Optimal in vitro culture conditions for murine predominant immature CD8a+ dendritic cells.

BACKGROUND: The prospects of using immature CD8a(+) dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers of predominant DC2 from murine bone marrow cells. METHODS: Three groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without endogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Flt3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml Flt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without additional cytokines. All cells were cultured at 37 degrees C under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies. RESULTS: The cytokine group exhibited higher proportions of typical immature CD8a(+) DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P<0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P<0.05). CONCLUSIONS: Culture in the presence of 5 ng/ml GM-CSF, 25 ng/ml Flt3L, 20 ng/ml IL-4 and 100 ng/ml SCF can rapidly induce large quantities of predominant immature CD8a(+) DC from murine bone marrow cells. Therefore, these represent optimal culture conditions for preparing murine immature DC2 in vitro.

High expression of PSM-E correlated with tumor grade in prostate cancer: a new alternatively spliced variant of prostate-specific membrane antigen.

BACKGROUND: Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) has been targeted for therapy and diagnosis of PCa. In the current study, PSMA cDNA was cloned from PCa tissue by RT-PCR. After sequencing, a new spliced variant of PSMA (PSM-E) was discovered and its specificity in PCa was evaluated. METHODS: PSM-E and PSMA mRNA were measured in LNCaP, PC-3 and prostate or nonprostatic malignancies. Following transfection of PC-3 with PSM-E cDNA in the pcDNA3.0 vector, PSM-E expression was measured by immunofluorescence and Western-blot. PSM-E and PSMA mRNA levels were quantified by a real-time PCR assay in normal prostate (n = 7), benign prostatic hyperplasia (BPH) (n = 22) and PCa (n = 41). The correlation between their levels and tumor grade was analyzed. RESULTS: PSM-E cDNA is identical to PSMA except for a 97-nucleotide region and a 93-nucleotide region. PSM-E and PSMA mRNA were detected in PCa and LNCaP, not in PC-3; PSMA could be detected in some nonprostatic tumors whereas PSM-E not. The expression of PSM-E protein was detected in transfected cells. Significant difference of PSM-E mRNA levels was observed among normal prostate, BPH and PCa (P < 0.001), and PSM-E levels increased with increasing Gleason score (r = 0.514, P < 0.001). PSMA mRNA levels were higher in BPH and PCa than in normal prostate (P < 0.001), but no difference between BPH and PCa, no significant correlation was observed between PSMA levels and Gleason score (r = 0.229, P = 0.057). CONCLUSIONS: PSM-E may be a potential prognostic indicator for PCa progression and may be a new target antigen for therapy of PCa.

[The effect of cell killing and apoptosis by human herpes simplex virus- thymidine kinase/ganciclovir system combined with allitride in BIU87 cells].

OBJECTIVE: To study the killing effect of human herpes simplex virus-thymidine kinase/ganciclovir (HSV-TK/GCV) system combined with allitride and the possible apoptosis mechanism in BIU87 cells. METHODS: The cytotoxicity after combination were estimated by theamine blue tetrazolium bromide (MTT). The morphological changes were observed with inverted microscope and in-situ cell apoptosis detection kit. Changes of apoptosis rate and cell cycle were assessed by flow cytometry. B-cell lymphoma-2 (bcl-2), bax, caspase-3 (cysteine aspartate specific proteinase) mRNA changes were detected by reverse transcriptase polymerase chain reaction, and caspase-3 activity was estimated with colorimetry. RESULTS: For combination group, the cell killing rate was raised to 72.50% to compare with 35.00% of GCV and 37.00% of allitride separately and there was a synergistic effect between these two drugs. The cell apoptosis was induced in all three groups and for the combination group the time of S-phase and G(2)-phase arrest were earlier than other two groups. Both drugs could inhibit the expression of bcl-2 and promote the expression and activity of caspase-3. CONCLUSIONS: The combination of HSV-TK/GCV system with allitride can inhibit the proliferation of BIU87 cells congenerously through apoptosis, which may be correlated with S- and G(2)-phase arrest, down-regulation of bcl-2 and increased caspase-3 expression and its activity.