Rho kinase inhibitor Y-27632 downregulates IL-1ß expression in mice with experimental autoimmune myocarditis.

Autoimmune myocarditis is the limited or diffuse inflammation of the myocardium due to dysfunctional cellular and humoral immunity mechanisms. We constructed mouse models of experimental autoimmune myocarditis (EAM) using peptide MyHC-α614-629. On the day after secondary immunization, the mice were intraperitoneally injected with Rho kinase (ROCK) inhibitor Y-27632. On day 21, the cardiac tissues were harvested and weighed. The hearts of EAM mice were significantly enlarged and whitened. Furthermore, body weight (BW) slowly increased during the treatment period, the heart weight (HW) and the ratio of HW/eventual BW were increased, and inflammatory infiltration and fibrosis were aggravated in the myocardial tissue. Y-27632 treatment improved the aforementioned phenotypic and pathological features of EAM mice. Mechanistic analysis revealed a significant increase in Notch1, Hes1, Jag2, Dil1, Toll-like receptor (Tlr) 2, and interleukin (IL)-1ß expression in the myocardial tissue of EAM mice. Notably, IL-1ß expression was correlated with that of Notch1 and Tlr2. Following Y-27632 treatment, the expression of key target genes of the Notch signaling pathway (Notch1, Hes1, Dil1, and Jag2) and Tlr2 were obviously decreased. Y-27632 treatment also decreased the number of monocytes in the spleen of EAM mice. Thus, ROCK inhibitor Y-27632 exerted a protective effect in EAM mice by downregulating IL-1ß expression. This study aimed to provide a reference point for the future treatment of myocarditis in clinical settings.

Effect of adoptive transfer of CD4+CD25+Foxp3+ Treg induced by trichostatin A on the prevention of spontaneous abortion.

OBJECTIVE: To investigate whether epigenetic modification of CD4+CD25- T-cells in vitro can make up for the inadequacy of CD4+CD25+Foxp3+ Treg in animal model of spontaneous abortion and prevent immune response-mediated spontaneous abortion. METHODS: Trichostatin A (TSA) was applied to inhibit histone deacetylases (HDACs) and thereby to epigenetically modify the special location of Foxp3 gene in CD4+CD25- T-cells of CBA/J mice. The expressions of CD25, Foxp3, CTLA-4 and PD-1 of CD4+ T cells isolated from spleen of mice were characterized by flow cytometric analysis. Concentrations of transforming growth factor- ß (TGF-ß) and IL-10 in the supernatants of cultured Treg were measured using ELISA. The purified CD4+ T cells treated with different reagents were injected into pregnant CBA/J mice mated with DBA/2J males on Day 1 and 4 of pregnancy, respectively. The embryo resorption rate was assessed on Day 14 of pregnancy. RESULTS: TSA treatment significantly increased the population of CD4+CD25+Foxp3+ iTreg. Those TSA induced Treg expressed high levels of PD-1 and CTLA-4, and secreted high levels of TGF-ß and IL-10. Adoptive transfer of those iTreg at both early stage and implantation of stage of pregnancy significantly increased population of CD4+CD25+Foxp3+ Treg in spleens of recipient miscarriage prone mice and significantly reduced resorption in those mice. CONCLUSION: Epigenetic regulation of Foxp3 can generate functional regulatory T-cells. Adoptive transfer of TSA- induced CD4+CD25+Foxp3+ Treg at an early stage of pregnancy can induce maternal-fetal immune tolerance and reduce embryo resorption in miscarriage prone mice.

CYP1B1 enhances the resistance of epithelial ovarian cancer cells to paclitaxel in vivo and in vitro.

Ovarian cancer (OC) is the most frequent cause of mortality among gynecological malignancies, with a 5-year survival rate of approximately 30%. The standard regimen for OC therapy includes a platinum agent combined with a taxane, to which the patients frequently acquire resistance. Resistance arises from the oxidation of anticancer drugs by CYP1B1, a cytochrome P450 enzyme overexpressed in malignant OC. The aim of the present study was to determine the role of CYP1B1 expression in the drug resistance of OC to the taxane, paclitaxel (PTX). Immunohistochemical staining was used to assess CYP1B1 expression in a panel of ovarian samples (53 primary cancer samples, 14 samples of metastastic cancer, 30 benign tumor samples and 19 normal tissue samples). Semi-quantitative RT-PCR was also performed to determine CYP1B1 expression in several OC cell lines. Finally, we used proliferation and toxicity assays, as well as a mouse xenograft model using nude mice to determine whether α-naphthoflavone (ANF), a CYP1B1 specific inhibitor, reduces resistance to PTX. CYP1B1 was overexpressed in the samples from primary and metastatic loci of epithelial ovarian cancers. In some cell lines, PTX induced CYP1B1 expression, which resulted in drug resistance. Exposure to ANF reduced drug resistance and enhanced the sensitivity of OC cells to PTX in vitro and in vivo. The expression profile of CYP1B1 suggests that it has the potential to be a useful diagnostic marker and prognostic factor for malignant OC. The inhibition of CYP1B1 expression by specific agents may provide a novel therapeutic strategy for the treatment of patients resistant to PTX and may improve the prognosis of these patients.

[Predictive value of placenta-derived RASSF1A sequence expression in maternal plasma for pre-eclampsia].

OBJECTIVE: To investigate the expression of placenta-derived RASSF1A gene in maternal plasma during first and second trimesters, and to explore its value for the prediction of pre-eclampsia. METHODS: For 325 pregnant women of the first trimester, free DNA of plasma samples was extracted at 7-12, 13-18, and 19-24 gestational weeks, respectively. Methylation-sensitive restriction enzyme digestion followed by fluorescence quantitative PCR (MSRE+ PCR) was employed for analyzing the concentrations of hypermethylated RASSF1A gene. Blood pressure, proteinuria and clinical feature were monitored at the same time. Those who had subsequently developed pre-eclampsia were selected as the pre-eclamptic group, 30 normal pregnant women were selected as the control group. Hypermethylated RASSF1A gene in maternal plasma was retrospectively analyzed. The relationship between clinical classification, type of pre-eclampsia and concentrations of the gene were further analyzed. RESULTS: Twenty-six out of the 325 pregnant women developed pre-eclampsia as their only complication. At 13-18 gestational weeks, the mean concentrations of fetus-specific RASSF1A sequences were 141.62 copies/mL in maternal plasma of pre-eclamptic pregnancies, which was significantly greater than that of the controls (98.90 copies/mL). Fetus-derived RASSF1A levels were 2.03 fold higher in pre-eclamptic subjects than controls at 19-24 gestational weeks. There was a significant difference in the level of hypermethylated RASSF1A gene between the mild and severe pre-clamptic subjects at 13-24 gestational weeks (P< 0.05). The concentrations of the sequences were significantly higher in early-onset severe pre-eclampsia than late-onset severe pre-eclampsia at 19-24 gestational weeks (P< 0.05). CONCLUSION: Altered expression of hypermethylated RASSF1A gene may be detected in maternal plasma during second trimester, which has important significance for early prediction of pre-eclampsia.

Quantification and application of the placental epigenetic signature of the RASSF1A gene in maternal plasma.

OBJECTIVES: The aims of the detection of hypermethylated RASSF1A gene in maternal plasma from all three trimesters of pregnancy were to show its feature of cell-free fetal DNA and to make up deficients of genetic markers. This study also aimed at investigating of its application value in pre-eclampsia compared with third trimester. METHODS: Eighty pregnant women (7-41 gestational weeks) including normal pregnant women (60 cases) and pre-eclamptic pregnant women (20 cases) were selected as study groups and 20 normal non-pregnant women were selected as control group. Free DNA of plasma samples was extracted, and RASSF1A levels before and after double-methylation-sensitive restriction enzyme digestion of HinP1I and HhaI were determined to measure total and fetal cell-free DNA, respectively. beta-Actin gene was detected as a control to confirm complete enzyme digestion. RESULTS: The median concentrations of hypermethylated RASSF1A gene were 46 copies/mL in first trimester, 96 copies/mL in second trimester, 527 copies/mL in third trimester and 1947 copies/mL in pre-eclampsia. There was positive correlation between fetal-derived hypermethylated RASSF1A levels and the severity of pre-eclampsia. CONCLUSION: Hypermethylated RASSF1A gene may be considered as an epigenetic marker to detect the fetal DNA in maternal plasma and expands the clinical application of non-invasive prenatal diagnosis.

[Quantitative detection of the hypermethylated RASSF1A gene in maternal plasma of pre-eclampsia].

OBJECTIVE: To investigate the level of hypermethylated ras association domain family 1A (RASSF1A) gene in maternal plasma of pre-eclampsia and its clinical value. METHODS: Sixty pre-eclampsia women including 30 mild and 30 severe cases were selected, 60 women with normal pregnancy were studied as control. Free DNA from plasma samples was extracted, fluorescence quantitative polymerase chain reaction (FQ-PCR) was used to detect the concentrations of RASSF1A gene before and after methylation-sensitive restriction digestion. Meanwhile, beta-actin gene was detected as a control to confirm complete enzyme digestion. RESULTS: The median concentration of hypermethylated RASSF1A gene was 3.31-fold higher in samples from pre-eclamptic pregnancies than that in controls. There was significant difference between the mild and severe pre-eclamptic subjects (P<0.05), with the median concentrations of 1659 copies/mL and 2036.50 copies/mL, respectively. CONCLUSION: Hypermethylated RASSF1A gene in pre-eclampsia plasma was significantly increased and the concentrations were related to the severity of pre-eclampsia.

Prevalence and type distribution of human papillomavirus infection in women from Datong, China.

This survey of 931 cervical specimens in women from Datong, China indicates that the overall human papillomavirus (HPV) prevalence was 18.6%, and the most prevalent high-risk HPV types were 16, 58, 18, 52 and 33. This study demonstrates the epidemiology of HPV infection in Datong and the potential impact of vaccination in this region.