Cyclosporin A inhibits the early phase of NF-kappaB/RelA activation induced by CD28 costimulatory signaling to reduce the IL-2 expression in human peripheral T cells.

Cyclosporin A (CsA) inhibits interleukin (IL)-2 production, activation and proliferation of human peripheral T cells (HPTC) costimulated with simultaneous engagement of T cell receptor (TCR)/CD3 and CD28. We demonstrated that 10 ng/ml CsA, which reduced the proliferation of HPTC costimulated with anti-CD3 and anti-CD28 by half, prevented NF-AT and NF-kappaB from migrating into the nucleus. Whereas CsA added even 30 min after the costimulation caused NF-AT to remain in the cytoplasm, the delayed addition of CsA could not prevent NF-kappaB from translocating into the nucleus. CsA, which was added to HPTC simultaneously with the engagement of both CD3 and CD28 or the 1-h-delayed engagement of CD28 after prior TCR/CD3-triggering, inhibited NF-kappaB p65/RelA from binding to the target DNA fragment, followed by reduction of HPTC proliferation in response to the costimulation. When CsA was added 30 min after the delayed engagement of CD28 following the prior engagement of TCR/CD3, these inhibitory effects were diminished. Antisense NF-kappaB p65/RelA oligonucleotides inhibited p65/RelA mRNA expression, diminished IL-2 mRNA expression in the costimulated HPTC and reduced HPTC proliferation to the same extent as CsA added simultaneously with the costimulation. The CsA- or antisense p65/RelA oligonucleotide-induced reduction in the proliferation of costimulated HPTC was overcome by the addition of exogenous IL-2. These findings indicate that the major effects of CsA on the early phase of CD28-mediated costimulation in the presence of TCR/CD3 signaling are to inhibit NF-kappaB/RelA from translocating into the nucleus and binding to the target DNA sequence in the IL-2 gene promoter region, which induces IL-2 expression leading to HPTC proliferation.

Expression of human scavenger receptor B1 on and in human platelets.

OBJECTIVE: The abundance of HDL particles correlates inversely with the incidence of coronary heart disease. The human scavenger receptor B1 (hSR-B1/CLA-1) is a receptor for HDL. Expression of hSR-B1/CLA-1 mRNA and protein in human platelets was determined using reverse transcriptase-polymerase chain reaction and Western blot, respectively. Presence of the protein on the surface of platelets was shown using flow cytometry. METHODS AND RESULTS: Immunochemical staining for hSR-B1/CLA-1 showed that it was expressed in megakaryocytes, the platelet precursors of human bone marrow. These findings prompted us to ask whether hSR-B1/CLA-1 was differentially expressed on platelets obtained from patients with atherosclerotic disease compared with those in control subjects. Our findings showed that abundance of hSR-B1/CLA-1 was significantly reduced on the surface of platelets from patients with atherosclerotic disease. The reduced levels of hSR-B1/CLA-1 were associated with increased cholesterol ester content in platelets from patients with atherosclerotic disease compared with control subjects. A negative correlation existed between hSR-B1/CLA-1 expression and platelet aggregation. In summary, our studies show that the HDL receptor hSR-B1/CLA-1 is expressed in platelets and their precursor, the megakaryocyte. The levels of hSR-B1/CLA-1 expression correlate inversely with cholesterol ester content and platelet aggregation. CONCLUSIONS: These findings suggest that determining the level of hSR-B1/CLA-1 expression on the platelets may be a useful clinical marker for atherosclerotic diseases.

Expression of cytokeratin 8 in lung cancer cell lines and measurement of serum cytokeratin 8 in lung cancer patients.

It has been reported that cytokeratin 8 (CK8) can be expressed in several cancers and expression of CK8 is correlated with increased invasiveness of the tumor in vitro and in vivo. In the present study, we investigated expressions of CK8 in human lung cancer cell lines. In addition, we also evaluated the clinical significance of CK8 measurements in sera of patients with lung cancer. Expression of mRNA for CK8 was semi-quantitatively evaluated by the competitive reverse transcriptase-polymerase chain reaction (competitive RT-PCR), using human lung cancer cell lines. The level of CK8 protein in culture supernatants of lung cancer cell lines and 70 sera of patients with lung cancer was measured by enzyme-linked immunosorbent assay (ELISA). Levels of serum CK8 according to clinical parameters were also examined. The level of expression of CK8 mRNA in non-small cell lung cancer (NSCLC) cell lines was significantly high compared with that of small cell lung cancer (SCLC) cell lines (P<0.05). The level of CK8 in culture supernatants in NSCLC was significantly high compared with that of SCLC. The level of serum CK8 in patients with NSCLC was significantly high compared with that of normal non-smokers and compared with that of SCLC (P<0.05). Patients with a CK8 value of 50.0 ng/ml, or higher, had a statistically significant diminished survival compared with those patients whose CK8 values were lower. In conclusion, CK8 was preferentially expressed in NSCLC. Increasing values of CK8 were significantly associated with tumor progression and decreased survival in patients with NSCLC. Therefore, CK8 in sera may become a novel tumor marker in patients with lung cancer.