Peripheral blood mononuclear preparation

Rininger, J.A. et al., Immunopharmacological activity of Echinacea preparations following simulated digestion on murine macrophages and human peripheral blood mononuclear cells, J Leukoc Biol, 68, 503, 2000. 

Prepare peripheral blood mononuclear cells (PBMC) from heparinized blood, by Ficoll-Hypaque density centrifugation (21) as follows ... 

Although the blood is an easily accessible tissue to study, because red blood cells outnumber leukocytes by about 1000 to 1 in the peripheral circulation, the analysis of leukocytes by any technique is difficult unless the red cells can be removed. Techniques for removing red cells usually involve either density gradient centrifugation (pelleting red cells and neutrophils, leaving lymphocytes and monocytes behind to be collected from a layer as a peripheral blood mononuclear cell preparation [PBMC]) or differential lysis of red blood cells, leaving intact the more robust lymphocytes, monocytes, and neutrophils. 

Fig. 6.2. The FSC and SSC signals resulting from the cells in different blood preparations (whole peripheral blood whole peripheral blood after erythrocyte lysis and peripheral blood mononuclear cells [PBMCs] with granulocytes removed by density gradient centrifugation). The bottom panels indicate the five clusters into which the scatter signals fall.

Fig. 6.12. Back-gating from CD14/CD45 fluorescence to determine the scatter characteristics of lymphocytes. Such back-gating facilitates the placing and then evaluation of a lymphocyte scatter gate within a peripheral blood mononuclear cell preparation.

Fig. 6.13. Difficulties in placing a pure and inclusive lymphocyte scatter gate on a peripheral blood mononuclear cell preparation from a transplant patient with few and blasted lymphocytes.

Our initial findings that NPI-0052 inhibited all three proteolytic functions of the IDS proteasome vide supra) led us to compare its profile with other proteasome inhibitors (Figure 12.1) such as bortezomib,14 carfilzomib (PR-171)31 and CEP-187 70.30 These agents inhibit the CT-L activity to a similar degree as NPI-0052 but exhibit different inhibition profiles for the T-L and C-L activities. In addition, NPI-0052 exhibits a different recovery profile of proteasome functions in whole blood, normal organs, tumour and peripheral blood mononuclear cell preparations compared with other agents.52... 

Preparation of Human Peripheral Blood Mononuclear Cells Using Density Gradient Centrifugation (2,4)... 

As lymphocytes, monocytes, and macrophages are the primary targets for viral infection, the penetration of antiviral agents is important. Lymphocytes and monocytes are indicated as peripheral blood mononuclear cells (PBMC). The preparation of control PBMC from blood was reported in considerable detail by Jemal et al. [38], In addition, a validated assay for the determination of ATA in PBMC was developed. The determination of protease inhibitors in hmnan PBMC was reported by several groups [39-40]. After LLE, the analytes were analysed by LC-MS. Both methods enable the intra-cellular determination of the analytes and can be applied for TDM and pharmacokinetic studies. 

This chapter describes the procedures that can be used to determine compounds that have antiviral activity against HIV. These include maintenance of lymphoblastoid cell lines, preparation of peripheral blood mononuclear cells (PMMCs), and determination of the infectivity of the HIV stock-supernatant and antiviral assays. The assays described use both acutely and chronically infected cells. Toxicity of compounds is assessed by measuring 14C uptake. These protocols are used for the evaluation of compounds that can be carried out by a single individual in a Category 3 containment laboratory. The number of compounds analyzed would be about 10, which is a convenient number to fill a single 96-well p24 enzyme-linked immunosorbent assay (ELISA) plate. 

The studies mentioned above indicate that bromelain has a certain cytotoxic potential. It remains an open question whether the observed antineoplastic effects of bromelain preparations reside in the proteolytic enzyme or in some or more other components of the mixture. Before the work of Maurer et al. [104] and of Batkin et al. [105], the antitumor activity of bromelain was explained primarily by its fibrinolytic and platelet aggregation inhibitory activity, which is believed to interfere with the fibrin and coagulation features of tumor cells [2], More recently, Desser and Rehberger [107] demonstrated that bromelain stimulates the production of alpha tumor necrosis factor in human peripheral blood mononuclear cell cultures in a time-dependent manner. Immunomodulation, especially the release of cytokines, is believed to be responsible for the possible therapeutic potential of bromelain. However, further experimental evidence is necessary, first, to prove the antineoplastic action of the proteolytic enzyme, and second, to demonstrate that bromelain in vivo is a valuable therapeutic agent in humans. 

In theory deoxycytidine should act as a competitive substrate for deoxycytidine kinase, but parenteral deoxycytidine in S.Y. produced no additional benefit. The presence of high dATP and dADP levels in the peripheral blood mononuclear cells in one child but not the other is difficult to explain. As discussed elsewhere (Goday et al, this S3rmposium) the preparations contained both platelets and nucleated red cells and this may account for the discrepancy. 

Resuspend in 10 ml RPMI/FCS and count the cells. The cell preparation—peripheral blood mononuclear cells (PBMQ—will be a mixture of B lymphocytes. T Ijnnpho-cytes, NK cells, and monotytes. and some platelets. 

The heparin Immobilized polyurethane surfaces (prepared by plasma grafting of 1 acryloyl benzotrlazole and subsequent hydrolysis/amlnatlon) are effective In suppressing thrombus formation. The adhesion of peripheral blood mononuclear cells was also lower on such modified surfaces [72]. Heparin immobilized on vinyl pyridine grafted styrene butadiene-styrene block copolymers also shows good biocompatiblllty. The adsorption of albumin and fibrinogen are reduced with the increasing graft levels and heparin content [41]. 

Jemal, M. Rao, S. Gatz, M. Whigan, D. Liquid chromatography-tandem mass spectrometric quantitative determination of the HIV protease inhibitor atazanavir (BMS-232632) in human peripheral blood mononuclear cells (PBMC) practical approaches to PBMC preparation and PBMC assay design for high-throughput analysis, J.Chromatogr.B, 2003, 795, 273 - 289. 

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