Preliminary evidence of the association between DNAm and orbital volumetry in GO.

BACKGROUND: The pathogenesis underlying the alterations of orbital architecture in Graves' orbitopathy (GO) is not yet fully understood. The present study aimed to investigate the association of DNA methylation in peripheral blood and orbital volumetry in Chinese patients with GO. METHODS: A total of 35 GO subjects (70 orbits) were subjected to computed tomography (CT) scan. The total cross-sectional area of the extraocular muscles (orbital muscles, OM), total orbit area (TOA), and the exophthalmometry were measured, and OM/TOA ratio was calculated. Targeted bisulfite sequencing was performed on seven candidate genes. RESULTS: No significant correlation was established between the DNA methylation levels of these genes and exophthalmometry. The MBP methylation level was found to be correlated with OM/TOA ratio (P<0.05). Multiple linear regression analysis on parameters, including age, sex, TRAb, duration of GO, and DNA methylation levels of seven genes with OM/TOA ratio confirmed that MBP and OM/TOA ratio had a significant correlation (P<0.05). The partial least squares analysis showed that the top three genes with the highest loadings were MBP, BOLL, and BECN1, and OM/TOA ratio affected the DNA methylation block than exophthalmometry. CONCLUSIONS: This study provided preliminary evidence that MBP is a potential gene associated with OM enlargement in GO patients according to the combination of DNA methylation sequencing and orbital CT measurement.

[Transduction of dendritic cells by recombinant adeno-associated virus (rAAV) carrying hepatitis B virus antigens].

OBJECTIVE: To test the efficiency of infection of recombinant adeno-associated virus (rAAV) carrying hepatitis B virus S, C or X antigen, rAAV-HBV-S, C, X to human dendritic cells. METHODS: Recombinant AAV plasmids containing HBV-S, C or X gene were constructed and packaged into rAAV in 293 cells. Monocytes were isolated from healthy donor and pulsed by rAAV-HBV-S, X, C or 293 lysate as control at the first day of isolation, then the dentritic cells were cultured for 7 days in vitro. The transcription and expression of HBV-S, C or X gene were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) or intracellular staining with fluorescence activated cell sorter (FACS), respectively. RESULTS: The titer of rAAV-HBV-S, C, X virus was approximately 10(-7) copies per ml. After infection the HBV-S, C or X transcription expression could be seen by RT-PCR in the infected dendritic cells, the efficiency was about 90 percent by FACS. CONCLUSION: rAAV-HBV-c can effectively infect and pulse dendritic cells.

[Classification and choice of surgical procedures for chronic pancreatitis].

OBJECTIVE: To explore the classification, choice of surgical procedures and the clinical outcome of surgical management for chronic pancreatitis. METHODS: 54 patients with chronic pancreatitis undergoing operation in our hospital from 1983 to 2004 were analyzed retrospectively, who were divided into chronic calcifying pancreatitis and chronic obstructive pancreatitis according to the clinical manifestations. RESULTS: There were 41 men (76%) and 13 women (24%) with a mean age of 54 years. The cause of chronic pancreatitis was alcohol related in 25 cases (46%), cholelithiasis in 21 (39%), and previous episodes of acute pancreatitis in 18 (33%). Clinical manifestations included abdominal pain in 38 cases (70%), obstructive jaundice in 27 cases (50%). There existed a significant difference in some clinical materials between the two groups of chronic calcifying pancreatitis and chronic obstructive pancreatitis, which might mean the different pathologic basis in the two kinds of chronic pancreatitis. A total of 34 patients underwent nine different operations without perioperative deaths. Both the Puestow procedure and the pancreatoduodenectomy was safe and achieved pain relief in a large percentage of patients, which could also improve the exocrine function whereas the endocrine function remained unchanged. Addition of biliary bypass to the Puestow procedure was suitable for the patients with stenosis of common bile duct. Jaundice was the main manifestation in the patients with the inflammatory mass in the head of the pancreas and Whipple's procedure or other resectional procedures should be performed for them. Only drainage of bile duct had a better outcome for the relief of jaundice, but its effect to pancreas need to be further evaluated. CONCLUSION: The clinicopathologic characteristics of obstructive chronic pancreatitis was more variable and the surgical management should be also different for individuals.

[Binding and inhibition of adeno-associated virus Rep78 protein with hepatitis B virus C promoter].

OBJECTIVES: Adeno-associated virus (AAV) Rep78 is known for its inhibitory effects on replication of several viruses and oncogenes transformations. The study was to investigate the effect of Rep78 on hepatitis B virus C (HBV-C) gene and the mechanism of it. METHODS: HBV-C promoter and HBV-C gene with its promoter were amplified by PCR and labeled with 32P-ATP. Electrophoretic mobility shift assay (EMSA) and in vitro transcription were utilized to detect the binding of MBP-Rep78 with HBV-C promoter and the transcription of HBV-C gene. RESULTS: EMSA showed that by increasing the amount of Rep78 protein from 0.1 microg to 1.0 microg, the binding bands got stronger in a dose-dependent manner. In addition, Rep78 antibody was used to certify the specificity of this binding. The compound of Rep78, Rep78 antibody and HBV-C promoter were seen as super shift bands in EMSA. Meanwhile, HBV-C gene transcription was significantly inhibited by in vitro transcription which meant that Rep78 could not only bind with HBV-C promoter, but also could inhibit the transcription of HBV-C gene. CONCLUSION: AAV Rep78 could inhibit the transcription of HBV-C gene through its binding with HBV-C promoter.

[Recombinant adeno-associated virus carrying hepatitis B virus C gene (rAAV-HBV-C) effectively pulses dendritic cells (DC) without affecting their maturity].

OBJECTIVE: Recombinant virus pulsated dendritic cells (DCs) may affect their survival, growth and maturity. This study is to test the infection efficiency of recombinant adeno-associated virus carrying hepatitis B core antigen (rAAV-HBV-c) to DCs and the growth and maturity of them. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors. Adherent monocytes were pulsed by rAAV-HBV-c and 293 lysate as controls on the first day of isolation. DCs were cultivated in AIM-V media with 1000 u/ml granulocyte macrophage stimulating factor (GM-CSF), 1000 u/ml interleukin-4 (IL-4) and 50 ng/ml tumor necrosis factor-alpha (TNFalpha) separately in vitro. DCs were examined at different times and the expressions of several clusters of differentiations (HLADR, CD14, CD80, CD83, CD86) were studied using FACS after being cultured for 7 days. The transcription and expression of HBV-C gene were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and intracellular staining fluorescence activated cell sorter (FACS), respectively. RESULTS: The rAAV-HBV-c infected and uninfected monocytes gradually matured and their morphology had no significant differences. The CDs expressed on the surfaces of the two groups of DCs were also similar (HLADR: 96.1% vs. 94.5%; CD86: 87.7% vs. 89.8%; CD83: 75.6% vs. 78%; CD80: 52% vs. 54.3%; CD14: 6.4% vs. 4.5%). HBV-C gene mRNA expression was measured using RT-PCR and 89.5% of the rAAV-HBV-c infected DCs showed their protein expression using FACS. CONCLUSION: rAAV-HBV-c can effectively pulse DCs without affecting the growth and maturity of them.

[Sodium butyrate induces rat hepatic oval cells differentiating into mature hepatocytes in vitro].

OBJECTIVE: To elucidate the effects of sodium butyrate on rat hepatic oval cell differentiation in vitro. METHODS: Hepatic oval cells were isolated from rats fed with a choline-deficient diet supplemented with 0.1% (w/w) ethonine for 4 to 6 weeks. The cultured hepatic oval cells were identified by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). After hepatic oval cells were treated with sodium butyrate, the morphological changes were studied through Giemsa staining and the albumin expression level was tested by Western blot. RESULTS: Immunohistochemical results showed the isolated cells were positive for both mature hepatocyte marker albumin and bile duct cell marker cytokeratin-19. Furthermore, RT-PCR results showed that the cells expressed stem cell marker c-kit, but not hematopoietic stem cell marker CD34. In short, the isolated cells were rat hepatic oval cells. 0.75 mmol/L sodium butyrate induced obvious phenotype changes of hepatic oval cells, including enlargement of the oval cells, a decrease in nucleus to cytoplasm ratio, and a 50% increase in the number of binucleated cells. Western blot results showed that 0.75 mmol/L sodium butyrate markedly raised the expression of albumin. CONCLUSION: Sodium butyrate, a differentiation promoting agent, can induce rat hepatic oval cells (liver progenitor cells) to differentiate into mature hepatocytes in vitro.

[Isolation and prelimary characterization of a kind of rat liver potential progenitor cells small round hepatic cells].

Liver undergoes profound regeneration usually after hepatic damage. It has been shown in recent study that two kinds of liver stem cells, which are mainly oval cells (OVCs) and small hepatocytes, are involved in the process of liver regeneration as they differentiated into premature liver cells. However, the origination of oval cells as well as its differentiation property is not quite understood. In this study, we isolated a novel potential liver progenitor cells, namely small round cells (SRCs). The cellular features of the cells such as morphological appearance, surface marker, growth curve, and differentiation induced by DMSO were analyzed. SRCs and OVCs were obtained by using discontinuous digestions and isopyknic centrifugation, respectively. With non-radioactive measurement of MTT, the cell growth was assayed. Meanwhile, SRCs were stained with antibodies against CK19, ALB and AFP antibody to characterize their tissue-specificities. The results showed that SRCs appeared in round-shaped, but irregular in size. The nuclei of SRCs were relative small with rich nucleolus visible. SRCs were semi-floated during primary culture. Even cultured in F12:DMEM (1:1) mixed-medium supplemented with 15% fetal calf serum, viability of the cells could merely be expanded over 7 days. SRCs were positively stained by CK19, ALB and AFP within initial 3 days. After DMSO stimulation, SRCs were not only morphologically changed into OVCs, but also expressed those markers identical to OVCs. It was suggested that SRCs could be considered as an potential candidate of liver progenitors which were related to OVCs.

[A quantitative assay for telomerase activity in peripheral blood mononuclear cells from patients with acute leukemia].

To establish a quantitative assay for telomerase activity and analyze the telomerase activity in peripheral blood mononuclear cells (PBMNC) from patients with acute leukemia, a fluorescent dye, PicoGreen, was added to the products after telomere repeat amplification protocol. The samples were excited at 480 nm and the fluorescence emission intensity was measured at 520 nm using a spectrofluorometer. Telomerase activity was detected in PBMNCs from 20 cases of normal individuals and 25 patients with acute leukemia. The results showed that the fluorescence of PicoGreen binding to double-stranded DNA specifically was enhanced with increase of DNA quantities. In conclusion, the met hod is rapid, simple and quantitative, the telomerase activities of PBMNCs from acute leukemia patients are significantly higher than that of the normal controls.