DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B / 中国结合医学杂志

OBJECTIVE@#To explore the molecular bases of Chinese medicine (CM) syndrome classification in chronic hepatitis B (CHB) patients in terms of DNA methylation, transcription and cytokines.@*METHODS@#Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients (DNA methylation: 15 cases; serum cytokines: 62 cases) with different CM syndromes, including dampness and heat of Gan (Liver) and gallbladder (CHB1, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan stagnation and Pi (Spleen) deficiency (CHB2, DNA methylation: 5 cases, serum cytokines: 15 cases), Gan and Shen (Kidney) yin deficiency (CHB3, DNA methylation: 5 cases, serum cytokines: 16 cases), CHB with hidden symptoms (HS, serum cytokines:16 cases) and healthy controls (DNA methylation: 6 cases). DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples. Genome-wide DNA methylation was detected using Human Methylation 450K Assay and furthered verified using pyrosequencing. Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay (ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively.@*RESULTS@#Totally 28,667 loci, covering 18,403 genes were differently methylated among CHB1, CHB2 and CHB3 (P<0.05 and |Δβ value| > 0.17). Further validation showed that compared with HS, the hg19 CHR6: 29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1 (P<0.05). However, they remained unaltered in CHB2 (P>0.05). Levels of Interleukin (IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups (P<0.05). Levels of macrophage inflammatory protein (MIP)-1α and MIP-1β were higher in CHB3 than other groups and leukemia inhibitory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups (P<0.05). IL-12, MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F (HLA-F) mRNA expression (R2=0.238, P<0.05; R2=0.224, P<0.05; R=0.447, P<0.01; respectively). Furthermore, combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1, CHB2 and HS.@*CONCLUSIONS@#Demethylation of CpG loci in 5' UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12, MIP-1α and MIP-1β levels, indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.@*REGISTRATION NO@#ChiCTR-RCS-13004001.

Non-association of IL-12 +1188 and IFN-gamma +874 polymorphisms with cytokines serum level in occult HBV infected patients

Occult hepatitis B infection [OBI] is identified as a form of hepatitis in which despite the absence of detectable HBsAg, HBV-DNA is observed in peripheral blood of patients. The main aim of this study has been to investigate the association between polymorphisms in +874 of IFN-gamma and +1188 of IL-12 with their serum level in patients suffering from OBI. In this experimental study, plasma samples of 3700 blood donors were tested for the presence of hepatitis B surface antigen [HBsAg] and anti-HBc by ELISA. The HBsAg[-]/anti-HBc[+] samples were selected and screened for HBV-DNA by PCR. HBV-DNA positive samples were assigned as OBI cases and ARMS-PCR techniques were performed to examine the two known polymorphisms within IL-12 and IFN-gamma. In addition, the serum levels of IL-12 and IFN-gamma were also determined by ELISA. Results of this study demonstrated that, 352 [9.5%] out of 3700 blood samples were HBsAg[-]/anti-HBc[+]and HBV-DNA was detected in 57/352 [16.1%] of HBsAg[-]/anti-HBc[+] samples. Our results showed that groups showed significant difference in CC allele of +1188 region of IL-12 and no difference was observed in the other evaluated genes. Our results also showed that the alleles of +1188 region of IL-12 and alleles of +874 of IFN-gamma were also not associated with serum level of cytokines. According to the results of this study, it may be concluded that the polymorphisms in +1188 region of IL-12 and +874 region of IFN-gamma would not affect the expression of both cytokines at serum level in OBI patients

Co-Immunization of Plasmid DNA Encoding IL-12 and IL-18 with Bacillus Calmette-Guerin Vaccine against Progressive Tuberculosis

PURPOSE: Bacillus Calmette-Guerin (BCG) vaccine has widely been used to immunize against tuberculosis, but its protective efficacy is variable in adult pulmonary tuberculosis, while it is not efficiently protective against progressive infection of virulent Mycobacterium tuberculosis strains. In this study, the protective effects of plasmid DNA vaccine constructs encoding IL-12 or IL-18 with the BCG vaccine were evaluated against progressive infection of M. tuberculosis, using mouse aerosol challenge model. MATERIALS AND METHODS: Plasmid DNA vaccine constructs encoding IL-12 or IL-18 were constructed and mice were immunized with the BCG vaccine or with IL-12 DNA or IL-18 DNA vaccine constructs together with the BCG vaccine. RESULTS: The BCG vaccine induced high level of interferon gamma (IFN-gamma) but co-immunization of IL-12 or IL-18 DNA vaccine constructs with the BCG vaccine induced significantly higher level of IFN-gamma than a single BCG vaccine. The BCG vaccine was highly protective at early stage of M. tuberculosis infection, but its protective efficacy was reduced at later stage of infection. The co-immunization of IL-12 DNA vaccine constructs with the BCG vaccine was slightly more protective at early stage of infection and was significantly more protective at later stage infection than a single BCG vaccine. CONCLUSION: Co-immunization of IL-12 DNA vaccine with the BCG vaccine induced more protective immunity and was more effective for protection against progressive infection of M. tuberculosis.

[Evaluation the relationship between alleles of+1188 in region of IL-12 with serum level of cytokine in patients with occult HBV infection]

Occult hepatitis B infection [OBI] is a form of hepatitis, which in despite of absence of detectable HBsAg, HBV-DNA is present in peripheral blood of patients. Evaluation the relationship between alleles of+1188 in region of IL-12 with serum level of cytokine in patients with occult HBV infection. In this study, the plasma samples of 3700 blood donors were tested for HBsAg and anti-HBc by ELISA. The HBsAg negative ve and anti-HBc positive samples were selected and screened for HBV-DNA by PCR. HBV-DNA positive samples assigned as OBI cases and PCR-SSP and ELISA were performed to examine the polymorphisms in region of [+1188 and serum level of IL-12] respectively. The results showed that there is a significant difference in CC allele of+1188 region of IL-12 in two groups and no difference in the other evaluated genes. There is not any significant difference in serum level of IL-12 between OBI patients and controls. Our results also showed that there isn't any significant statistically relation between alleles of+ 1188 region of IL-12 with serum level of cytokine. According to the results of this study it could be concluded that OBI patients unable to produce enough quantity of IL-12 and it may be related to different IL-12 gene. CC allele was associated with OBI, hence, it seems that +1188 region of IL-12 gene has an important role in expression of IL-12 gene. Evaluation of relation between polymorphisms in +1188 region of IL-12 gene and its expression. In vitro and under mitogene affect can useful because no association was seen between serum level of IL-12 and alleles of this region

Transfected human mesenchymal stem cells do not lose their surface markers and differentiation properties

In this study, we evaluated the biological properties of human mesenchymal stem cells transfected (hMSC) with a plasmid vector expressing human cytokine interleukin-12 (IL-12). Surface markers were analysed by immunophenotyping using flow cytometry. Differentiation capability was evaluated towards adipogenesis and osteogenesis. We demonstrated that successfully transfected hMSC retained their surface immunophenotypes and differentiation potential into adipocytes and osteocytes. These results indicate that hMSC may be a suitable vehicle for gene transduction.

Administration of multiple cytokine genes with anti-tumor activity inhibits both tumor incidence and tumor growth

The finding of reporter gene expression in muscle cells after intramuscular injection of a reporter gene containing DNA has suggested that injection of a certain gene in its naked form could induce an expression of the injected gene. The result proposed the concept, namely DNA or genetic vaccine technology, that injection of an antigen gene could induce a specific immune response against the antigen. Although the concept was initially applied to vaccination technology, the result also means that administration of cytokine genes with anti-tumor activity could exert their functions when they are applied as a naked form of DNA. To test the possibility, plasmid vector containing granulocyte macrophage-colony stimulation factor (GM-CSF) and interleukin-12 (IL-12) genes, which are known as one of the most potent anti-tumor cytokines, were constructed and injected into mice together with syngeneic tumor cells. When the cytokine gene containing plasmid was injected on the same day of tumor cell injection, a tumor mass developed in 4 out of 5 mice tested. Even among the 4 mice, the tumor mass of a mouse disappeared 2 weeks after tumor development. In addition, tumor generation was significantly delayed in cytokine gene injected mice and the average tumor size was about 51.5% that of vector control injected mice. These results suggested that tumor treatment through the injection of multiple cytokine genes with potent anti-tumor activity significantly inhibits tumor development and growth, and that the method could be considered as one of the tools for efficient tumor treatment.