Implication of delayed TNF-alpha exposure on dendritic cell maturation and expansion from cryopreserved cord blood CD34+ hematopoietic progenitors.

Most currently used systems for dendritic cell (DC) production from progenitors entail tumor necrosis factor alpha (TNF-alpha) at the onset of cell culture, based on the notion that TNF-alpha might be required in the early stages of DC development. To optimize conditions for DC expansion from cryopreserved cord blood (CB) CD34+ hematopoietic progenitors, we took a dynamic approach to define the timing of TNF-alpha exposure to the culture. We cultured cord blood CD34+ cells in RPMI-1640 with 10% human AB plasma, stem cell factor (days 1-6), granulocyte-macrophage colony-stimulating factor (days 1-18), interleukin-4 (days 6-18) and varying schedules of TNF-alpha (0-144 h after thawing). Expression of the DC-associated markers, including CD83/CD1a, HLA DR/CD86/CD80, CD14/CD40, was monitored every 3 days. Our data demonstrate that delayed TNF-alpha exposure by 48-72 h after thawing gave rise to two- to three-fold increase in the yield of CD83+ DCs that were highly active in stimulating allogeneic T-cell proliferation compared to immediate TNF-alpha exposure. Thus, the immediate exposure of cryopreserved cord blood CD34+ cells to TNF-alpha, potentially compromising DC expansion, should be avoided. This finding should be of significant consideration when using cryopreserved CD34+ progenitor cells as a source of immunologically competent DCs in a clinical setting.

[Inhibition of lectin induced peripheral blood lymphocyte proliferation by colorectal cancer extract: a preliminary report of three cases].

BACKGROUND: Tumor infiltrating lymphocytes (TIL) in colorectal cancer are a manifestation of local, cell mediated immune response to the malignant tumor. Tumor progression is due to impairment of the host ability to control tumor growth. Several studies suggested possible causes for such impairment, however, the precise factor(s) underlying such malfunction is uncertain. AIM: To compare the possible effects of colorectal cancer (CRC) and normal colonic mucosa extracts on lectin induced blastogenesis of the same patients' peripheral blood lymphocytes (PBL) proliferation. METHODS: CRC and normal mucosa extracts were obtained from 3 patients undergoing curative surgery for colon adenocarcinoma. Proliferation assays used PBL from the CRC patients, incubated with Concanavalin A (ConA) and Phytohemoglobin (PHA) in the presence of CRC or normal mucosa extract and in medium alone. Proliferation was measured by H3 Thymidine incorporation following 48 hours on incubation. RESULTS: Exposure of ConA induced PBL proliferation assay to CRC extract yielded a 98.7% inhibition measured by counts/minute (cpm) of incorporated H3 Thymidine compared to normal colonic mucosa extract (1,214 +/- 594 cpm vs. 95,335 +/- 6,997 cpm respectively, p = 0.0018). PHA stimulated proliferation exposed to CRC extract showed a 99.7% decrease in blastogenic activity compared to normal mucosa extract (362 +/- 175 cpm vs. 62,375 +/- 16,591 cpm respectively, p = 0.0234). CONCLUSIONS: These preliminary results suggest that CRC extract contain factor(s) capable of profoundly inhibiting lectin induced proliferation of PBL. Shedding of suppressor substances may be one of the possible mechanisms by which the tumor evades the effector arm of the cell-mediated immune response. Characterization of such factors may aid in intratumor, local and systemic cellular immune response reconstitution.

Plasminogen activator inhibitor-1 promotes formation of endothelial microparticles with procoagulant potential.

BACKGROUND: Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state. METHODS AND RESULTS: Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and alpha(V)beta3 integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1-deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin. CONCLUSIONS: The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction-elevated levels of PAI-1 and propensity toward thrombosis.