar, Madison, WI) and injected intravenously into naïve BALB/c recipient mice (5 × 10 6 CD4 + cells/mouse).

Techniques: Expressing, Injection, Transgenic Assay, Clinical Proteomics, Plasmid Preparation

Journal:

Article Title: Systemic protein delivery by muscle-gene transfer is limited by a local immune response

doi: 10.1182/blood-2004-03-0848

Figure Lengend Snippet: In vitro cytokine secretion, activation, and proliferation by lymphocyte cultures from DO11.10 TCR transgenic BALB/c mice at 10 days after vector administration. Splenocytes or LN cells from naive controls or AAV-CMV-OVA intramuscularly injected mice were cultured in 6-well plates (5 × 106 cells/well) for 3 days and stimulated with 100 μg/mL OVA or mock stimulated. Conditioned media were collected at day 3 of cell culture and analyzed by cytokine-specific ELISA for IL-2 (A,D), IL-10 (B,E), and IFN-γ (C,F). (A-C) Splenocyte cultures. (G) Summary of percent CD69+ of CD4+TCR+ cells for lymphoid organs of naive controls and vector-injected mice as determined by flow cytometry. Spleens from each animal were processed and analyzed individually. Results are average (5/group) ± SD. Results from LN cells represent average ± SD for 3 counts by flow cytometry with each count representing LN cells pooled from 1 to 2 animals. Splenocytes (H) and LN cells (I) were also cultured (5 × 105 cells/well in 96-well plates) overnight with OVA stimulation (100 μL/mL) or without OVA stimulation (mock), and pulsed with 3H-thymidine for an additional 12 hours. Results for 3H-thymidine incorporation are shown in counts per minute (CPM). All cell cultures were set up in quadruplicate. LNs were inguinal and popliteal LNs. D-LN indicates draining LNs of vector-injected leg muscle; ND-LN, nondraining LNs of noninjected contralateral leg. Results from D-LNs are marked by vertical arrow. Spleens from each animal were processed and analyzed individually. Results are average (5/group) ± SD. Results from LN cells represent average ± SD for 3 wells with each well representing LN cells pooled from 1 to 2 animals.

Article Snippet: Purified CD4 + T cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinmidyl ester (CFSE flow kit; Renovar, Madison, WI) and injected intravenously into naïve BALB/c recipient mice (5 × 10 6 CD4 + cells/mouse).

Techniques: In Vitro, Activation Assay, Transgenic Assay, Plasmid Preparation, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal:

Article Title: Systemic protein delivery by muscle-gene transfer is limited by a local immune response

doi: 10.1182/blood-2004-03-0848

Figure Lengend Snippet: In vitro cytokine secretion, activation, and proliferation by lymphocyte cultures from DO11.10 TCR transgenic BALB/c mice at 30 days after vector administration. Splenocytes or LN cells from naive controls or AAV-CMV-OVA intramuscularly injected mice were cultured in 6-well plates (5 × 106 cells/well) for 3 days and stimulated with 100 μg/mL OVA or mock stimulated. Conditioned media were collected at day 3 of cell culture and analyzed by cytokine-specific ELISA for IL-2 (A,D), IL-10 (B,E), and IFN-γ (C,F). (A-C) Splenocyte cultures. (D-F) LN cell cultures. (G) Summary of percentage CD69+ of CD4+TCR+ cells for lymphoid organs of naive controls and vector-injected mice as determined by flow cytometry. Splenocytes (H) and LN cells (I) were also cultured (5 × 105 cells/well in 96-well plates) overnight with OVA stimulation (100 μL/mL) or without OVA stimulation (mock), and pulsed with 3H-thymidine for an additional 12 hours. Results for 3H-thymidine incorporation are shown in CPM. LNs were inguinal and popliteal LNs. D-LN indicates draining LNs of vector-injected leg muscle; ND-LN, nondraining LN of noninjected contralateral leg. Results from D-LNs are marked by vertical arrow. All experimental conditions for panels A-I (including sizes per group and determination of average ± SD) were as described for Figure 3.

Article Snippet: Purified CD4 + T cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinmidyl ester (CFSE flow kit; Renovar, Madison, WI) and injected intravenously into naïve BALB/c recipient mice (5 × 10 6 CD4 + cells/mouse).

Techniques: In Vitro, Activation Assay, Transgenic Assay, Plasmid Preparation, Injection, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal:

Article Title: Systemic protein delivery by muscle-gene transfer is limited by a local immune response

doi: 10.1182/blood-2004-03-0848

Figure Lengend Snippet: Quantitation of OVA-specific cytokine responses in DO11.10 TCR transgenic BALB/c mice by ELISPOT. Mice were naive controls or had been injected intramuscularly with AAV-CMV-OVA vector (3 × 1012 vg/animal). Panels A, C, E, and G show numbers of IFN-γ–secreting cells as spot-forming units (SFUs) per 106 cells after mock or OVA stimulation. Panels B, D, F, and H show numbers of IL-4–secreting cells as SFUs per 106 cells after mock or OVA stimulation. (A-B) Splenocytes, day 10 after gene transfer. (C-D) LN cells, day 10. (E-F) Splenocytes, day 30. (G-H) LN cells, day 30 after gene transfer. LNs were inguinal and popliteal LNs. D-LN indicates draining LNs of vector-injected leg muscle; ND-LN, nondraining LNs of noninjected contralateral leg. Results from D-LNs are marked by vertical arrow. All ELISPOT cultures were set up in quadruplicate. Spleens from each animal were processed and analyzed individually. LN cells were pooled for experimental groups (5 animals/group). Results are average of quadruplicate measurement ± SD.

Article Snippet: Purified CD4 + T cells were labeled with 5-(and-6)-carboxyfluorescein diacetate succinmidyl ester (CFSE flow kit; Renovar, Madison, WI) and injected intravenously into naïve BALB/c recipient mice (5 × 10 6 CD4 + cells/mouse).

Techniques: Quantitation Assay, Transgenic Assay, Enzyme-linked Immunospot, Injection, Plasmid Preparation