nonhuman primates serve as key animal models for a variety of

nonhuman primates serve as key animal models for a variety of viral infections. This treatment also depleted 80C90% of CD3C CD159A+ lymphocytes, putative NK cells, from blood for at least 1 week and was associated with the loss of NK-cell cytotoxicity when evaluated by assays. Using this method, transient depletion of NK cells from two rhesus monkeys chronically infected with simian immunodeficiency virus failed to cause changes in virus replication. These studies describe a non-human primate model for Rosiglitazone NK-cell depletion Rosiglitazone and suggest a limited role for cytotoxic CD16+ NK cells in controlling AIDS virus replication during chronic infection. for 3 min and then incubated at 37 in 5% CO2 for 4 hr. The spontaneous release of calcein was determined by incubating loaded target cells in medium alone and maximal release was determined by adding 2% Triton-X to lyse all the target cells. After completion of incubation, tube strips were centrifuged at 400 for 8 min, and 100 l supernatant from each sample was transferred to a 96-well plate (Optiplate? 96F, Perkin Elmer, Fremont, CA) and fluorescence was measured on a fluorometer (Victor-3, Perkin Elmer) at an excitation wavelength of 494 nm and emission wavelength of 517 nm. The median value for each triplicate was used in the calculation of cytotoxicity. Cytotoxicity, measured as per cent specific release of calcein, was calculated using the following formula: Effector cell preparation and fractionation The PBMC were isolated from fresh, heparinized blood specimens obtained from normal rhesus macaques by density gradient centrifugation. They were either maintained unfractionated for use in cytotoxicity assays or were fractionated into NK-cell-enriched and NK-cell-depleted fractions by incubation with phycoerythrin (PE)-conjugated anti-CD16 (3G8, BD Biosciences) and anti-CD159A (NKG2A, Z199, Beckman Coulter) antibodies, washed and incubated with anti-PE magnetic beads. Cells were then sorted using an autoMACS (Miltenyi Biotechnology, Auburn, CA) into CD16/CD159A-enriched or CD16/CD159A-depleted cell fractions. Some PBMC were incubated with only anti-PE magnetic beads but otherwise processed similarly through the autoMACS system. These cells served as a sham-sorted control cell population. To confirm the size of the NK-cell subset in each cell fraction, cells were stained with anti-CD3-allophycocyanin (SP34, BD Biosciences) and anti-CD8-ECD (7PT-3F9) antibodies in addition to those described above. Detection of circulating mouse antibody and anti-mouse immunoglobulin antibody To detect the persistence of 3G8 in the blood, plasma Mouse monoclonal to CD53.COC53 monoclonal reacts CD53, a 32-42 kDa molecule, which is expressed on thymocytes, T cells, B cells, NK cells, monocytes and granulocytes, but is not present on red blood cells, platelets and non-hematopoietic cells. CD53 cross-linking promotes activation of human B cells and rat macrophages, as well as signal transduction. specimens from antibody-treated monkeys were incubated with normal rhesus PBMC and then stained with a secondary goat anti-mouse PE-conjugated antibody (Jackson ImmunoResearch, West Grove, PA) to detect 3G8 binding to NK cells. Samples were analysed by flow Rosiglitazone cytometry as described above. This assay had a limit of 3G8 detection in plasma of 100 ng/ml. To detect anti-mouse immunoglobulin antibodies in monkey plasma, 96-well enzyme-linked immunosorbent assay (ELISA) plates were coated with 3G8 and incubated overnight at 4. Plates were blocked with blocking reagent buffer (Pierce, Rockford, IL) for 15 min at room temperature. Plasma samples from antibody-treated rhesus monkeys were diluted in dilution buffer (PBS/05% non-fat dry milk), applied to the wells in serial dilutions, incubated at room heat for 1 hr and Rosiglitazone washed with washing buffer (PBS/05% non-fat dry milk/001% Tween-20). Goat anti-human IgGChorseradish peroxidase (Jackson ImmunoResearch), at 1:30 000 in dilution buffer, was added to each well, incubated at room heat for 1 hr and washed with washing buffer. Tetramethylbenzidine microwell peroxidase substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to each well, incubated at room heat for 10C30 min and then the reaction was stopped with H2SO4 (Stop Answer, Kirkegaard & Perry Laboratories). Optical density readings were measured on an ELISA reader at 450 nm. The titre of anti-mouse immunoglobulin antibody.

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