Peripheral blood culture

Peripheral blood cultures are stimulated to divide by the addition of a T cell mitogen such as phytohaemagglutinin (PHA) to the culture medium. Mitotic activity is at a maximum at about 3 days but begins at about 40 h after PHA stimulation and the chromosome constitution remains diploid during short-term culture (Evans and O Riordan, 1975). Treatments should commence at about 44 h after culture initiation. This is when cells are actively proliferating and cells are in all stages of the cell cycle. They should be sampled about 20 h later. In a repeat study the second sample time should be about 92 h after culture initiation. Morimoto et al. (1983) report that the cycle time for lymphocytes averages about 12-14 h except for the first cycle. 

Peripheral blood cultures are established in medium containing BrdU and PHA. Cocemid is added 1-2 h before harvest and the cells are harvested between 60 and 70 h post-PHA stimulation. Cell harvest and slide preparations are conducted according to routine cytogenetic methods. 

A new generation of antiinflammatory agents having immunosuppressive activity has been developed. The appearance of preclinical and clinical reports suggest that these are near entry to the pharmaceutical market. For example, tenidap (CP-66,248) (12) has been demonstrated to inhibit IL-1 production from human peripheral blood monocytes in culture (55). Clinically, IL-1 in synovial fluids of arthritic patients was reduced following treatment with tenidap. Patients with rheumatoid or osteoarthritis, when treated with tenidap, showed clinical improvement (57,58). In addition to its immunological effects, tenidap also has an antiinflammatory profile similar to the classical NSAIDs (59). Other synthetic inhibitors of IL-1 production are SKF 86002 (20) andE-5110 (21) (55). 

L Vova TS. 1984. [Study of the mutagenic effect of 5 promising pesticides in mouse bone marrow cultured human peripheral blood lymphocytes, and in the yeast Saccharomyces-cerevisiae.] Tsitol Genet 18 455-457. (Russian)... 

Chao CC, Molitor TW, Close K, Hu S, Peterson PK (1993) Morphine inhibits the release of tumor necrosis factor in human peripheral blood mononuclear cell cultures. Int J Immunopharmacol 15 447 53... 

KULLING s E, ROSENBERG B, JACOBS E, METZLER M (1999) The phytoestrogens coumestrol and genistein induce structural and chromosomal aberrations in cultured human peripheral blood lympocytes. Arch Toxicol. Ti 50-54. 

Blood cultures x 2 from each access site (peripheral and central), urinalysis, urine culture, chest x-ray, sputum cultures... 

Growing clinical data also points to the importance of IL-8 in atherogenesis. IL-8 has been found in atheromatous lesions from patients with atherosclerotic disease including carotid artery stenosis (103), CAD (118), abdominal aortic aneurysms (AAA) (103,104,114), and peripheral vascular disease (PVD) (104). Furthermore, studies using plaque explant samples have yielded more direct evidence for IL-8 involvement. Media from cultured AAA tissue induced IL-8-dependent human aortic endothelial cell (HAEC) chemotaxis (122). Homocysteine, implicated as a possible biomarker for CAD, is also capable of inducing IL-8 (123-125) by direct stimulation of endothelial cells (123,124) and monocytes (125). When patients with hyperhomocysteinemia were treated with low-dose folic acid, decreases in homocysteine levels correlated with decreases in IL-8 levels (126). Statins significantly decrease serum levels of IL-6, IL-8, and MCP-1, as well as expression of IL-6, IL-8, and MCP-1 mRNA by peripheral blood monocytes and HUVECs (127). Thus, IL-8 may be an underappreciated factor in the pathogenesis of atherosclerosis. 

One of the most sensitive biological effects of ionizing radiation is to increase the frequency of normally observed chromosome aberrations (but not to induce qualitatively special abnormalities). Peripheral blood lymphocytes are the most feasible cells for chromosome investigations, as blood samples are easy to obtain and the techniques to stimulate the lymphocytes to proliferate within a culture medium and to prepare suitable chromosome slides for microscopic analyzation have their routine protocoil (e. g. Yunis, 1965 Lloyd et al, 1982). 

Horton JE, Raisz LG, Simmons HA, Oppenheim JJ, Mergenhagen SE (1972) Bone resorbing activity in supernatant fluid from cultured human peripheral blood leukocytes. Science 177 793-795... 

Peterson, P.K., Sharp, B., Gekker, G., and Keane, W.F., Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity, J. Immunol., 138,3907, 1987. 

Ernst, M., et. al., Immune cell functions in industrial workers after exposure to 2,3,7,8-tetrachlorodibenzo-/ -dioxin Dissociation of antigen-specific T cell responses in cultures of diluted whole blood and of isolated peripheral blood mononuclear cells, Environ. Health Perspect. 106 (Suppl. 2), 701, 1998. 

Chao, C.C. et al., Morphine potentiates transforming growth factor-beta release from human peripheral blood mononuclear cell cultures, J. Pharmaco.l Exp. Ther., 262, 19, 1992. 

Recent studies with BN 52021 have demonstrated that PAF can modulate various immune processes. BN 52021 can inhibit the suppressive effect of PAF on T-lymphocyte proliferation and cytokine production. When PAF is added to human peripheral blood lymphocyte cultures (containing 5-10% monocytes) stimulated with phytohaemaglutinin (PHA) or concanavalin A (Con A), a non-toxic concentration-dependent inhibition of lymphocyte proliferation is... 

Established cell lines, cell strains or primary cell cultures may be used. The most often used are Chinese hamster cell lines and human peripheral blood lymphocytes. The merits of these two cell lines have been reported (Ishidate and Hamois, 1987 Kirkland and Gamer, 1987). The cell system must be validated and consistently sensitive to known clastogens. 

For primary isolation of HIV, patient peripheral blood mononuclear cells (PBMC) are collected, the usual inoculum being 106-107 cells. This is the most productive specimen, although virus has been cultured from plasma, semen, tears, saliva, breast milk, and brain tissue. The virus can be cultured from patient specimens at any time in the course of disease, during which the titer changes. Blood contains approximately 60 TCID50% (50% tissue culture infective dose) per milliliter when a person is asymptomatic, and about 7000TCID50/ml in later stages of HIV disease. 

To characterize IRIV-elicited immune responses in vitro, we addressed cell proliferation and cytokine expression in peripheral blood mononuclear cell (PBMC) cultures, as well as IRIV effects on dendritic cells (DC). In all experiments, PBMC were obtained from healthy donors and, if needed, further separated into different cell subsets. Finally, cells were cultured in the presence or absence of IRIV as indicated. 

Figure 1 Continued on next page) Immunopotentiating reconstituted influenza virosomes (IRIV) induced antigen specific proliferation of CD4+CD45RO+ cells. (A) Peripheral blood mononuclear cells (PBMQ from healthy donors n=3) were cultured in the absence of stimuli (Neg), in the presence of IRIV (V), and in the presence of control liposomes (L) at the indicated dilutions. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (B) Cord blood mononuclear cells from two donors were cultured in the absence of stimuli (Neg) or in the presence of phytohaemag-glutinin (PHA), concanavalin A (ConA), IRIV (V) or L at the indicated concentrations. Proliferation was measured on day 3 of culture for PHA and ConA cultures and on day 6 for IRIV and L stimulated cultures. (Q Purified CD4+ or CD8+ cells were cocultured with autologous irradiated PBMC in the absence of stimuli (Neg) and in the presence of IRIV (V) at the indicated concentrations. Proliferation was measured on day 6 of culture by H-thymidine incorporation. (D) Purified CD4/CD45RA+ cells and CD4/CD45RO-I-cells were isolated from PBMC of one healthy donor and cocultured with autologous irradiated PBMC in the presence of IRIV (V) or L at the indicated concentration. Proliferation was measured on day 6 of culture by H-thymidine incorporation. Source From Ref 6.

Figure 2 Cytokine gene expression in immunopotentiating reconstituted influenza virosomes (IRIV) stimulated peripheral blood mononuclear cells (PBMC). PBMC were cultured in the presence or absence of IRIV. On days 1 and 2, culture cells were harvested and total cellular RNA was extracted and reverse transcribed. The cDNAs thus obtained were tested in real time polymerase chain reaction assays in the presence of primers specific for the indicated cytokine genes. Source From Refs. 6 and 9.

Figure 3 Cytokine secretion in immunopotentiating reconstituted influenza viro-somes (IRIV)-stimulated peripheral blood mononuclear cells (PBMC). PBMC from a healthy donor were cultured in the absence of stimuli (Neg) or in the presence of IRIV (V, 1 50 diluted) or control liposomes (L, 1 50 diluted). On days 1, 2, and 4 supernatants were harvested and the concentrations of interferon-y (A), GM-CSF (B), TNF-a (C), and interleukin-4 (D) were determined by ELISA. Abbreviations GM-CSF, granulocyte monocyte colony stimulating factor TNF-a, tumor necrosis factor-a. Source From Ref. 6.

Figure 6 Immunopotentiating reconstituted influenza virosomes (IRIV) adjuvant effects in the induction of tumor associated antigen-specific cytotoxic T cell. CD14-negative cells from a healthy donor peripheral blood mononuclear cells were cocultured with autologous immature dendritic cells (iDC) in the presence of Melan-A/Mart-l27-35, alone (A) or supplemented with either control liposomes (B) or IRIV (1 50, C). On day 7, culture cells were restimulated with Melan-A/MART-127-35 pulsed iDC and cultured for six further days [see Materials and Methods ]. On day 7 after restimulation cells were stained with fluorescein isothiocyanate-conjugated anti-CD8 and phosphatidylethanolamine-conjugated HL A-A0201 /Melan-A/MART -127-3 5 tetramers. Source From Ref. 6.

Primary Culture 57 D.1.3 Isolation of Peripheral Blood Lymphocytes Requirements ... 

A variety of cell lines, strains, or primary cell cultures, including human cells, may be used (e.g., Chinese hamster fibroblasts, human or other mammalian peripheral blood lymphocytes). Cell cultures are exposed to the test substance both with and without metabolic activation and at predetermined intervals after exposure, they are treated with a metaphase-arresting substance (e.g., colchicine), harvested, stained, and metaphase cells are analyzed microscopically for the presence of structural chromosome aberrations. At least three concentrations should be used. 

Cultured cells (from human peripheral blood lymphocytes or from Syrian hamster embryo (SHE)) or cell lines (CHO, V79, CHL/IU, L5178 Y) are exposed to the test substances both with and without an exogenous source of metabolic activation unless primary cells with metabolizing capability are used. After exposure to the test substance, cell cultures are grown for a sufficient period to... 

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