fab182p mccr1 r d systems  (R&D Systems)

Journal: The Journal of General Virology

Article Title: C-C chemokine receptor type 5 (CCR5) utilization of transmitted and early founder human immunodeficiency virus type 1 envelopes and sensitivity to small-molecule CCR5 inhibitors

doi: 10.1099/vir.0.025270-0

Figure Lengend Snippet: Sensitivity to small-molecule CCR5 inhibitors of T/F HIV-1 Envs in cell–cell fusion. QT6 effector cells were prepared by infection with vTF1.1 for 1 h followed by transfection with Env expression constructs. Target QT6 cells were transfected with CD4 and CCR5 in pcDNA3, and a construct encoding luciferase under the transcriptional control of T7 promoter. At 16–18 h post-transfection, target cells were pre-treated with serially diluted CCR5 inhibitor maraviroc (a), CMPD-167 (b) or SCH-412147 (c) for 30 min before mixing with effector cells. The luciferase activity of cell lysates was determined approximately 8 h later. The fusogenic activity in medium alone was arbitrarily set to 100 % for each Env. Data are representative of three independent experiments, with each determination performed in triplicate (mean± sd ).

Article Snippet: PE-conjugated mouse anti-human CCR5 mAb 45531 and isotype control were purchased from R&D Systems.

Techniques: Infection, Transfection, Expressing, Construct, Luciferase, Control, Activity Assay

Journal: The Journal of General Virology

Article Title: C-C chemokine receptor type 5 (CCR5) utilization of transmitted and early founder human immunodeficiency virus type 1 envelopes and sensitivity to small-molecule CCR5 inhibitors

doi: 10.1099/vir.0.025270-0

Figure Lengend Snippet: Sensitivity to small-molecule CCR5 inhibitors of T/F HIV-1 Envs in pseudotyped-virus infection. Stocks of pseudotyped reporter viruses were prepared by co-transfecting 293T cells with Env expression constructs and plasmid pHIVΔEnv for 48 h and then harvested. TZM-bl cells were pre-treated with serially diluted CCR5 inhibitor maraviroc (a), CMPD-167 (b) or SCH-412147 (c) for 30 min before pseudotyped viruses were added. The luciferase activity of cell lysates was determined approximately 24 h later. The infectivity in medium alone was arbitrarily set to 100 % for each Env. Data are representative of three independent experiments, with each determination performed in triplicate (mean± sd ).

Article Snippet: PE-conjugated mouse anti-human CCR5 mAb 45531 and isotype control were purchased from R&D Systems.

Techniques: Virus, Infection, Expressing, Construct, Plasmid Preparation, Luciferase, Activity Assay

Journal: The Journal of General Virology

Article Title: C-C chemokine receptor type 5 (CCR5) utilization of transmitted and early founder human immunodeficiency virus type 1 envelopes and sensitivity to small-molecule CCR5 inhibitors

doi: 10.1099/vir.0.025270-0

Figure Lengend Snippet: Mechanisms of CCR5 inhibition and inhibitor resistance. (a) Binding epitopes of CCR5 inhibitors. CHO-CCR5 stable transfectants were treated with 1 μM of each respective CCR5 inhibitor for 1 h, washed extensively and stained with PE-conjugated anti-CCR5 mAb 3A9, 2D7, 45531 or with isotype-matched control IgG before analysis by flow cytometry. hCCR5, human CCR5; mCCR5, mouse CCR5. (b–d) Fusogenic activity of T/F HIV-1 Envs with CCR5/CXCR2 chimeras (b), with CCR5 in the presence of CCR5 inhibitor TAK-779 (c) and with CCR5/CXCR2 chimeras in the presence of the CCR5 inhibitor maraviroc, CMPD-167, SCH-412147 or TAK-779 (d). QT6 effector cells were prepared by infection with vTF1.1 for 1 h, followed by transfection with Env expression constructs. Target QT6 cells were transfected with CD4 and CCR5 or CCR5/CXCR2 chimera in pcDNA3, and a construct encoding luciferase under the transcriptional control of the T7 promoter. The effector and target cell populations were mixed at 16–18 h after transfection, and the luciferase activity of cell lysates was determined approximately 8 h later. In experiments using CCR5 drugs, target cells were pre-treated with serially diluted CCR5 inhibitor for 30 min before mixing. The fusogenic activity with CCR5 was arbitrarily set to 100 %. Data are representative of three independent experiments, with each determination performed in triplicate (mean± sd ).

Article Snippet: PE-conjugated mouse anti-human CCR5 mAb 45531 and isotype control were purchased from R&D Systems.

Techniques: Inhibition, Binding Assay, Staining, Control, Flow Cytometry, Activity Assay, Infection, Transfection, Expressing, Construct, Luciferase

Journal: Journal of Inflammation (London, England)

Article Title: CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

doi: 10.1186/1476-9255-6-22

Figure Lengend Snippet: Human chemokines and their receptors targeted by early HHV-6A infection

Article Snippet: Cells were then permeabilized, by treatment with 0.05% saponin in 0.5% BSA-PBS for 10 minutes and then labelled with a buffer containing tetramethylrhodamine B thioisocyanate, TRITC-anti-human caveolin polyclonal antibody (Sc894, Santa Cruz Biotechnology Inc., CA, USA) and FITC-anti-human CCR5 monoclonal antibody (R&D Systems, UK) as described [ ].

Techniques:

Journal: Journal of Inflammation (London, England)

Article Title: CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

doi: 10.1186/1476-9255-6-22

Figure Lengend Snippet: Competitive binding of U83A to signalling receptors CCR1 and CCR5 displaces human chemokines . COS-7 cells transfected with CCR1 (A) or CCR5 (B, C) were incubated with [ 125 I]CCL3 (A and B) or [ 125 I]CCL5 (C) in the presence of increasing concentration of cold chemokine. IC 50 obtained were for (A) U83A: 0.4 nM, (B) U83A: 0.01 nM and (C) U83A-N: 0.1 nM. Binding curves were fitted by nonlinear regression and IC 50 values were calculated using Graphpad Prism.

Article Snippet: Cells were then permeabilized, by treatment with 0.05% saponin in 0.5% BSA-PBS for 10 minutes and then labelled with a buffer containing tetramethylrhodamine B thioisocyanate, TRITC-anti-human caveolin polyclonal antibody (Sc894, Santa Cruz Biotechnology Inc., CA, USA) and FITC-anti-human CCR5 monoclonal antibody (R&D Systems, UK) as described [ ].

Techniques: Binding Assay, Transfection, Incubation, Concentration Assay

Journal: Journal of Inflammation (London, England)

Article Title: CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

doi: 10.1186/1476-9255-6-22

Figure Lengend Snippet: U83A specifically chemoattracts CCR5 bearing primary human leukocytes . PBMCs were plated out on a Neuroprobe chemotaxis apparatus. Lower chambers were filled with a range of chemokines. After 90 minutes incubation, cells remaining on top of the filter were removed and the migrated cells, in the wells below the filter, collected and stained for CCR2 and CCR5. Red peak denotes the background cell migration (buffer only) and green denotes the test peptide/chemokine. CCR2 antibody used was linked to PE and CCR5 antibody linked to FITC. The top, second and third panels show flow cytometry results for cells exposed to 1 nM of CCL2, U83A-N or U83A, respectively. Migration of CCR2 bearing cells were shown towards wells containing CCL2, and CCR5 bearing cells towards those with U83A. Experiments were repeated three times, one representative result shown. For the U83A treatment, the CCR5 specific results from two further donors are shown in the bottom panel. There was no specific chemotaxis detected using the negative control with migration buffer only.

Article Snippet: Cells were then permeabilized, by treatment with 0.05% saponin in 0.5% BSA-PBS for 10 minutes and then labelled with a buffer containing tetramethylrhodamine B thioisocyanate, TRITC-anti-human caveolin polyclonal antibody (Sc894, Santa Cruz Biotechnology Inc., CA, USA) and FITC-anti-human CCR5 monoclonal antibody (R&D Systems, UK) as described [ ].

Techniques: Chemotaxis Assay, Incubation, Staining, Migration, Flow Cytometry, Negative Control

Journal: Journal of Inflammation (London, England)

Article Title: CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

doi: 10.1186/1476-9255-6-22

Figure Lengend Snippet: U83A treatment results in long-term delays of CCL4 driven internalisation of CCR5 in the absence of re-stimulation . CCR5 expression in MAGI-CCR5 cells was monitored by flow cytometry after incubation with 100 nM U83A in buffer (plain line) or control, buffer only (dashed line) for 10 minutes, then washed, incubated for the indicated time (x axis, in minutes), and stimulated with 100 nM CCL4 for 10 minutes, cells were then stained with FITC-CCR5 antibody and fixed. CCR5 surface levels were then determined by flow cytometry. Results were normalised in reference to CCR5 level of expression at time 0, as 100% values, and are a combination of two experiments each run in duplicate. P values for mean differences are 0.029, 0.066 and 0.016 for 30, 60 and 120 minutes time points, respectively (unpaired T test).

Article Snippet: Cells were then permeabilized, by treatment with 0.05% saponin in 0.5% BSA-PBS for 10 minutes and then labelled with a buffer containing tetramethylrhodamine B thioisocyanate, TRITC-anti-human caveolin polyclonal antibody (Sc894, Santa Cruz Biotechnology Inc., CA, USA) and FITC-anti-human CCR5 monoclonal antibody (R&D Systems, UK) as described [ ].

Techniques: Expressing, Flow Cytometry, Incubation, Control, Staining

Journal: Journal of Inflammation (London, England)

Article Title: CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

doi: 10.1186/1476-9255-6-22

Figure Lengend Snippet: Percentage of CCR5 surface expression after U83A or U83A-N treatment followed by CCL4 stimulation

Article Snippet: Cells were then permeabilized, by treatment with 0.05% saponin in 0.5% BSA-PBS for 10 minutes and then labelled with a buffer containing tetramethylrhodamine B thioisocyanate, TRITC-anti-human caveolin polyclonal antibody (Sc894, Santa Cruz Biotechnology Inc., CA, USA) and FITC-anti-human CCR5 monoclonal antibody (R&D Systems, UK) as described [ ].

Techniques: Expressing

Journal: Journal of Inflammation (London, England)

Article Title: CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

doi: 10.1186/1476-9255-6-22

Figure Lengend Snippet: U83A delays internalisation of CCR5 and effects on localisation of caveolin-1 . Localisation of CCR5 and caveolin-1 were monitored by confocal microscopy in the absence of chemokine stimulation, buffer only (a, d, g), and when stimulated for 10 minutes with CCL4 (b, e, h) or U83A (c, f, i). DAPI nucleus staining was used as a control, which gave nuclear staining of all cells indicated (not shown). After stimulation, cells were fixed and permeabilised, then reacted with CCR5 antibody linked to FITC, green channel (a, b, c) showing internalisation induced by CCL4 (b), and caveolin-1 antibody linked with TRITC, red channel (d, e, f), showing coalescence of punctuate staining induced by U83A (f). The merged staining (g, h, i) shows for U83A treatment, examples of punctae of caveosome-like structure, with yellow merged fluorescence as indicated in the enlarged insets (i). Representative 0.39 micron slices are from the z-stack from three independent assays.

Article Snippet: Cells were then permeabilized, by treatment with 0.05% saponin in 0.5% BSA-PBS for 10 minutes and then labelled with a buffer containing tetramethylrhodamine B thioisocyanate, TRITC-anti-human caveolin polyclonal antibody (Sc894, Santa Cruz Biotechnology Inc., CA, USA) and FITC-anti-human CCR5 monoclonal antibody (R&D Systems, UK) as described [ ].

Techniques: Confocal Microscopy, Staining, Control, Fluorescence

Journal: Journal of Inflammation (London, England)

Article Title: CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

doi: 10.1186/1476-9255-6-22

Figure Lengend Snippet: U83A specific induction of co-localisation of CCR5 with caveolin-1 . Localisation of CCR5 and caveolin-1 was monitored by confocal microscopy, in absence of stimulation (a), when stimulated with 30 minutes with 100 nM CCL4 (b) or U83A (c). CCR5 is labelled with antibody linked to FITC (green) and caveolin-1 with TRITC (red), co-localisation appears as yellow.

Article Snippet: Cells were then permeabilized, by treatment with 0.05% saponin in 0.5% BSA-PBS for 10 minutes and then labelled with a buffer containing tetramethylrhodamine B thioisocyanate, TRITC-anti-human caveolin polyclonal antibody (Sc894, Santa Cruz Biotechnology Inc., CA, USA) and FITC-anti-human CCR5 monoclonal antibody (R&D Systems, UK) as described [ ].

Techniques: Confocal Microscopy