However, after 72 h of treatment with 20 g/ml antiCIL-13 neutralizing antibody, the proliferation of HDLM2 cells (which secrete moderate levels of IL-13) was suppressed to 27% of that of untreated control cells (Fig. a logical objective for future therapeutic Pentostatin strategies. were used as quantitation standards. The hybridization was performed as previously described (7). Northern Blot Analysis. Total RNA was isolated using Trizol reagent (GIBCO BRL). 15 g total RNA was separated on a 1% formaldehyde agarose gel. Samples were run in 0.02 M MOPS (3-[N-morpholino]propanesulfonic acid), pH 7.0, 8 mM sodium acetate, 1 Rabbit Polyclonal to TRAF4 mM EDTA. The gel was blotted overnight in 20 SSC on a Hybond N+ nylon membrane (Amersham Pharmacia Biotech) and cross-linked using UV irradiation. Membranes were hybridized at 65C to [-32P]dATP-labeled (Mutiprime DNA Labeling System, Amersham Pharmacia Biotech) cDNA fragments specific for the human Notch2, NF-IL3, urokinase, IL-13, and human -actin genes. Membranes were washed first in 0.1% SDS/2 SSC (30 min, 65C) and then in 0.1% SDS/0.2 SSC (30 min, 65C). Cytokine Production. Supernatants of cell cultures (105 cells/ml) were recovered 48 h after medium exchange and assayed for IL-5, IL-13, and GM-CSF production by specific ELISA using Quantikine-kits (R&D Systems). Fixation of Biopsied Material. Freshly biopsied lymph nodes were fixed in 10% formalin for 24 h before overnight processing Pentostatin and paraffin embedding. In Situ Hybridization. For in situ hybridization, paraffin sections were mounted and fixed according to standard protocols. The IL-13 probe used was a 388-bp human cDNA probe synthesized using reverse transcriptase PCR and IL-13Cspecific primers as follows: Pentostatin 5 GTTGACCACGGTCATTGCTCTCACT and 3 TTCAGTTGAACCGTCCCTCGCGAA. The cDNA was cloned into the pCRII vector (TA Cloning Kit; Invitrogen). Sense and antisense probes were synthesized from the linearized vector with SP6 or T7 polymerase, labeled with [33P]UTP, and processed as previously described (10). The sections were counterstained with toluidine blue using a standard protocol. Immunohistochemistry. Immunohistochemistry was performed using mAbs against CD30 (Dako) and CD15 (Becton Dickinson). Formalin-fixed paraffin sections were digested with pepsin and incubated (1 h) with the primary antibody. The slides were washed in PBS and incubated with biotinylated rabbit antiC mouse IgG (20 min) and with streptavidinCbiotin complex labeled with horseradish peroxidase (20 min) after a second washing step (Ultra Streptavidin Detection System; Pentostatin Signet). The enzyme reaction was developed with AEC (3-amino-9-ethyl carbazole) and the slides were counterstained with hematoxylin. Analysis of Cell Proliferation after Neutralization of IL-13. HD-derived L428, KMH2, and HDLM2 cells and LCL control cells (2 104/well) were cultured in 96-well flat-bottomed plates for 24, 48, or 72 h in the presence of antiCIL-13, antiCIL-5, or isotype control antibodies (PharMingen) at 5, 10, 20, 30, 50, 100, or 150 g/ml. Cells were treated either with 0.5, 1, 5, 10, 50, 100, and 200 ng/ml IL-13 (R&D Systems) or with these doses of IL-13 combined with 20 g/ml antiCIL-13. [3H]thymidine (1 Ci/ well) was added to each well for an 8-h incubation. The cells were harvested on filters and the incorporation of [3H]thymidine into cellular DNA was measured as previously described (11). Results and Discussion Of 950 genes displayed on the microchip, the genes showing a greater than threefold difference in expression in both HD-derived cell lines were IL-13; IL-5; ornithine decarboxylase (ODC); ICAM-3; urokinase plasminogen activator (UPA); IgE Fc receptor II; insulin-like growth factor (IGF) II; NF-IL3/E4BP4; Notch2; GM-CSF; interferon regulatory factor (IRF)-1, IRF-5, and IRF-6; nitric oxide synthase (NOS) 3; and the TCR chain (Fig. ?(Fig.11 B). IL-13 expression Pentostatin in HD-derived L428 and KMH2 cells was increased compared with that in control LCL-GK cells by 26.7- and 17.1-fold, respectively, and IL-5 expression was increased by 14.2- and 18.5-fold (Fig. ?(Fig.11 B). Open.
- T-cell epitopes are peptides derived from antigens and identified by the T-cell receptor (TCR) when bound to MHC molecules displayed within the cell surface of APCs
- Cloning of gene fragments encoding diagnostic antigens
- Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope)
- Spleens were harvested in 1 (C) or 2 wpi (B, C) and cells were analyzed by movement cytometry in comparison to na?ve mice
- [PMC free article] [PubMed] [Google Scholar] 19