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

Article Title: IL-16 promotes leukotriene C(4) and IL-4 release from human eosinophils via CD4- and autocrine CCR3-chemokine-mediated signaling.

doi: 10.4049/jimmunol.168.9.4756

Figure Lengend Snippet: FIGURE 3. Receptors involved in IL-16-induced activation of eosino- phils. A, CD4 mediates the activation of eosinophils by IL-16. As indi- cated, eosinophils were pretreated for 30 min with inhibitors of IL-16/CD4 interaction (including sCD4 and Fab of OKT4 anti ()-CD4 Ab (or irrel- evant control W6/32 anti-HLA Ab)) or with the Gi protein inhibitor PTX and then were incubated with IL-16 (100 nM) for 1 h for induction of lipid bodies, priming for enhanced LTC4 production (fluid-phase assays), and IL-4 release (solid-phase matrix assays). B, Involvement of Gi protein- coupled CCR3 receptors. As indicated, eosinophils were pretreated for 30 min with antagonists of PAF (CV6209 and BN52021) and CCR3 (Met- RANTES (MetR) and anti-CCR3 neutralizing mAb (or isotype control rat IgG2a)) receptors and then were activated with IL-16. Results are means SD from three independent assays. and , p 0.05 compared with nonstimulated and IL-16-stimulated eosinophils, respectively. B, Values represent the calculated percentage of inhibition with antagonists in com- parison with stimulated increases in lipid body numbers, LTC4 production, and IL-4 release above baselines.

Article Snippet: Eosinophils were stimulated with IL-16 (0.01–100 nM; R&D Systems), RANTES, or eotaxin (6 nM; R&D Systems) for time periods ranging from 5 min to 3 h. For inhibitor studies, cells were pretreated for 30 min with pertussis toxin (PTX) (20 ng/ml; Calbiochem, La Jolla, CA), recombinant soluble CD4 (sCD4) (50 ng/ml; R&D Systems), Fab of anti-CD4 (clone OKT4; American Type Culture Collection, Manassas, VA) and anti-HLA class I (clone W6/32; Sigma-Aldrich) mAbs (1 g/ml), anti-CCR3 mAb (clone 61828.111; R&D Systems), or isotype control rat IgG2a at 10 g/ml (BD PharMingen, San Diego, CA), met-RANTES (60 nM; R&D Systems), the platelet-activating factor (PAF) receptor antagonists CV6209 and BN52021 (10 M; Biomol, Plymouth Meeting, PA), brefeldin A (BFA) (0.1 and 1 g/ml; Biomol), or their vehicles, as indicated.

Techniques: Activation Assay, Control, Incubation, Inhibition

Journal:

Article Title: Maturation and Trafficking Markers on Rotavirus-Specific B Cells during Acute Infection and Convalescence in Children

doi: 10.1128/JVI.78.20.10967-10976.2004

Figure Lengend Snippet: Representative flow cytometry analysis of large IgD− B cells from acutely infected children (left lower dot plots) and children in convalescence (right lower dot plots) for the expression of the indicated maturation markers. B cells were negatively selected with magnetic beads and then stained with monoclonal antibodies against IgD, α4β7, CD38, CD27, or CCR6 and with GFP-VLP. For analysis, cells were gated on large (top left dot plot), IgD− (top right dot plot) lymphocytes. Based on this subpopulation, the lower dot plots were created showing expression of CCR6, CD38, and CD27 and GFP-VLP fluorescence. The percentages of IgD− GFP-VLP+ (RV-sIg) cells expressing each marker are indicated in the right quadrants.

Article Snippet: Then the cells were washed with PBS-0.5% bovine serum albumin (Sigma)-0.02% sodium azide (Mallinckrodt Chemicals, Paris, Ky.) (staining buffer) and stained with different combinations of monoclonal antibodies against the following human proteins: α4β7 phycoerythrin-conjugated monoclonal antibody Act-1, mouse IgG1 (Charles Mackay Leukosite Inc., Cambridge, Mass.); purified CCR10 monoclonal antibody 1B5, mouse IgG2a (Millenium Pharmaceuticals Inc., Cambridge, Mass.); purified CCR9 monoclonal antibody 96-1, mouse IgG1 (Millenium Pharmaceuticals); purified CCR6 monoclonal antibody 53103, mouse IgG2b (R&D Systems, Minneapolis, Minn.); and purified CD38 monoclonal antibody HB7, mouse IgG1 (BD Immunocytometry Systems, San Jose, Calif.).

Techniques: Flow Cytometry, Infection, Expressing, Magnetic Beads, Staining, Fluorescence, Marker

Journal:

Article Title: Maturation and Trafficking Markers on Rotavirus-Specific B Cells during Acute Infection and Convalescence in Children

doi: 10.1128/JVI.78.20.10967-10976.2004

Figure Lengend Snippet: Mean and standard error of the mean of IgD− (a) and IgD− RV-sIg (b) large B cells that express the maturation markers CD38high, CD27high and CCR6 in children during acute infection (n = 6 to 12 children) and convalescence (n = 4 to 8 children). The frequencies of cells expressing each marker in individual children were obtained as described for Fig. ​Fig.2.2. *, significant differences between acute infection and convalescence for each marker (P < 0.01, Mann-Whitney test).

Article Snippet: Then the cells were washed with PBS-0.5% bovine serum albumin (Sigma)-0.02% sodium azide (Mallinckrodt Chemicals, Paris, Ky.) (staining buffer) and stained with different combinations of monoclonal antibodies against the following human proteins: α4β7 phycoerythrin-conjugated monoclonal antibody Act-1, mouse IgG1 (Charles Mackay Leukosite Inc., Cambridge, Mass.); purified CCR10 monoclonal antibody 1B5, mouse IgG2a (Millenium Pharmaceuticals Inc., Cambridge, Mass.); purified CCR9 monoclonal antibody 96-1, mouse IgG1 (Millenium Pharmaceuticals); purified CCR6 monoclonal antibody 53103, mouse IgG2b (R&D Systems, Minneapolis, Minn.); and purified CD38 monoclonal antibody HB7, mouse IgG1 (BD Immunocytometry Systems, San Jose, Calif.).

Techniques: Infection, Expressing, Marker, MANN-WHITNEY

Journal:

Article Title: Maturation and Trafficking Markers on Rotavirus-Specific B Cells during Acute Infection and Convalescence in Children

doi: 10.1128/JVI.78.20.10967-10976.2004

Figure Lengend Snippet: Coexpression of maturation markers and trafficking receptors on large IgD− RV-sIg B cells. Shown are representative experiments of the study of the coexpression of CD38 and CD27 (a) and CD38, CCR6, CCR9, CCR10, and α4β7 (b) on large IgD− RV-sIg B cells from children during acute infection (left dot plots) and convalescence (right dot plots). Analysis was done with the basic strategy described for Fig. ​Fig.2.2. Dot plots show expression of each indicated marker and/or receptor on cells with a gate on large IgD− RV-sIg B cells. The percentages of cells in all the quadrants are indicated.

Article Snippet: Then the cells were washed with PBS-0.5% bovine serum albumin (Sigma)-0.02% sodium azide (Mallinckrodt Chemicals, Paris, Ky.) (staining buffer) and stained with different combinations of monoclonal antibodies against the following human proteins: α4β7 phycoerythrin-conjugated monoclonal antibody Act-1, mouse IgG1 (Charles Mackay Leukosite Inc., Cambridge, Mass.); purified CCR10 monoclonal antibody 1B5, mouse IgG2a (Millenium Pharmaceuticals Inc., Cambridge, Mass.); purified CCR9 monoclonal antibody 96-1, mouse IgG1 (Millenium Pharmaceuticals); purified CCR6 monoclonal antibody 53103, mouse IgG2b (R&D Systems, Minneapolis, Minn.); and purified CD38 monoclonal antibody HB7, mouse IgG1 (BD Immunocytometry Systems, San Jose, Calif.).

Techniques: Infection, Expressing, Marker

Journal: Cancer gene therapy

Article Title: Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection.

doi: 10.1038/sj.cgt.7700605

Figure Lengend Snippet: Figure 1 Construction of the chimeric MGMT-Apn1 construct. The MGMT and Apn1 cDNA sequences were amplified from bacterial clones and the chimeric molecule was created using the overlapping PCR technique.3 The fusion product was subcloned into the pcDNA 3.0 vector, sequenced and used to transfect K562 cells.

Article Snippet: DNA isolation, RNA isolation, Northern and Western blot analyses, and sodium dodecyl sulfate-polyacrylamide eletrophoresis (SDS-PAGE) were performed as has been previously described.3 Western blot analysis was performed with the mouse anti-MGMT monoclonal antibody (MT 23.2; Novus Biologicals, Littleton, CO) and the goat anti-mouse IgG antibody (Chemicon International, Inc., Temecula, CA).

Techniques: Construct, Amplification, Clone Assay, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: Human-yeast chimeric repair protein protects mammalian cells against alkylating agents: enhancement of MGMT protection.

doi: 10.1038/sj.cgt.7700605

Figure Lengend Snippet: Figure 2 Analysis of K562 cells containing chimeric transgene. (a) The MGMT-Apn1 K562 clone and pcDNA vector-transfected control cells were analyzed by Northern blot analysis. Both the radiolabeled MGMT and Apn1 cDNAs hybridized with a 1.75 kb message from total RNA of MGMT-Apn1-transfected cells, but not of vector-control cells. The radiolabeled GAPDH cDNA hybridized to a 1 kb transcript from total RNA of both control cells and MGMT-Apn1-transfected cells. (b) Western blot analysis: The MGMT monoclonal antibody detected the 64 kDa MGMT- Apn1 fusion protein of transfected K562 cells, but not of control cells (lane 3). The antibody also detected the 21 kDa MGMT protein from MGMT- transfected K562 cells (lane 2). (c) MGMT activity analysis: The 18-mer O6-methyl guanine oligonucleotide assay was performed with 50 mg of protein from sonicated K562 cell extract. MGMT activity was detected in both MGMT- and MGMT-Apn1-transfected K562 cells. Presence of the 8-mer fragment indicates MGMT activity. (d) AP endonuclease activity assay: The 26-mer oligonucleotide assay was performed with 5 mg of total protein from sonicated cell extract in the presence of 20 mM EDTA. Apurinic endonuclease activity was detected in MGMT-Apn1-transfected cells, but not control or MGMT-transfected cells. Presence of the 14-mer indicates cleavage of the artificial AP site. Apn1 is Mg2+ independent, EDTA resistant whereas the endogenous mammalian Ape1 protein requires Mg2+ and is inactive in the presence of EDTA.

Article Snippet: DNA isolation, RNA isolation, Northern and Western blot analyses, and sodium dodecyl sulfate-polyacrylamide eletrophoresis (SDS-PAGE) were performed as has been previously described.3 Western blot analysis was performed with the mouse anti-MGMT monoclonal antibody (MT 23.2; Novus Biologicals, Littleton, CO) and the goat anti-mouse IgG antibody (Chemicon International, Inc., Temecula, CA).

Techniques: Plasmid Preparation, Transfection, Control, Northern Blot, Western Blot, Activity Assay, Oligonucleotide Assay, Sonication