Objective To observe the immunologic effect of lactoferrin on repeated respiratory infection(IRRI) in children.Methods Ninety-eight cases of IRRI were divided into two groups randomly.The control group (48 cases)were treated with routine therapy.The treatment group(50 cases) were treated with lactoferrin based on routine therapy for 2-3 months.T cell subgroup,immunoglobulin and complements were determined before and after treatment.Results Total effective rates in treatment group and control group were 86% and 22.9% respectively.The therapeutic efficacy in treatment group was significantly higher than that in control group(P

Animals ; Animals, Newborn ; Bifidobacterium ; growth & development ; metabolism ; Gastrointestinal Tract ; microbiology ; Liver Function Tests ; Parenteral Nutrition ; methods ; Rabbits

OBJECTIVETo construct a eukaryotic expression vector for expressing hepatitis B virus (HBV) recombinant HBsAg-EGFP fusion protein and obtain a stable transfected Chang Liver cell line.

METHODSThe coding region of HBsAg gene of HBV was amplified by PCR and was digested by BamH I/EcoR I . This fragment was inserted into pEGFPN1 with T4 ligase and transformed E-coli TG1. The positive recombinant plasmid was selected, then the recombinant plasmid was transfected into Chang Liver cell by Lipofectamine 2000 cells containing stable transformants were selected by the ability of resistance to G418 and isolated with a limited dilution. The stable transfected cell line expressing high level HBsAg-EGFP fusion protein was obtained.

RESULTSThe eukaryotic expression vector named pEGFPN1-HBsAg was successfully constructed and the stable transfected Chang Liver cell line expressing pEGFPN1-HBsAg fusion protein was obtained.

CONCLUSIONThe stable transfected Chang Liver cell line could express pEGFPN1-HBsAg fusion protein, could be used to screen the proteins differentially expressed in HBsAg expression Chang Liver cells, which brought some new clues for studying the potential molecular mechanism of HBsAg protein.

Cell Line ; Gene Expression ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatitis B Surface Antigens ; genetics ; metabolism ; Hepatitis B virus ; genetics ; metabolism ; Humans ; Liver ; cytology ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; methods

OBJECTIVEThe present aimed to observe the effect of phosphatase inhibitor cyclosporine A on the subcellular location and on expression of HBcAg in HepG2.2.15 cells.

METHODSThirty micrograms/ml of cyclosporine A (CSA) was added into HepG2.2.15 cell culture system and on days 2 and 4 HBcAg and HBsAg were respectively stained with fluorescent immunocytochemistry and observed under confocal microscope. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling method.

RESULTSHBcAg was mostly expressed in cytoplasm in the control HepG2.2.15 cells. After 2 days CSA administration of the expression of HBcAg and HBsAg in cytoplasm significantly decreased and the signals of HBcAg in nucleus increased , whereas the HBcAg was still mainly expressed in nucleus in about 1/4 of the cells. Cell apoptosis was observed in about 30% of the cells.

CONCLUSIONCSA improves the nuclear entry of core protein. The increase of HBcAg in nucleus was likely to be related with it's phosphorylation and cell aging or apoptosis.

Active Transport, Cell Nucleus ; Apoptosis ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cyclosporine ; pharmacology ; Cytoplasm ; metabolism ; Hepatitis B Core Antigens ; analysis ; metabolism ; Hepatitis B Surface Antigens ; analysis ; Humans ; In Situ Nick-End Labeling ; Phosphorylation

OBJECTIVETo gain some insights into the critical events relating to HBV transcriptional regulation by comparing HBV replicative characteristics in different cell lines.

METHODSHepatic cell lines QSG-7701 and HepG2 were transfected with plasmid PUC18-HBV 1.2 by standard calcium phosphate precipitation method, and 1.0 microg pSEAP2-control vector was included in the transfection procedures to serve as an internal control monitoring the transfection efficiency. Hepatitis B surface antigen (HBsAg) in the medium was detected by ELISA method and HBV DNA was quantitated using fluorescent quantitative PCR. The intracellular HBsAg and HBcAg were detected with immunofluorescent staining. The gene expression profiles of QSG-7701 and HepG2 were compared using oligonucleotide microarray; partial differentially expressed genes were verified with quantitative RT-PCR.

RESULTSIn the medium of the cultured HepG2 cells, HBsAg and HBV DNA could be detected 6 days after the transfection, whereas in QSG-7701 cells, the HBsAg and HBV DNA could be detected for 2 weeks. The HBV DNA in the culture medium of QSG-7701 was about 50 times more than that of the HepG2 cells which were kept in 1 x 10(7)copies/ml(-3) x 10(7)copies/ml for 0 to 10 days after the transfection. On the 4th day after the transfection, 20% to 30% of the QSG-7701 cells were positive with HBsAg and HBcAg immunofluorescent staining. The gene microarray analysis showed that most transcription factors involved with HBV life cycle in QSG-7701 and HepG2 cells had similar levels, whereas some factors involved with HBV transcriptional regulation and core particle disassembly, such as interleukin-6 (R=5.1340), retinoid X receptor, alpha (R=5.1268), hepatic leukemia factor (R=3.2538), serine protease PRRS23 (R=2.8356), hepatitis B virus x interacting protein (R=0.4939), serine protease inhibitor Kazal type 1 (R=0.0740) and matrix metalloproteinase 3 (negative in QSG-7701) were all differentially expressed by HepG2 cells.

CONCLUSIONDifferent than HepG2 cells, the QSG-7701 cells could support a high level and relatively stable HBV replication after HBV DNA transient transfection. The HBV core particles were probably recycled in the QSG-7701 cells. The differential gene expressions between QSG-7701 and HepG2 might explain the mechanism of the different HBV replication patterns. Hepatic cell line QSG-7701 might serve as a useful tool for HBV transcriptional regulation research.

Cell Line ; Gene Expression Regulation, Viral ; Genetic Vectors ; Genome, Viral ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Humans ; Virus Replication

OBJECTIVETo explore the role of stem cell mobilization on regeneration of partially grafted livers.

METHODSRats models with cross-sex 50% PLTx (partial liver transplantation) were established. The rats were divided into three groups: PLTx, WLTx (whole liver transplantation) and sham operation groups. Bone marrow and liver samples were collected on days 1, 3, 5, 7 postoperatively (each n = 6). The quantitative variations of the cells with stem cell markers in the bone marrow, including beta2m-/Thy-1.1+, CD45+/CD34+, Flt2/3+ and c-kit+ markers, were detected using flow cytometry. Sry gene positive cells in donor livers were detected by fluorescent in situ hybridization (FISH), and the expressions of CD34, c-kit and Thy-1.1 were detected by immunohistochemistry technique.

RESULTSCompared with the WLTx and sham operation groups, beta2m-/Thy-1.1+, CD45+/CD34+ cells in bone marrows in the PLTx group increased on the first postoperative day and decreased on the following days. The CD34, c-kit and Thy-1.1 positive cells detected in portal tract areas peaked during the 3-5 postoperative days. CD34+/CD45+ positive cells could be detected. The expressions of CD34, c-kit and Thy-1.1 positive cells were rare in the WLTx and sham operation groups. Sry+ cells could be detected in portal tract areas and few Sry+/CD34+ and Sry+/Thy-1.1+cells were detected.

CONCLUSIONIn the PLTx group, the stem cells in the bone marrow were mobilized and stem cells in the liver were activated.

Animals ; Antigens, CD34 ; immunology ; Bone Marrow Cells ; cytology ; immunology ; Female ; Hematopoietic Stem Cell Mobilization ; Leukocyte Common Antigens ; immunology ; Liver Transplantation ; methods ; Male ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; immunology ; Thy-1 Antigens ; immunology

OBJECTIVESThe hepatitis B virus core protein has been found in nuclei, cytoplasm, or both of hepatocytes transfected with HBV DNA. It is still unclear whether intact core particles could pass through nuclear pores and what could be the mechanism regulating the subcellular localization of the core protein. This study on the distribution of core protein in hepatocytes and its translocation has a potential advantage to learn more about the HBV life cycle.

METHODSDimethyl sulphoxide (DMSO, 2%), which effects hepatic differentiation, and/or 1 micro mol/L heteroaryldihydropyrimidine Bay41-4109, which interferes with the assembly of core particles, were added into HepG2.2.15 cell culture system for 4 days. The hepatitis B virus core antigen (HBcAg) and hepatitis B virus surface antigen (HBsAg) were stained with fluorescent immunocytochemistry and then observed under a confocal microscope. HBcAg in cytoplasm and nuclei were respectively extracted and analyzed using Western blot. HBV covalently closed circular DNA (cccDNA) was detected by using selective PCR method.

RESULTSThe HBcAg was mostly expressed in the cytoplasm and weak signals of cccDNA were detected in the control HepG2.2.15 cells. After DMSO treatment, the expression of HBcAg in cytoplasm was increased about 2.5-fold; the expression of HBcAg and cccDNA in nuclei also increased. With the use of Bay41-4109, the signal of HBcAg in cytoplasm decreased 2/3, but it increased in the nuclei, and cccDNA decreased in the nuclei. When the HepG2.2.15 cells were treated both with DMSO and Bay41-4109, cord-liked distribution of HBsAg was observed in the cytoplasm. HBcAg in cytoplasm was decreased 1/2 but the HBcAg in the nuclei increased about 5-fold, whereas the cccDNA was almost negative.

CONCLUSIONIn HepG2.2.15 cells, the core protein is mainly assembled as a formation of core particles in the cytoplasm and they are blocked by the nuclear membrane. Bay41-4109 interferes with the assembly of core particles and the dissociated core proteins are able to enter the nuclei. DMSO promotes the nuclear entry of core protein/core particles and facilitates the formation of cccDNA.

Chromosome Positioning ; Dimethyl Sulfoxide ; pharmacology ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; Neoplasm Metastasis ; Pyridines ; pharmacology ; Pyrimidines ; pharmacology ; Viral Core Proteins ; metabolism ; Virus Assembly

BACKGROUNDOligosaccharides in human milk may protect infants by improving the intestinal micro-flora and fermentation. This study was to investigate effects of infant formula milk consisting of galacto-oligosaccharide (GOS) on intestinal microbial populations and the fermentation characteristics in term infants in comparison with that of human milk.

METHODSThe test formula (Frisolac H, Friesland, Netherland) was supplemented with GOS at a concentration of 0.24 g/dl. Human milk and another formula without oligosaccharides (Frisolac H, Friesland, Netherland) were used as positive and negative control respectively. Growth, stool characteristics, and side effects of the recruited infants were recorded after 3 and 6 months' follow-up, and the fecal species were collected for the analysis of intestinal micro-flora, short chain fatty acid (SCFA) and pH.

RESULTSAt the end of 3- and 6-month feeding period, intestinal Bifidobacteria and Lactobacilli were significantly increased in infants fed with GOS supplemented formula and human milk when compared with infants fed with negative control formula; however, there was no statistically significant difference between GOS supplemented formula and human milk groups. Stool characteristics were influenced by the supplement and main fecal SCFA (acetic), and stool frequency were significantly increased in infants fed with GOS supplemented formula and human milk, while the fecal pH was significantly decreased as compared with that of negative control (P < 0.05). Supplementation had no influence on incidence of side effects (including crying, regurgitation and vomiting).

CONCLUSIONSSupplementing infant formula with GOS at a concentration of 0.24 g/dl stimulates the growth of Bifidobacteria and Lactobacilli in the intestine and stool characteristics are similar to in term infants fed with human milk.

Bifidobacterium ; isolation & purification ; Dietary Supplements ; Galactose ; administration & dosage ; Humans ; Infant ; Infant Formula ; Infant, Newborn ; Intestines ; microbiology ; Lactobacillus ; isolation & purification ; Oligosaccharides ; administration & dosage

Animals ; Cattle ; Child Development ; Dietary Supplements ; Fatty Acids, Unsaturated ; administration & dosage ; blood ; Growth ; Humans ; Infant ; Infant Nutritional Physiological Phenomena ; Milk

OBJECTIVETo investigate the change in dendritic cells (DCs) in children with chronic immune thrombocytopenia (cITP) and the effect of glucocorticoid on DCs in children with cITP.

METHODSFifteen children with cITP and 20 healthy controls were included in the study. Flow cytometry was used to measure the DC subsets count in the 15 children with cITP before and after glucocorticoid treatment as well as the corresponding values in the 20 healthy controls. The DCs derived from peripheral blood monocytes in children with cITP were cultured in vitro and collected, and their immunophenotypes were determined by flow cytometry.

RESULTSBefore glucocorticoid treatment, the children with cITP showed no notable change in the absolute count of myeloid DCs (mDCs) but showed decreased absolute count of plasmacytoid DCs (pDCs) and increased mDC/pDC ratio compared with the healthy controls (P<0.05). After glucocorticoid treatment, the children with cITP demonstrated increased absolute count of pDCs and decreased absolute count of mDCs and mDC/pDC ratio compared with before treatment (P<0.05). Before glucocorticoid treatment, the children with cITP had significantly higher positive rates of HLA-DR, CD80, CD83 and CD86 on peripheral blood DCs than the healthy controls (P<0.01). All the positive rates were significantly decreased after glucocorticoid treatment (P<0.01), so that there was no significant difference from the healthy controls (P>0.05).

CONCLUSIONSDisproportion and functional disturbance of DC subsets is associated with the pathogenesis of cITP in children. Glucocorticoid can strengthen the immunosuppression of DCs in children with cITP, which may contribute to the effectiveness of glucocorticoid as a treatment.

Adolescent ; Child ; Child, Preschool ; Chronic Disease ; Dendritic Cells ; drug effects ; immunology ; Female ; Glucocorticoids ; pharmacology ; Humans ; Immunophenotyping ; Male ; Thrombocytopenia ; drug therapy ; immunology