Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Histamine release from the basophils of control and asthmatic subjects and a comparison of gene expression between "releaser" and "nonreleaser" basophils.

doi: 10.4049/jimmunol.178.7.4584

Figure Lengend Snippet: FIGURE 3. Histamine release from the five releasers and three nonre- leasers basophils on the same day as RNA purification. Percoll-enriched releaser and nonreleaser basophils were incubated for 30 min in various concentrations of anti-IgE or A23187 (5 ng/ml), and histamine levels were measured in the incubation supernatants using ELISA. Results, corrected for spontaneous release, show the range of duplicate assays. Anti-IgE in- duced no histamine release from NR116 and NR130.

Article Snippet: The IL-3 ELISA kit was from R&D Systems, and the human histamine kits were from Beckman Coulter.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Histamine release from the basophils of control and asthmatic subjects and a comparison of gene expression between "releaser" and "nonreleaser" basophils.

doi: 10.4049/jimmunol.178.7.4584

Figure Lengend Snippet: FIGURE 5. Time course and dose-response studies of anti-IgE-induced basophil chemokine release. In A–C, releaser (solid lines) or nonreleaser (dashed lines) basophils were incubated for 1, 2, or 4 h with 0.1 g/ml anti-IgE. In D–F, releaser (solid bars) or nonreleaser (striped bars) basophils were incubated with 0.01, 0.1, 1, and 5 g/ml anti-IgE or 500 ng/ml A23187 for 4 h at 37°C. Levels of IL-8 (A), MIP-1 (B), and MIP-1 (C) in the culture supernatants were analyzed by ELISA.

Article Snippet: The IL-3 ELISA kit was from R&D Systems, and the human histamine kits were from Beckman Coulter.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Oncoimmunology

Article Title: Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment

doi: 10.1080/2162402X.2017.1322238

Figure Lengend Snippet: Characterization of therapeutic DC.Tbet. Bone marrow-derived DC were generated in GM-CSF + IL-4 cultures for 5–6 d, before being infected for 48 h with rAd.mTbet or control rAd.EGFP or empty rAd.ψ5, or they were left untransduced, as indicated. In (A), DC were treated, as indicated, with the addition of LPS + IFNγ for the second 24 h of infection. Affymetrix gene array analyses were performed on DC.Tbet (A), with transcripts increased >5-fold compared with control DC reported. DC.Tbet generated from Tbet-ZsG (H-2b) reporter mice were then analyzed by IFM for intracellular expression of the Tbet reporter (green) and IL-36γ (red), with DAPI staining of nuclei (B). DC.Tbet or control DC were cultured at 4 × 105 cells/mL. After 48 h of infection, supernatant was harvested and mIL-12p70 (C) or mIL-36γ (D) production was analyzed by ELISA. **p < 0.05 for DC.Tbet vs. control DC (t-test). In E, DC.Tbet, control DC.ψ5, or PBS were then injected intratumorally into mice bearing d7 established s.c. MCA205 sarcomas on days 7 and 14 post-tumor inoculation. Tumor size was measured over time. **p < 0.05 for DC.Tbet vs. PBS or control DC.ψ5 treatment on days ≥ 11 (ANOVA). Data are representative of those obtained in two independent experiments performed in each case.

Article Snippet: Murine IL-36γ ELISA kit (Aviva Systems Biology Corporation, part number OKEH03002) was used per the manufacturer's instructions.

Techniques: Derivative Assay, Generated, Infection, Control, Expressing, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Injection

Journal: Oncoimmunology

Article Title: Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment

doi: 10.1080/2162402X.2017.1322238

Figure Lengend Snippet: DC.Tbet injected i.t. promote the rapid infiltration of lymphocytes and the development of PNAd+ blood vessels in association with enhanced locoregional induction of IL-36γ. MCA205 tumor-bearing wild-type C57BL/6 mice or Tbet-ZsG reporter mice were treated by i.t. delivery of 1 × 106 DC.Tbet or DC.ψ5 7 d post-tumor inoculation. In (A), tumors were harvested from Tbet-ZsG mice at 4h, 10h or 24h after treatment with DC.Tbet and tissue sections analyzed by IFM for CD4+ T cells, CD8+ T cells, CD19+ B cells, and PNAd+ HEV. In (B) and (C), expression and localization of peripheral node addressin (PNAd), CD11c+ DC, CD3+ T cells, and B220+ B cells were analyzed in tumor sections from Tbet reporter mice on days 5 (B) and 12 (C) post-treatment with DC.Tbet. In (D), day 5 (post-DC.Tbet treatment) tumor sections from Tbet-ZsG mice were analyzed by IFM for co-expression of the Tbet reporter (green) and IL-36γ (red, using a specific polyclonal antibody). In (E), IL-36γ protein levels were assayed by quantification of fluorescence from IFM, or transcript levels were assayed by real-time PCR in total tumor RNA isolated from wild-type C57BL6/J hosts, at the indicated time points following DC.Tbet or DC.ψ5 treatment. Data are representative of those obtained in two to three independent experiments performed in each case.

Article Snippet: Murine IL-36γ ELISA kit (Aviva Systems Biology Corporation, part number OKEH03002) was used per the manufacturer's instructions.

Techniques: Injection, Expressing, Fluorescence, Real-time Polymerase Chain Reaction, Isolation

Journal: Oncoimmunology

Article Title: Tbet and IL-36γ cooperate in therapeutic DC-mediated promotion of ectopic lymphoid organogenesis in the tumor microenvironment

doi: 10.1080/2162402X.2017.1322238

Figure Lengend Snippet: DC.IL36γ produce/secrete bioactive IL-36γ and upregulate intrinsic transcription of Tbet. Real-time PCR (A) and Western blot analysis (B) for IL-36γ were performed on lysates of DC.IL36γ/EGFP vs. control DC.null or DC.EGFP to confirm transduction efficacy. In (C), Affymetrix gene array analyses were performed as outlined in Materials and Methods, with transcripts increased >5-fold compared in DC.IL36γ vs. control DC.null reported. In (D) and (E), real-time PCR (D) and Western blot analysis (E) for Tbet were performed on lysates of DC.IL36γ/EGFP and/or DC.Tbet/EGFP and/or control DC.EGFP and/or control DC.null. In (A) and (D), mean ± SD data are reported; **p < 0.05 (t-test). In (F), DC were differentiated for 5 d in vitro, with CD11c+ cells then isolated and either transduced with rAd to express EGFP, mTbet, and/or mIL-36γ, or untransfected control DC were treated for 24 h with the indicated TLR agonists or agonist anti-CD40 (FGK45) antibody. Cell-free supernatants were then analyzed by mIL-36γ ELISA. *p < 0.05 for DC.Tbet and DC.IL36γ vs. all other treatment groups (ANOVA). In (G), cell-free supernatants were recovered from engineered DC and analyzed for bioactivity by addition to cultures of bulk splenocytes isolated from Tbet (ZsG) reporter mice; i.e., 106 splenocytes were cultured in 200 μL of basal media (negative control), media containing LPS + IFNγ (positive control) or cell-free media harvested from DC.null or DC.IL36γ cells 48 h after rAd infection. After overnight culture, splenocytes were analyzed by flow cytometry for upregulation of intracellular Tbet reporter expression. In (H), DC.IL36γ cells were analyzed by IFM as described in Fig. 1(B) to detect coordinate expression of Tbet and IL-36γ protein. All data are representative of those obtained in two to three independent experiments performed in each case.

Article Snippet: Murine IL-36γ ELISA kit (Aviva Systems Biology Corporation, part number OKEH03002) was used per the manufacturer's instructions.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Control, Transduction, In Vitro, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, Negative Control, Positive Control, Infection, Flow Cytometry, Expressing