[Demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on peripheral blood mononuclear cells in chronic hepatitis B patients].

OBJECTIVE: To observe the expression variations and influencing factors of programmed death one (PD-1) and DNA demethylation of PD-1 promoter on peripheral blood mononuclear cells (PBMCs) in chronic hepatitis B (CHB) patients and further investigate the relationship between the demethylation pattern of PD-1 gene in promoter region and the PD-1 expression on PBMC in CHB patients. METHODS: A total of 162 subjects, including 144 CHB patients and 18 healthy blood donors, were enrolled. The expression of PD-1 on PBMCs was detected by flow cytometry. And the serum HBV markers, HBV DNA load and liver function were also measured. DNA of PBMCs was treated with sodium bisulfite; the PD-1 promoter fragments were amplified by polymerase chain reaction (PCR) and then transformed into Escherichia coli. Positive clones were selected for sequencing and the methylation status of fragments of PD-1 promoter was examined. RESULTS: With the PD-1 expression in normal controls (10.8% ± 4.4%) as a baseline level, the expression of PD-1 in CHB patients significantly increased. In CHB patients, the serum expression of PD-1 in PBMCs from patients with positive HBeAg (27.1% ± 18.4%) was much higher than that from those with negative HBeAg (19.6% ± 15.6%). And the expression level of PD-1 was not correlated with serum HBV DNA load and serum level of alanine aminotransferase. The results of bisulfite genomic sequencing showed that demethylation probability of some CG points in PD-1 promoter region (-601, -553, -538, -483, -463, -317 bp) were significantly correlated with PD-1 expression level (P < 0.05). CONCLUSION: The demethylation pattern of PD-1 gene in promoter region is associated with the PD-1 expression on PBMC in CHB patients.

[Effects of 5-azaC on methylation pattern of the perforin promoter of the perforin gene in normal human T cells].

OBJECTIVE: To investigate the effects of DNA hypomethylation on mRNA and protein expression of perforin promotor in T cells. METHODS: T cells were isolated from the peripheral venous blood of healthy donors by density gradient centrifugation. CD4(+) and CD8(+) subsets were isolated using Miltenyi beads and protocols provided by the manufacturer. Where indicated the T cells were stimulated with PHA for 24 h, then treated with 5-azaC for an additional 72 h. Genomic DNA, mRNA, and protein were isolated from untreated and 5-azaC-treated T cells. Purified DNA was treated with sodium bisulfite, the desired sequences were amplified in sequential fragments using nested PCR. The amplified fragments were cloned into bacteria DH5 alpha and 5 independent clones for each of the amplified fragments were sequenced. The expression of perforin was determined using real time RT-PCR and Western blot. RESULTS: The perforin mRNA and protein in the CD4(+) and CD8(+) subsets treated with 5-azaC were significantly higher than those in the untreated subsets (P<0.05). The results of bisulfite genomic sequencing showed that the methylation of perforin promotor was significantly reduced in the treated cells compared with the untreated cells (P<0.05). CONCLUSION: The mRNA and protein expression of perforin significantly increases in the CD4(+) and CD8(+) T cells treated with 5-azaC,which is associated with DNA hypomethylation of perforin promoter in T cells.

[Serum levels of antibodies for IgG, IgA, and IgM against the fungi antigen in psoriasis vulgaris].

OBJECTIVE: To detect the serum levels of antibodies against Malassezia further and Candida albicans in patients with psoriasis vulgaris (PV), and to investigate the relationship between Malassezia furfur or Candida albicans and pathogenesis of PV. METHODS: The serum levels of antibodies for IgG, IgA, and IgM against the whole cell antigen Wag and soluble antigen (Sag) of Malassezia fufur and Candida albicans in 40 PV patients and 20 healthy controls were detected by indirect-ELISA, RESULTS: The serum levels of IgG against Wag of Malassezia furfur and Candida albicans were significantly higher in PV patients than those in the healthy controls (P < 0.01, P < 0.05, respectively), The serum levels of IgM against Sag of Malassezia furfur and Candida albican, IgA against Sag of Candida albicans were significantly lower in PV patients than those in the healthy controls (P < 0.05). CONCLUSION: There are some humoral immunity abnormalties in PV patients. Malassezia furfur and Candida albicans may be related to the pathogenesis of PV.