[Objective]To compare spontaneous correction of the unfused thoracic curves after selective anterior versus posterior fusion in Lenke5 adolescent idiopathic scoliosis(AIS). [Method]A total of 72 Lenke5 AIS patients were rescruited from May 1997 to October 2005.Out of them,40 received selective anterior fusion(group A) and 32 received selective posterior fusion(group B).All had a minimum of 2-year follow-up.[Result] No complication were found in both groups at the latest follow-up.The thoracic curve was corrected from 30? to 16? for group A,33? to 18? for group B.Both groups had a better spontaneous correction of the unfused thoracic curves.The correction rate had no significant difference between groups.However,the thoracic curve was increased in four patients(2 in each group;group C),which resulted in trunk imbalance.The thoracolumbar/lumbar thoracic(TL/L:T) Cobb's ratio averaged 1.09 in the four patients whereas 1.59 in other 68 patients(group D).The flexibility of the thoracic curve had significant difference in group C and D(34.2% vs 57.3%).[Conclusion]Both of the surgical treatments can get a better spontaneous correction of the unfused thoracic curves.It is important to evaluate the.thoracolumbar/lumbar–thoracic(TL/L:T) Cobb's ratio and the flexibility of the thoracic curve before selective fusion.

Objective:To purify a kind of deoxyribonuclease from earthworm Eisenia foetida (named earthworm DNase, EDNase) and study its characteristics. Methods: Acetone precipitation, ion-exchange chromatography, high performance liquid chromatography, SDS-PAGE, CapiUary electrophoresis isoelectric focusing and MALDI-TOP MS were used for the study. Results: This purified protocol improved 137-fold purification and 45.6 % recovery of enzyme activity. The molecular mass of EDNase was estimated to be 63 000. Mg2+ , Mn2+ and Ca2+ were strong inhibitors of EDNase, while Na+ slightly increasd the enzyme activity. The enzyme was completely stable in the pH range from 4. 4 to 5.2 and had a pH optimum of 4.8. The optimum temperature was 37℃ and the enzyme was stable up to 40 ℃. The pI of the enzyme was 6. 20. Km and Vmax for the enzyme were 1.52 g/L and 4. 89 mg/(mL ·min), respectively, with calf thymus DNA as substrate. The enzyme was able to degrade chromosemal DNA, linear λbacteriophage DNA as well as supereoiled plasmid DNA, but didn' t display any RNase activity. Conclusion: This kind of deoxyribonuclease possesses unique characteristics, which is different from the deoxyribonucleases which we have known before.

We developed vectors expressing two antigen of H5N1 influenza virus. Based on the human H5N1 avian influenza virus strain A/Anhui/1/2005 isolated in China, we amplified the matrix protein 2 (M2) and Hemagglutinin (HA) genes by PCR and subcloned them into pStar vector to construct two genes co-expressing recombinant DNA vaccine pStar-M2/HA. After transfection of 293 cells with the plasmid, we confirmed with indirect immunofluorescence assay (IFA) that M2 and HA genes cloned on plasmid pStar co-expressed successfully. Using Ad-Easy adenovirus vector system, by homologous recombination in bacteria and packaging in 293 cells, we constructed two recombinant adenoviruses, namely Ad-M2 and Ad-HA. After infection of 293 cells with the recombinant adenoviruses, we confirmed with IFA that M2 and HA genes cloned into adenoviruses expressed successfully. We then combined the recombinant DNA vaccine and adenoviral vector vaccines in immunization of BALB/c mice with a prime-boost regime. On day 0 and day 28, we immunized the mice with DNA vaccine and on day 14 and day 42, with recombinant adenovirus vaccines. We took blood samples before each injection and 14 days after the final injection. On day 56, we collected splenocytes from the mice. ELISA and hemagglutination inhibition (HI) assay showed that the vaccines successfully induced specific IgG antibodies against HA protein in serum of the immunized mice. ELISPOT confirmed that the vaccines successfully induced the special cellular immune response to M2 and HA protein of H5N1 influenza virus. The study on combined immunization with M2 and HA genes provided basis for development of novel influenza vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Genetic Vectors ; genetics ; Hemagglutinin Glycoproteins, Influenza Virus ; biosynthesis ; genetics ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Vaccination ; Vaccines, DNA ; immunology ; Viral Matrix Proteins ; biosynthesis ; genetics

In order to evaluate the response to vector-expressed M1 and HA genes of influenza virus in mice, we prepared recombinant plasmid pStar-M1/HA and recombinant adenovirus Ad-M1/HA containing both the full-length matrix protein 1(M1) and hemagglutinin (HA) genes of human H5N1 influenza virus strain A/Anhui/1/2005. We then combined the DNA vaccine and adenoviral vaccine in immunization of BALB/c mice with a prime-boost regime. We immunized the mice with DNA vaccine at day 0 and 28 and with recombinant adenoviral vaccines at day 14 and 42. We took blood samples before each injection and 14 days after the final injection for detection of humoral immune responses. At day 56, we sacrificed the mice and collected splenocytes for detection of cellular immune responses. ELISA and hemagglutination inhibition (HI) assay showed that specific IgG Abs against H5N1 influenza virus was induced in serum of the immunized mice. ELISPOT results confirmed that the specific cellular immune responses were successfully induced against the M1 and HA proteins of H5N1 influenza virus. This study provides new strategy for development of novel influenza vaccines.
Adenoviridae ; genetics ; metabolism ; Animals ; Antibodies, Viral ; blood ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Immunization ; Influenza A Virus, H5N1 Subtype ; immunology ; Influenza Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; Vaccines, DNA ; immunology ; Viral Matrix Proteins ; genetics ; immunology

Matrix protein 2(M2) is an integral tetrameric membrane protein of influenza A virus, which functions as ion channel. M2 sequence has shown remarkable conservation, so there has been growing interest in it as "universal" vaccine. In order to establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator, M2 gene was obtained by PCR amplification from the plasmid containing the segment 7 of influenza A virus strain A/PR/8/34 firstly. The PCR product was cloned into BamH I/Not I restriction site of pcDNA5/FRT/TO vector, and cotransfected with pOG44 which express Flp recombinase into Flp-In T-REx-293 cell. Integration of pcDNA5/FRT/TO-M2 into the cell genome at the Flp Recombination Target (FRT) site brought the SV40 promoter and the initiation codon in frame with the hygromycin resistance gene. Thus, stable cell lines were selected for hygromycin resistance. The expression of M2 protein from hygromycin-resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay (IFA). 16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator. The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.
Animals ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; Influenza A virus ; genetics ; metabolism ; Influenza Vaccines ; genetics ; Operator Regions, Genetic ; Recombinant Proteins ; biosynthesis ; genetics ; Tetracycline ; pharmacology ; Transfection ; Viral Matrix Proteins ; biosynthesis ; genetics

Objective To compare the immune effects induced with universal influenza A vaccine based on branched M2e polypeptides by two immunization routes in mice.Methods The M2e monomer and (M2e)4-tuftsin were synthesized using chemical method.One group of mice were immunized intramuscularly (i.m.) with 10 μg of (M2e)4-tuftsin plus aluminum adjuvant.And the other group of mice were intranasally immunized with the same amount of polypeptides plus JY adjuvant.Then the immune effects of different immune pathways were observed.Results Intramuscular route induced higher level of M2 specific antibody than intranasal route.Intramuscular injection of (M2e)4-tuftsin was the most effective in stimulating T cell responses.Both intramuscular and intranasal vaccinations (M2e) 4-tuftsin had protective effect,but intramuscular vaccination could provide the most efficient protective immunity.Conclusion The data indicated that the intramuscular vaccination could be the better way for immunization than intranasal vaccination.

Objective To construct the recombinant baculovirus co-expressing M1 and]NA genes of influenza H1N1 virus.Methods The M1 and NA genes of influenza virus (A/PR8/8/34) were amplified by PCR and then inserted into the pFastBacdual (pFBD)donor plasmid to construct the recombinant pFBDM1-NA.pFBD-M1-NA was transformed into DH10Bac E.coli competent cells containing bacmid vector to construct the recombinant shuttle vector rBacmid-M1-NA.Sf9 cells were transfected with rBacmid-M1-NA and then the recombinant baculovirus rBac-M1-NA was obtained.The virus titer was tested by plaque formation,M1 and NA genes in genome were identified by PCR,and the expression of M1 and NA gene by IFA.Results PCR result proved that pFBD-M1-NA and rBacmid-M1-NA were constructed correctly.The recombinant baculovirus rBac-M1-NA titer was 1 × 107pfu/ml.IFA analysis showed that M1 and NA genes were expressed in sf9 cells infected with rBac-M1-NA.Conclusion The recombinant baculovirus coexpressing M1 gene and NA gene of infuenza H1 N1 virus was successfully established.

Objective:To evaluate the effectiveness of three laboratory routine disinfection methods on influenza virus inactivation.Methods:The physical method of ultraviolet irradiation and two chemical methods of ethanol and "84 disinfectant" were used to inactivate the influenza virus respectively. After disinfecting the virus samples at different times or concentrations, MDCK cells were inoculated and the inactivation effect of influenza virus was identified by observing cytopathic effect (CPE).Results:When the influenza virus was irradiated by ultraviolet light for ≥1 min, treated with 75% ethanol for 0.25-32 min, the effective chlorine concentration of "84 disinfectant" was ≥400 mg/L, and the influenza virus was disinfected at room temperature for ≥1 min, no virus infectivity could be detected under the three conditions.Conclusions:The condition of inactivating influenza virus by routine disinfection methods in laboratory was explored, which provided reference data for establishing the protection specifications and the emergency treatment plans for influenza virus laboratories.

BACKGROUNDDiversity of orthopedic infections with various local environments affects the pattern and prevalence of pathogens. It is not well-characterized whether different pathogens have different propensity to cause different types of orthopedic infections. We aimed to investigate the frequency of different pathogens derived from orthopedic infections, and determine the relationship between the prevalence of clinical isolates and the type of orthopedic implants, especially focusing on staphylococci.

METHODSFrom January 2006 to December 2011, orthopedic infections were identified retrospectively from clinical microbiology laboratory and orthopedic medical records. The sources of orthopedic infections were divided into two main groups: those associated with implants and those not associated with implants. Implants-associated infections were further subdivided into five subgroups: arthroplasty, internal fixation, external fixation, internal and external fixation, and others. We analyzed microbiological spectrum in different groups and subgroups. Antibiotic susceptibility of staphylococci was analyzed.

RESULTSOnly coagulase-negative staphylococci (CoNS) was significantly more likely to be associated with implants-associated infections (P = 0.029). The overall pathogens prevalence of arthroplasty was significantly different from other subgroups (P < 0.05). 65% isolates from external fixation was Gram-negative bacteria. Some percentage (55%) of S. aureus and (83%) CoNS were resistant to methicillin. No resistance to glycopeptide was seen in all of staphylococci.

CONCLUSIONSStaphylococcus aureus was the most frequent isolates in orthopedic infections but was not associated with the presence or absence of implants. Only CoNS was implants-associated, especially for arthroplasty infection. Cefazolin alone is not enough for orthopedic surgery prophylaxis in settings with a high prevalence of methicillin-resistant staphylococci.