Objective To find out the importance of human bocavirus (HBoV) as an infectious agent for population in Beijing, China. Seroprevalence study was conducted by using expressed recombinant major capsid VP2 protein as an antigen.Methods Serum specimens collected from infants and children who visited the Children's Hospital Affiliated to the Capital Institute of Pediatrics for health check-up and adults visiting the Xuanwu Hospital, Beijing for diseases other than respiratory infections from April 1996 to March 1997 were used for the investigation. The major capsid protein VP2 from HBoV was expressed in E. coli strain BL21 (DE3) with the transformed PET30b vector inserted with full-length VP2 gene of HBoV and the specific antigenicity of this expressed protein was validated by previous study. Western blotting was used to detect specific IgG antibody against HBoV in collected serum specimens diluted to 1:200. Mock expressed protein was E. coli cells strain BL21 (DE3) with the transformed PET30b vector without insert. Anti-His monoclonal antibody and rabbit anti-HBoV VP2 polypeptides hyper-immune serum were used as positive control for antibody detection.Results Out of 677 serum specimens tested, 400 (59.1% ) were positive for HBoV by Western blotting. About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV antibodies, and antibody positive rates were decreased in age groups of 1 and 2 months (41.4% and 31.3%, respectively) then increased in the following ages from 6 months to 7 years old ( from 45.6% to 69.7% ). The antibody positive rates were maintained at a relatively constant level ( about 70% ) in the age groups from 7 years to 40 years of age and became lower ( 61.8% - 62. 8% ) in those over 50 years.Conclusions The high seroprevalence of antibody against recombinant HBoV VP2 protein and early age antibody acquisition indicate that HBoV has been circulating in population of Beijing, China as early as in 1996 and most of children had been exposed to HBoV by the age of 7 years. Infants under the age of 6 months were susceptible to this virus.

Background Our previous study determined that expressions of matrix metalloproteinases (MMPs) and tissue matrix metalloproteinase inhibitors (TIMPs)change in the conjuntival fibroblasts of conjunctivochalasis in vitro.To seek a suitable drug is very important in the prevention and treatment of conjunctivochalasis.Objective This study was to explore the effect of qijingmingmu soup on the expressions of MMPs and TIMPs in human conjunctival fibroblasts of conjunctivochalasis.Methods Twenty-four SD rats were randomized into two groups.Qijingmingmu soup was administration gastrically for consecutive 3 days,and normal saline solution was given in the same way in the control group.The blood was collected from aortaventralis and drug serum was prepared.Human conjunctival samples were obtained during the surgery of conjunctivochalasis relaxation and cultured in the DMEM containing 10％ fetal bovine serum,20％,15％,10％,5％ of drug serum and 8 ml/L epidermal growth factor(EGF) was added into the medium respectively.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expressions of MMP1,MMP3,TIMP1 and TIMP3in conjunctival fibroblasts.Results Cultured cells grew well with the fusiform shape and showed the positive response for vimentin.The expression value of MMP1 (A value)in the cells was declined after administration of qijingmingmu soup.A significant difference was found in the expression of MMP1 among the control group,20％,15％,10％,5％ drug serum groups and EGF group(F=466.664,P＜0.05),and that in the 10％ ([9.92±0.14] mg/L) and 20％ ([11.87 ±0.11] mg/L) drug serum groups was significantly lowed in comparison with the control group([16.31±0.10] mg/L)(t=99.974,87.394,P＜0.05).The expression value of the MMP3in the cells in the various drug serum groups,EGF group and the control group was significantly different(F=158.168,P＜0.05),with a lower value in the 20％ drug serum group compared with the control group ([3.50±0.03] mg/L vs.[4.44 ± 0.11] mg/L) (t =21.991,P ＜ 0.05).Also,the significantly different expressing value of TIMP1 was seen among all the groups (F=183.508,P＜0.05),and expressing value of TIMP1 in the 15％ drug serum group was(1.88±0.06)mg/L,which was lower than(3.20±0.32) mg/L of the control group(t=10.353,P＜0.05).Furthermore,the expressing value of the TIMP3 in the cells was significantly different among the various groups(F=54.503,P＜0.05),and that of the 20％ drug serum group was (1.743±0.065)mg/L and it was significantly higher than (1.54 ± 0.05) mg/L of the control group (t =5.046,P =0.004).However,the expressing value of TIMP3of the 15％,10％ and 5％ drug serum groups was lower than that of the control group,respectively all at(P＜0.05).Conclusions Qijingmingmu soup drug serum at the concentration of 20％ can down-regulate the expressions of MMP1,MMP3,TIMP1 and up-regulate the expression of TIMP3 in human conjunctivochalasis bulbar conjunctival fibroblastsin vitro,which probably plays preventive and therapeutic effects on conjunctivochalasis.

Objective To analyze the clinical features,diagnosis and treatment of 2 pedigrees (5 patients) with familial hemangioblastoma. Methods The detailed clinical and imaging data of 2 pedigrees (5 patients) with familial hemangioblastoma,admitted to our hospital from September 2005 to May 2010, were retrospectively analyzed; their diagnosis and treatment were concluded. Results Among the 3 patients of the first pedigree,2 patients displayed cystic and solid tumor and 1 cystic tumor under cranial MRI; both 2 patients of the second pedigree displayed cystic solids.All the 5 patients had mutations and could be diagnosed as having yon Hippel-Lindau (VHL).No patients were combined with other parts of the lesion; after microscopic total/sub-total resection, no patients existed postoperative occurrence and no patient died. Conclusion MRI is the most important detective method in the diagnosis of patients with familiar hemangioblastomas; microsurgery is still the most important therapy method to familiar hemangioblastomas.

OBJECTIVETo isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.

METHODSWe detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.

RESULTSThe isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.

CONCLUSIONCD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

Adult Stem Cells ; cytology ; Biomarkers ; metabolism ; Cell Separation ; methods ; Cell Shape ; Cell Size ; Coculture Techniques ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; metabolism ; Humans ; Immunohistochemistry ; Male ; Receptors, G-Protein-Coupled ; metabolism ; Sertoli Cells ; Spermatogonia ; cytology ; Testis ; metabolism ; Thy-1 Antigens ; isolation & purification ; metabolism ; Ubiquitin Thiolesterase ; metabolism

The aim of this study was to obtain the capsid protein VP2 of human bocavirus (HBoV) identified in Beijing recently and construct virus-like particles (VLPs) in insect cells for further study of this virus. The full-length VP2 gene of HBoV from BJ3722 was inserted into the baculovirus expression transfer vector (pFastBac 1) to obtain the recombinant Bacmid, and generation of recombinant baculoviruses was followed by transfection of the recombinant Bacmid into insect cells. Then the recombinant VP2 protein was recognized by SDS-PAGE using Coomassie-blue staining and Western blot using hyper-immune serum against VP2 of HBoV from rabbit. The recombinant baculoviruses were harvested and amplified to gain large amounts of viruses with high titers to infect insect cells at a multiplicity of infection (MOI) of 0. 5. After 7-10 days or 4-5 days of the infection, the supernatants of culture or the cell lysates treated with lysing solution were harvested, and ultracentrifuged twice through 40% sucrose cushion to obtain purified VLPs, which were followed by Western blot and IFA for VLPs' composition and specificity analysis, by electron microscopy for VLPs' morphologic structure. The recombinant VP2 protein with molecular weight of approximately 61 kD expressed in recombinant baculoviruses was recognized by SDS-PAGE using Coomassie-blue staining and Western blot. The presence of VP2 on VLPs was demonstrated by Western blot and IFA from samples collected during the purification of VLPs from the supernatants of culture or the cell lysates, and the expression of VP2 in insect cells led to the formation of VLPs which formed the typical icosahedral appearance of parvoviruses with a diameter of approximately 20 nm. In conclusion, the recombinant baculoviruses were constructed, the HBoV VP2 protein was expressed in insect cells with high specific antigenicity and VLPs was formed successfully.
Animals ; Blotting, Western ; Capsid Proteins ; genetics ; metabolism ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Fluorescent Antibody Technique, Indirect ; Human bocavirus ; genetics ; metabolism ; Microscopy, Electron, Transmission ; Polymerase Chain Reaction ; Spodoptera ; Virion ; genetics ; metabolism ; ultrastructure

OBJECTIVEAn outbreak of acute respiratory infections in children occurred in Beijing from November to December, 2002. To investigate the etiological agents of affected children who were in day care centers and primary schools.

METHODSThroat swab specimens were collected from one primary school children with acute respiratory infections visiting one outpatient department. After centrifuging, supernatant from the specimens were inoculated into MDCK and Hep-2 cells for virus isolation and pallets for viral antigen detection and using indirect immunofluorescent assay on common respiratory viruses. Nested polymerase chain reaction was used at the same time for detection of respiratory viruses and Mycoplasma pneumoniae (Mp).

RESULTSA total number of 80 specimens were collected during the outbreak. Among them influenza B virus were detected from 18 specimens, with a positive rate of 22.5% (18/80) while Mp were detected from 13 specimens, with a positive rate of 16.3% (13/80). Influenza A3 were also detected from 2 patients (2.5%, 2/80). However, influenza A1, RSV, adenovirus and parainfluenza viruses were not found from these specimens. Influenza B virus and Mp were detected simultaneity in two specimens and influenza A3 virus and Mp were detected in one specimen.

CONCLUSIONThe outbreak of acute respiratory infection in children during the period of investigation was caused by both influenza B virus and Mp.

Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Disease Outbreaks ; Female ; Humans ; Infant ; Influenza B virus ; isolation & purification ; Influenza, Human ; epidemiology ; Male ; Mycoplasma Infections ; epidemiology ; Mycoplasma pneumoniae ; isolation & purification ; Respiratory Tract Infections ; epidemiology ; microbiology

OBJECTIVETo develop a rapid, sensitive and specific method in identifying and typing on adenovirus from clinical specimens.

METHODSPrimers were designed using hexon gene of adenovirus as target. One primer pair was designed as universal primers for amplifying a 1278 bp gene fragment located at the hexon gene of adenovirus from all types. Four primer pairs located within the region of this 1278 bp were specifically designed for amplifying types 3, 7, 11 and 21 of adenoviruses, which were used for multiplex nest-PCR in a single tube. The products from this multiplex nest-PCR were 502 bp (for type 3), 311 bp (for type 7), 880 bp (for type 11) and 237 bp (for type 21), respectively. Type of the adenovirus tested could then be determined after agarose electrophoresis analysis of the PCR products.

RESULTSPCR products with predicted sizes were visualized in the agarose gel for prototype strains of adenovirus types 3, 7, 11 and 21, but not for other respiratory viruses, indicating that the technique was specific without cross reaction with other viruses. Out of the 118 clinical specimens which had been proved to be adenovirus positive by tissue culture and/or immunofluerescence assay, 76 belonged to adenovirus type 3 (76/118, 64.4%), 37 to adenovirus type 7 (37/118, 31.4%), 3 to adenovirus type 11 (3/118, 2.5%) but no adenovirus type 21 was detected. Two of the 118 positive specimens which were positive by both tissue culture and immunofluerescence could not be identified, suggesting that these 2 strains (1.7%) were with the types other than types 3, 7, 11 and 21. Out of the 33 specimens which were negative by both tissue culture and immunofluerescence, 3 showed positive by this multiplex PCR (2 of type 3 and 1 of type 7), suggesting this method was more sensitive than tissue culture and immunofluerescence.

CONCLUSIONThis multiplex nest-PCR method had the benefit of rapid,sensitive and specific nature so could be used for identifying types of adenoviruses in the clinical specimens.

Adenoviridae ; classification ; isolation & purification ; Adenovirus Infections, Human ; virology ; DNA Primers ; DNA, Viral ; analysis ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; Sequence Analysis, DNA

OBJECTIVETo understand the relationship between human rhinovirus (HRV) and acute respiratory infections in infants and young children in Beijing.

METHODSThroat swab/nasopharyngeal aspirates were collected from 3292 infants and young children with acute respiratory tract infections in Beijing from November 2002 to November 2006. Primers derived from the highly conserved 5'-noncoding region of human rhinovirus were used to detect HRV from clinical specimens by nested RT-PCR for which the sensitivity and specificity had been determined previously.

RESULTSOut of these 3292 specimens, 507 were (15.4%, 507/3292) HRV positive with RT-PCR method. HRV were detected from 220 out of 1315 outpatients and 287 out of 1977 inpatients with positive rates as 16.7% and 14.5% respectively. HRV was detected from 50.0% (8/16) of the patients with pharyngitis. Among 280 specimens collected from patients with acute bronchitis, 43 (15.4%) were HRV positive, including 14 from 80 patients with wheezy bronchitis (17.5%). High positive rates were also found in specimens from patients with pneumonia (12.6%, 150/1189), bronchiolitis (16.0%, 42/262) and asthma (12.8%, 10/78). In 53 patients with initial diagnosis as hematic disease or other complicate respiratory infections, 14 were HRV (26.4%, 14/53) positive. As for the seasonal distribution, HRV were detected in most of the months during thie period of research. The highest positive rate of HRV in each year fell in September (32.6%), February (24.2%) of 2004, February of 2005 (35.3%) and March (31.3%) from 2003 to 2006, respectively. Among these HRV positive patients, 44.8% were under 1 year of age (227/507), 15.4% (78/507) were 1 to 2 years old and 12.4% (63/507) were 2 to 3 years old.

CONCLUSIONHRV was associated with acute upper respiratory infections and lower respiratory infections including bronchitis, pneumonia and bronchiolitis in pediatric patients. Patients with lower immunity such as those with hematic diseases, were more susceptible to be infected by HRV. HRV could be detected in all age groups in this study, but the positive rates were decreasing with the increase of patients' age. Infants under 1 year of age seemed to be more likely to get HRV infection.

Acute Disease ; Adolescent ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Picornaviridae Infections ; epidemiology ; virology ; Respiratory Tract Infections ; epidemiology ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; Rhinovirus ; classification ; genetics ; pathogenicity ; Seasons

OBJECTIVETo develop a rapid, sensitive and specific method for detection human rhinovirus (HRV) from clinical specimens.

METHODSPrimers derived from the highly conserved 5'noncoding region of human rhinovirus were used to develop a nested RT-PCR for detecting HRV. The sensitivity and specificity of the RT-PCR were determined using various RNA while DNA viruses were used as control. Seven hundred and seventy-one specimens collected from children with symptoms of acute respiratory infections from Nov. 2002 to Oct. 2003 were analyzed for HRV by RT-PCR as well as for other respiratory viruses through isolation of virus and indirect immunofluorescent assay.

RESULTSOnly the cDNA from HRV was positive by RT-PCR, indicating the nested RT-PCR was specific. With RT-PCR, HRV were detected in 148 out of 771 specimens (19.2%). As for HRV positive rates, it was found 53.3% in pharyngitis patients; 43.8% in laryngitis patients and 28.7% in bronchitis patients. In Sep. 2002 and from Aug. 2003 to Oct. 2003, HRV positive rates were high (21.6% - 32.6%), with Sep. 2003 in particular--32.6%. From Mar. 2003 to Jul. 2003, HRV positive rates maintained from 16.0% to 19.1%.

CONCLUSIONHRV was one of the important agents for acute respiratory infections in infants and young children in Beijing.

5' Untranslated Regions ; Acute Disease ; Child, Preschool ; Conserved Sequence ; DNA, Viral ; analysis ; Humans ; Infant ; RNA, Viral ; analysis ; Respiratory Tract Infections ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rhinovirus ; genetics ; isolation & purification ; Sensitivity and Specificity

OBJECTIVETo understand the relationship of parainfluenza virus (PIV) and acute respiratory infections in infants and young children in Beijing, occurred in recent years.

METHODS3141 throat swab/nasopharyngeal aspirate specimens were collected from infants and young children with acute respiratory tract infections in Beijing from Jan 2001 to Dec 2003. All of these 3141 specimens were inoculated into MDCK cells for influenza virus and PIV isolation, since PIV had been isolated in MDCK cells in this laboratory from preliminary studies. Out of 3141 specimens, 702 were inoculated into MDCK as well as Vero cells to compare the sensitivity on virus isolation of these cell lines by micro plate method. Growth of PIV in cell culture were identified by haemoagglutination test and indirect immunofluorescent assay.

RESULTSThe PIV positive cases in Vero cells were also positive in MDCK cells, indicating that the sensitivity for PIV isolation in MDCK was equal to Vero cells. Out of these 3141 specimens, 94 (3.0%) were PIV positive, including 35 (35/1191, 2.9%) of PIV1, 11 (11/1191, 0.9%) of PIV3 in upper respiratory tract infections; 15 (15/1634, 0.9%) of PIV1, 24 (24/1634, 1.5%) of PIV3 in lower respiratory tract infections; 3 (3/207, 1.4%) of PIV in asthma; 1 (1/38) of PIV in patients with fever; 5 (5/71) of PIV in others. Data indicated that among upper respiratory tract infections caused by PIV, PIV1 was more commonly seen than PIV3.

CONCLUSIONMDCK cells could be used for PIV isolation from clinical samples while PIV was one of the important pathogenic viruses causing acute respiratory tract infections in infants and young children in Beijing for the recente years.

Acute Disease ; Animals ; Cell Line ; Cercopithecus aethiops ; Child, Preschool ; China ; Humans ; Infant ; Parainfluenza Virus 1, Human ; isolation & purification ; pathogenicity ; Parainfluenza Virus 3, Human ; isolation & purification ; pathogenicity ; Paramyxoviridae Infections ; diagnosis ; Respiratory Tract Infections ; virology ; Sensitivity and Specificity ; Vero Cells ; Virus Cultivation ; methods