Adult ; Female ; Graft vs Host Disease ; diagnosis ; Hepatitis, Autoimmune ; diagnosis ; Humans

AIM:To observe the pathological changes of the lower segment of nasolacrimal duct mucosa in rabbits at different stages after retrograde lachrymal dilated drainage tube implantation.
METHODS:Totally 14 New Zealand rabbits were used in the present study. One side of nasolacrimal duct was obstructed to produce an experimental model and operated the reverse implantation of nasolacrimal duct intubation. Histological changes of the lower segment of nasolacrimal duct mucosa were observed by routine light microscope at 2, 4, 6, 8, 10, 12 and 14wk after the operation.
RESULTS: Compared with the control side, the group of 2 and 4wk after surgery presented the inflammatory cytokine. The group of 12wk after the operation presented isolated granuloma. Group 12 and 14wk presented scattered granuloma. The size of the granulomas was smaller and the density of epithelioid cell and fibroblast were lower in group 12wk than those in group 14wk by HE and Masson trichrome stain.
CONCLUSION:Recurrent Silicone Tube is used to treat nasolacrymal duct obstruction. Nasolacrimal duct can be narrowed and blocked again by granuloma, progressive fibrosis and adhesion of surrounding tissues when tube is in the duct more than 12wk.

OBJECTIVETo prepare a specific high molecular weight polymer contrast agent capable of specifically targeting hepatocarcinoma cells (HCC) and to investigate its affinity in vitro using HepG2 cells.

METHODSThe high molecular weight polymer polylactic-co-glycolic acid (PLAG)-COOH was prepared by the double emulsion technique. PLAG-COOH microbubbles were combined with glypican-3 (GPC3) antibody to generate HCC targeting high molecular polymer ultrasound contrast agents by the carbodiimide method. The affinity for HCC cells was confirmed by measuring attachment to cultured HepG2 cells by flow cytometry and comparing the results with the properties observed for non-targeted high molecular weight polymer ultrasound contrast agents.

RESULTSThe average diameter of the targeted high molecular weight polymer ultrasound contrast agents was (800+/-10) nm. In vitro targeting of HepG2 cells showed that many of the targeted high molecular weight polymer ultrasound contrast agents attached tightly to the cell surface and that the GPC3-PLGA has a particularly strong targeting ability.

CONCLUSIONA HCC-specific high molecular contrast agent, GPC3-PLGA, was synthesized and evidenced a strong targeting ability towards HepG2 cells in vitro. This new agent may be exploited to improve diagnosis of liver cancer at the molecular level.

Carcinoma, Hepatocellular ; Contrast Media ; Humans ; Liver Neoplasms ; Microbubbles ; Molecular Weight

OBJECTIVETo prepare a glypican-3 (GPC3)-targeting hepatocellular carcinoma MR molecular probe and to evaluate its targeting specificity using HepG2 cells.

METHODSPoly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared by a double emulsion solvent evaporation method, and the surfaces were connected with anti-GPC3 mono-antibody and paramagnetic substance Gd3+. The physical properties of the probes were investigated using fluorescence microscopy, electron microscopy, Malvern particle size analysis, inductively coupled plasma atomic emission spectroscopy (ICP-AES) and 1.5T MR imaging. The specificity of the probes to target cultured HepG2 cells was determined by laser confocal microscopy. The signal characteristics, including signal-to-noise ratio (SNR), after co-incubation with HepG2 cells were analyzed by 1.5T MR imaging. Significance of differences between multiple groups (target group, non-target group, and control group) was assessed by one-way analysis of variance, and between two groups was assessed by the LSD-t test. A difference was considered to be statistically significant at P less than 0.05.

RESULTSThe GPC3-targeting hepatocellular carcinoma MR molecular probes were successfully prepared. The nanoparticles had a spherical shape, size of 495 +/- 17.5 nm, uniform size distribution, good dispersibility, no obvious aggregation, and could significantly increase the T1 signal. Using the ICP-AES measurement, 1 mol PLGA carried about 12 mol Gd3+, and as the Gd3+ concentration increased, the T1 signal increased. The prepared MR molecular probes could specifically target HepG2 cells, and could increase the T1 signal. The SNR value of the target group was 3.45 +/- 0.21, of the non-target group was 1.43 +/- 0.07, and of the control group was 1.12 +/- 0.03. The SNR value of the target group was significantly higher than that of the non-target group and the control group (P less than 0.05); there was no significant difference between the non-target group and the control group (P more than 0.05).

CONCLUSIONPLGA nanoparticles, anti-GPC3 mono-antibody and paramagnetic Gd3+ can be used to successfully prepare GPC3-targeting hepatocellular carcinoma MR molecular probes which are capable of specifically targeting HepG2 cells in vitro and being detected by 1.5T MR imaging. These MR molecular probes may represent a useful noninvasive imaging method for detecting early hepatocellular carcinoma in vivo.

OBJECTIVETo explore the effects of the exogenous and endogenous reactive nitrogen metabolites (RNM) as NK cell inhibitors on NK cell-mediated killing of K562 cells and the influence of Tiopronin (TIP), glutamylcysteinylglycine (GSH) and histamine dihydrochloride (DHT) as RNM scavengers on reversing the suppressing effect of RNM.

METHODSThe exogenous ONOO(-) was administered in the NK+K562 culture system, then the RNM scavengers were added in the NK+K562+ONOO(-) culture system, respectively. The concentrations of RNM, TNF-beta and IFN-gamma, K562 cell inhibition rate (KIR) and the percentage of living NK cells were examined. IL-2+PHA were used as monocyte (MO) activators in the culture system of MO+NK+K562. Then TIP, GSH and DHT were administered and the parameters of NK cell activity were analyzed.

RESULTSAfter exogenous ONOO(-) was administered in NK+K562 culture system, the percentage of living NK cells was decreased from (93.17 +/- 2.57)% to (71.87 +/- 1.02)% (P < 0.01) and KIR was decreased from (67.47 +/- 2.64)% to (43.44 +/- 2.87)% (P < 0.01). When TIP, GSH and DHT were administered into the systems, the percentage of living NK cells was increased to (91.13 +/- 3.67)% (P < 0.05), (88.03 +/- 1.46)% (P < 0.05), (73.60 +/- 2.76)% (P > 0.05), respectively; KIR was increased to (61.58 +/- 1.89)% (P < 0.05), (60.68 +/- 2.07)% (P < 0.05) and (45.26 +/- 3.31)% (P > 0.05), respectively. When IL-2/PHA were administered in the NK+K562+MO culture system, RNM products was increased from (82.10 +/- 6.60) micromom/L to (193.65 +/- 5.95) micromom/L(P < 0.01);KIR was decreased from (90.64 +/- 3.06)% to (61.29 +/- 2.22)% (P < 0.01). When the TIP, GSH and DHT were administered in the systems, RNM products were decreased to (91.32 +/- 6.81) micromom/L (P < 0.05), (84.66 +/- 5.99) micromom/L (P < 0.05) and (188.92 +/- 5.00) micromom/L (P > 0.05), respectively; KIR was increased to (84.31 +/- 4.56)%(P < 0.05), (81.65 +/- 3.09)% (P < 0.05) and (72.20 +/- 4.10)% (P < 0.05), respectively.

CONCLUSIONNK Cell-mediated killing of K562 cells can be suppressed by exogenous and endogenous RNM administration. Both of TIP and GSH can protect NK cells by scavenging RNM and enhance the antineoplasmic activity of NK cells.

Cells, Cultured ; Coculture Techniques ; Glutathione ; pharmacology ; Histamine ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-2 ; immunology ; pharmacology ; K562 Cells ; Killer Cells, Natural ; cytology ; immunology ; metabolism ; Lymphotoxin-alpha ; metabolism ; Monocytes ; cytology ; Peroxynitrous Acid ; pharmacology ; Reactive Nitrogen Species ; antagonists & inhibitors ; metabolism ; Tiopronin ; pharmacology

OBJECTIVETo evaluate the efficacy and safety of entecavir (ETV) maleate versus ETV in Chinese patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB).

METHODSThis was a randomized, double-blind, double-dummy, controlled, multicenter study. Patients were randomly assigned to receive 48 weeks of treatment with 0.5 mg/day ETV (group A; n = 26) or 0.5 mg/day ETV maleate (n = 31). Hepatitis B virus (HBV) DNA levels were measured at weeks 12, 24, and 48 by the Roche Cobas Ampliprep/Taqman PCR assay. Adverse events (AE) were recorded.

RESULTSBaseline characteristics were similar between the two groups. At weeks 12, 24, and 48, the mean HBV DNA level had similarly decreased from baseline in both groups (A: by 4.24, 4.61 and 4.88 log10 IU/mL vs. B: 4.01, 4.50 and 4.99 log10 IU/mL, respectively; all P more than 0.05). Patients who achieved undetectable levels of serum HBV DNA (less than 20 IU/mL) at week 48 were similar in the two groups (A: 69.23% vs. B: 80.65%; P more than 0.05). Both groups achieved similar normalization of ALT at week 48 (A: 96.00% vs. B: 83.87%; P more than 0.05). The overall AE incidence was similar for the two groups (A: 22.22% vs. B: 9.38%; P more than 0.05).

CONCLUSIONEntecavir maleate and entecavir showed similar efficacy and safety in patients with HBeAg-negative CHB.

Adult ; Antiviral Agents ; adverse effects ; therapeutic use ; Double-Blind Method ; Female ; Guanine ; adverse effects ; analogs & derivatives ; therapeutic use ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Male ; Maleates ; adverse effects ; therapeutic use ; Middle Aged ; Treatment Outcome

OBJECTIVETo establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses.

METHODSObtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance.

RESULTSSuccessfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity.

CONCLUSIONSuccessfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.

Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; Influenza, Human ; diagnosis ; virology ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVERegarding the strong antigen-presenting abilities of dendritic cells (DC), this study was carried out based on the induction and proliferation of DC derived from human umbilical cord blood; the anti-HBV effect of cytotoxicity T lymphocytes (CTL) activated by those DC pulsed with HBsAg was also carried out to explore a new way to activate the HBsAg-specific CTL.

METHODSCord blood was collected from the cord veins of normal placentae after Cesarean sections, from which cord blood mononuclear cells (CBMC) were separated through density gradient centrifugation. The CBMC were cultured in RPMI 1640 with a cytokine cocktail. Pulsed with HBsAg, the DC were prepared to activate the HBsAg-specific CTL among the CBMC. The cytotoxic effect of CBMCs activated by the DC primed with HBsAg was assayed through the killing of those HepG2-S target cells.

RESULTSTypical DC could be induced from CMBC cultured with a cytokine cocktail. DC pulsed by HBsAg activated HBsAg-specific CTL, which killed the target HepG2-S cells to some extent.

CONCLUSIONDC can be induced from CMBC with the cytokine cocktail and they show a strong antigen-presenting ability. DC produced in this way and pulsed by HBsAg can activate HBsAg-specific CTL in vitro. This might mean that it could be a new way to break the tolerance to HBV in chronic HBV-infected patients.

Cell Culture Techniques ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; Hep G2 Cells ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; immunology ; Humans ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo investigate the expression of OPCML gene in ovarian epithelial carcinoma and determine the relationship between mRNA expression and methylation of their promoters.

METHODTwenty normal ovarian tissues and 89 ovarian epithelial tumor specimens (72 malignant, 17 benign), as well as 3 ovarian carcinoma cell lines (SKOV-3, CAOV3, and 3AO), were collected for detection of OPCML gene expression by reverse transcription-polymerase chain reaction and for detection of promoter methylation by restriction enzyme cut analysis from 7. 1999 to 7. 2003.

RESULTSAmong ovarian epithelial carcinoma 19.4% expressed OPCML mRNA, while 85% of normal ovarian tissue and 76.5% of benign ovarian tumor. The ratio of expression of OPCML mRNA in ovarian epithelial carcinoma was significantly lower than those of normal (chi2 = 30.108, P = 0.0000) and benign tumors (chi2 = 21.162, P = 0.000). No OPCML mRNA expression was found in SKOV-3 and CAOV3, but was found in 3AO. Methylations were detected in 44.4% of cancer cells promoter, while 0% in normal ovarian tissue and benign ovarian tumors. The ratio of methylation of ovarian epithelial carcinoma was significantly higher than those of normal (chi2 = 13.630, P = 0.0000) and benign tumors (chi2 = 11.797, P = 0.000). Methylation was found in SKOV-3 and CAOV3, but not in 3AO. The relationship between gene expression and promoter methylation was correlated (r = 11.589, P = 0.002), especially at Hap I1 site (r = 11.640, P = 0.004). Methylation was also found in SKOV-3 and CAOV3 cell lines, but not in 3AO cell line.

CONCLUSIONDeletion of OPCML gene exists in ovarian epithelial carcinoma cell. The gene promoter methylations, especially Hap II motif, may be one of pathways that contribute the inhibition of OPCML expression.

Adult ; Aged ; Cell Adhesion Molecules ; genetics ; Cell Line, Tumor ; CpG Islands ; genetics ; DNA Methylation ; Female ; GPI-Linked Proteins ; Gene Deletion ; Humans ; Middle Aged ; Ovarian Neoplasms ; genetics ; pathology ; Promoter Regions, Genetic ; genetics ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the cytotoxic T lymphocyte (CTL) response induced by dendritic cells (DC) transduced with recombinant adenovirus vector bearing hepatitis B virus surface antigen (HBsAg) gene in hepatocellular carcinoma HepG2. 2. 15 cells in vitro.

METHODSFull length HBsAg cDNAs were subcloned into pIND vector, followed by being cloned into pShuttle vector. The HBsAg gene fragments resulted from the pShuttle-S digested with PI-Sce and I-Ceu were linked to the linear adeno-X virus DNA. After packaged with HEK293 cells, the adenovirus expression vector was obtained. Then the recombinant adenovirus expression plasmid AdVHBsAg was transfected into human monocyte-derived dendritic cells, to construct AdVHBsAg hepatocarcinoma tumor vaccine. The effectiveness of transfection was detected by Western blot. Surface molecules of AdVHBsAg-DC were detected by FACS. Autologous T cell proliferation stimulated by AdVHBsAg-DC was detected by 3H-TdR assay. Cytotoxic CTL activity induced by AdVHBsAg-DC in vitro was detected by LDH assay.

RESULTSHBsAg gene in the inserted DNA of AdVHBsAg was confirmed by PCR, and predictive fragments proved by restriction enzyme digestion analysis were exhibited. Cell pathological changes appear after 10 days HEK293 cells transfected AdVHBsAg. Western blot analysis showed that HBV surface antigen gene was expressed in transfected DC, indicating that the transfection was effective. AdVHBsAg-DC was able to upregulate CD1a, CD11c, CD80, CD86 and HLA-DR. Autologus T cell proliferation induced by AdVHBsAg-DCs was significantly higher than that in DC control group and LacZ-DC group (P < 0.05). AdVHBsAg-DC activated CTL presented the specific killer ability to the hepatocellular carcinoma cells expressing HBsAg.

CONCLUSIONDC transduced with recombinant adenovirus HBsAg can express HBV-related hepatocellular carcinoma antigen (HBsAg), and AdVHBsAg-DC can induce potent immune response against HBsAg-positive hepatocellular carcinoma cells in vitro.

Adenoviridae ; genetics ; Antigens, CD1 ; metabolism ; CD11c Antigen ; metabolism ; Cancer Vaccines ; immunology ; Carcinoma, Hepatocellular ; immunology ; pathology ; virology ; Cell Line, Tumor ; Cell Proliferation ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; metabolism ; Genetic Vectors ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Humans ; Liver Neoplasms ; immunology ; pathology ; virology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; cytology ; immunology ; Transfection