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

To understand the effectiveness of prokaryotic expression of fusion protein (F) of human metapneumovirus (hMPV) and its application as antigen, F proteins from different genotypes of hMPV were expressed in prokaryotic expression system and purified by Ni-NTA affinity chromatography column. According to the hydrophobicity, antigen index and surface probability of F protein, the subunit 1 (F1) region of F protein was generated and expressed in E. Coil. BL21(DE3). The 6-His-F1 proteins with molecular weight of approximately 37 kD generated from hMPV of two genotypes were expressed efficiently mainly in inclusion body. The antigenicity and specificity of the expressed proteins were tested and confirmed by Western Blot using polyclonal antibody against hMPV and one serum specimen from a patient with confirmed hMPV acute infection,and polyclonal antibodies against human respiratory syncytial virus and parainfluenza virus 2 and 3. The results of preliminary use of the expressed proteins for detecting antibodies against hMPV in 457 serum specimens collected from different age groups in Beijing indicated that 66%-67% of sera in all age groups were positive. The positive rate of antibodies declined in children in age groups from birth to 2-year-old and then rose along with the increase in age, in which the lowest was in age group from 1 to 2-year-old and the highest in newborn and people older than 60 years. The data indicated the existence of maternal transferred antibodies against hMPV in infants and the risk of hMPV infections in children younger than 2 years old.
Adult ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Escherichia coli ; genetics ; Gene Expression ; Humans ; Immunoglobulin G ; blood ; immunology ; Infant ; Infant, Newborn ; Metapneumovirus ; genetics ; Middle Aged ; Plasmids ; genetics ; Protein Engineering ; Protein Subunits ; genetics ; immunology ; Viral Fusion Proteins ; genetics ; immunology

To characterize the genomic sequence and arrangement of WU polyomavirus (WU virus) identified in clinical specimens collected from children with acute respiratory infections in Beijing, China, the sequences of capsid proteins VP1, VP2, and the large tumor antigen (LTAg), as well as the 5'-terminal sequence of WU virus, were amplified from the clinical specimen with ID number of BJF5276 which was determined as WU virus positive by PCR amplification. The PCR amplicons were sequenced, and genomic sequence analysis was performed by using the software DNAStar. In addition, VP2 coding-region sequences were amplified from other 21 clinical specimens identified as WU virus positive to investigate the gene diversity of WU virus. The genomic sequence of WU virus BJF5276 with accession number of HQ218321 in GenBank was 5,229 base pairs in length with 3 major coding domain sequences (CDS) sited on one strand coding for capsid proteins VP2, VP3 and VP1, and two CDS sited on the complementary strand coding for small tumor antigen (STAg) and LTAg; These 22 VP2 CDS sequences including 5 sequences submitted to GenBank were compared with 64 corresponding sequences downloaded from GenBank by MegAlign of DNAStar software, indicated that these sequences coming from children in Beijing shared high homology (over 98.8%) with those from GenBank. Phylogenetic analysis of these VP2 CDS by using Neighbor-joining (NJ) analyses with 2,000 bootstraps (Mega 4.0) showed that 20 sequences out of 22 belonged to clade Ia, and other 2 of them belonged to clade III, including 1 clustered in IIIa and 1 in a novel cluster proposed as IIIc. In conclusion, the genomic sequence of WU polyomavirus detected from clinical specimens from children in Beijing is closely related to other WU polyomaviruses in the feature of genomic coding region arrangement. Overall variation of VP2 CDS was very low, and there were different clades circulating in Beijing with a dominant clade Ia, which is different from dominated Ib circulating in other parts of the world reported previously, and a novel clade IIIc was proposed.
Acute Disease ; Child, Preschool ; China ; Female ; Genome, Viral ; Humans ; Infant ; Male ; Molecular Sequence Data ; Phylogeny ; Polyomavirus ; classification ; genetics ; isolation & purification ; Respiratory Tract Infections ; virology ; Viral Proteins ; genetics

OBJECTIVETo obtain isolated human metapneumovirus (HMPV) strains from clinical specimens collected from infants and children in Beijing and to promote the investigation on this important respiratory pathogen.

METHODClinical specimens including throat swabs from outpatients and nasopharyngeal aspirates from hospitalized children were collected from infants and children visited the affiliated children's hospital for acute respiratory infections during May 2008 to April 2009. HMPV positive specimens identified by RT-PCR and/or direct immunofluorescent assay with monoclonal antibody against HMPV were inoculated to LLC-MK(2) cells and incubated at 37°C and 33°C, respectively. The replication of the virus in the cells was detected by direct immunofluorescent assay followed by RT-PCR. The genotypes of the isolated virus strains were identified by RT-PCR.

RESULTOut of 1092 clinical specimens, 81 were HMPV positive by RT-PCR, the positive rate was 7.4% (81/1092). Among these positive specimens, 33 were inoculated to LLC-MK(2) cells and the replication of HMPV was revealed by antigen detection and RT-PCR from 5 out of these 33 inoculates. These isolated viruses could be passed in LLC-MK(2) cells and were not cross-reacted with other common respiratory viruses, such as ADV, RSV and Parainfluenza viruses 1/2/3 by monoclonal antibodies against these viruses in direct immunofluorescent assay. The HMPV was more likely to be isolated from fresh specimens within 24 hours after the collection of specimens which were not frozen. Four of the 5 isolated strains were identified as genotype A and 1 as genotype B. Unlike other respiratory viruses, these isolated HMPV did not show specific CPE in cell culture and the replication of the virus was identified by antigen detection and RT-PCR.

CONCLUSIONHMPV of both genotypes were isolated from infants and children with acute respiratory infections in Beijing which will accelerate the investigation of this important virus.

Acute Disease ; Child ; China ; Genes, Viral ; genetics ; Genotype ; Humans ; Infant ; Metapneumovirus ; isolation & purification ; Respiratory Tract Infections ; virology

OBJECTIVETo explore clinical and laboratory factors associated aspirin response, and the association between gastrointestinal bleeding and aspirin response in aged patients.

METHODSA total of 136 patients aged 60 and over [mean age (74.9 ± 7.0) years] with ischemic heart disease and at high risk for ischemic heart disease were included. Arachidonic acid induced platelet aggregation (AA-Ag) was measured before and at 7(th) day after taking aspirin (100 mg/d). Patients were followed for 6 months and incidence of gastrointestinal bleeding was obtained.

RESULTSPost-treatment AA-Ag was significantly reduced compared to baseline (13.29% ± 5.52% vs. 73.20% ± 7.32%, P < 0.05). A heterogeneous distributed post-treatment AA-Ag was observed (range 0.42% to 30.50%). Post-treatment AA-Ag was positively correlated with baseline AA-Ag (r = 0.493, P < 0.01). The level of post-treatment AA-Ag was significantly higher in the fourth quartile group at baseline than in the others quartile groups at baseline. Patients aged 80 years and over had significantly lower post-treatment AA-Ag (10.25% ± 4.68%) compared with patients of 60 - 69 years (13.96% ± 5.20%) and of 70 - 79 years (13.73% ± 5.48%, all P < 0.01). The incidence of patients in the lowest quartile of post-treatment AA-Ag was significantly higher in patients ≥ 80 years (38.24%) than in patients of 60 - 69 years (11.1%) and of 70 - 79 years (24.0%). Multiple variable analysis revealed post-treatment AA-Ag was significantly influenced by baseline AA-Ag, ≥ 80 years old, diabetes mellitus and acute coronary syndrome. We observed 4 (2.9%) mild gastrointestinal bleeding during follow up. Post-treatment AA-Ag was in the lowest quartile in 3 patients with mild gastrointestinal bleeding.

CONCLUSIONSIncreased baseline platelet reactivity as well as diabetes mellitus and acute coronary syndrome are associated with low aspirin response in the aged patients. Aspirin response is significantly higher in very old patients.

Acute Coronary Syndrome ; Aged ; Aged, 80 and over ; Arachidonic Acid ; Aspirin ; adverse effects ; therapeutic use ; Coronary Artery Disease ; drug therapy ; Gastrointestinal Hemorrhage ; chemically induced ; Humans ; Middle Aged ; Myocardial Ischemia ; drug therapy ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; adverse effects ; therapeutic use ; Platelet Function Tests ; Ticlopidine

OBJECTIVETo 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.

METHODSSerum 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 visited the Xuanwu Hospital, Beijing for diseases other than respiratory infections from April 1996 to March 1997 were used for 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 blot 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.

RESULTSOut of 677 serum specimens tested, 400 (59.1%) were positive by Western blot. About 45.3% (34/75) of the newborns under 1 month of age had anti-HBoV antibodies, and antibody positive rates were lower in the age groups of 1 and 2 months (41.4% and 31.3%, respectively) and were higher in the following ages from 6 months to 7 years (from 45.6% to 69.7%). The antibody positive rates were at a relatively constant level (about 70%) in the age groups from 7 years to 40 years and became lower (61.8% - 62.8%) in groups of age over 50 years.

CONCLUSIONThe high seroprevalence against recombinant HBoV VP2 protein and early age antibody acquisition indicate that HBoV has been circulating in 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 infection with this virus.

Adolescent ; Adult ; Antibodies, Viral ; blood ; Blotting, Western ; Bocavirus ; immunology ; Capsid Proteins ; immunology ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Immunoglobulin G ; blood ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Parvoviridae Infections ; epidemiology ; immunology ; Prevalence ; Seroepidemiologic Studies ; Young Adult

OBJECTIVETo characterize the prevalence of influenza B virus infection in infants and young children in Beijing.

METHODSMDCK cell culture, indirect fluorescence assay (IFA) and hemagglutination inhibition (HI) assay were used to isolate and identify type B influenza viruses from clinical samples collected from outpatients and inpatients who visited the Affiliated Children's Hospital because of acute respiratory infections from Nov. 2000 to Jun. 2006.

RESULTSOut of 10,770 clinical samples collected during this surveillance period, 384 (3.57%, 384/10,770) were positive for influenza B viruses. Circulation of influenza B viruses was revealed in the later epidemic season of influenza viruses each year. The detection rate for influenza B virus was higher than 10% each year during the survey, except in the period from 2003--2004 which was 2.91%. The highest detecting rate was 23.69% of the specimens collected in Mar. 2006. During the period of this study, most of the influenza B virus were identified from children who visited the outpatient department of the Affiliated Children's Hospital. Among those outpatients who were positive for influenza B, 77.6% (264/340) were older than 3 years of age, whereas the inpatients positive for influenza B, 66.0% (29/44) were under 3 years of age. Coinfection of influenza B virus with other respiratory viruses was not common, only one of the influenza B virus positive specimen was found also positive for influenza A3. There was no significant difference in positive rate between influenza virus B and A3. A significantly higher positive rate of influenza B virus than that of influenza A3 virus was seen from Sep. 2005 to May 2006 (23.9% vs 1.1%). B/Yamagata/16/168 lineage viruses were dominant during 2000--2002, and B/Victoria/2/87 lineage viruses became dominant during 2002--2003. After 2003, co-circulation of Victoria and Yamagata lineages of influenza B viruses was identified with predominance of Yamagata lineage viruses, while Victoria lineage viruses predominated during the 2005--2006 epidemic season.

CONCLUSIONInfluenza B viruses were identified from February to May in every influenza season during this surveillance period of 2000--2006. Most of the positive specimens were those collected from outpatient department. Victoria and Yamagata lineages of influenza B viruses co-circulated in Beijing, China in recent years.

Adolescent ; Age Distribution ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Infant ; Influenza B virus ; classification ; isolation & purification ; Influenza, Human ; epidemiology ; virology ; Male ; Prevalence

OBJECTIVETo establish a rapid, sensitive and specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for detecting human respiratory syncytial virus (RSV) in respiratory samples collected from children with acute respiratory infections.

METHODAccording to the conserved matrix gene sequences of respiratory syncytial virus subtypes A and B downloaded from GenBank, primers were designed and RT-LAMP assay was developed to detect RNA of RSV sensitivity of the RT-LAMP method was evaluated by using ten-fold serially diluted in vitro-transcribed matrix RNA fragments from RSV A and RSV B, respectively. Specificity of the RT-LAMP method was tested through cross-reaction with other RNA and DNA viruses. Then 5 RSV strains isolated from clinical specimens using tissue cultures were tested by RT-LAMP assay. A total of 101 nasopharyngeal aspirates from hospitalized patients with acute respiratory infections which had been tested by direct immunofluorescence assay (DFA), including 40 positive for RSV and 61 negative for RSV, were tested by RT-LAMP assay and by RT-nested PCR.

RESULTSensitivity analysis indicated that this RT-LAMP method was able to detect 1 copy/µl of RSV A and RSV B RNA, no amplification was shown in RT-LAMP with DNA or cDNA from other viruses in 60 min, revealed that the RT-LAMP assay is highly specific. Five RSV isolates confirmed as 4 RSV A and 1 RSV B previously were detected by RT-LAMP method as positive in 30 min. For those 101 specimens tested, 37 were RSV positive determined by RT-LAMP assay, as well as 35 RSV positive by RT-nested PCR. The total coincidence rate of RT-LAMP assay with DFA and RT-nested PCR in detecting RSV is 95.0%, 94.1% with Kappa value 0.895 and 0.871, respectively.

CONCLUSIONA new, sensitive, accurate and rapid method, RT-LAMP assay for detecting human respiratory syncytial viruses from nasopharyngeal aspirates was developed, which should be helpful in rapid detection of RSV from respiratory tract samples of children.

Acute Disease ; Child ; Child, Preschool ; DNA Primers ; Humans ; Infant ; Molecular Diagnostic Techniques ; Nasopharynx ; virology ; Nucleic Acid Amplification Techniques ; RNA, Viral ; isolation & purification ; Respiratory Syncytial Virus Infections ; diagnosis ; Respiratory Syncytial Virus, Human ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity