Objective To determine the involvement of DNA demethylation in tumor necrosis factor-α(TNF-α)-induced matrix metalloproteinase 9 ( MMP9) expression in human epidermal keratinocytes. Methods Real-time RT-PCR, Western blot, and enzyme-linked immuno sorbent assay (ELISA) were performed to determine the mRNA and protein levels of MMP9 after human keratinocyte cell line (HaCaT) cells were treated with 10 ng/ ml TNF-α or 2. 5 μmol/ L DAC/ 300 nmol/ L TSA. Bisulfite sequencing PCR ( BSP) and Methylation-sensitive high-resolution melt analysis ( Ms-HRM) were used to detect significantly differentially demethylated CpG sites in the human MMP9 promoter region in cells exposed to TNF-α. Different sites methylation constructs of promoter-luciferase reporter gene were made to detect the influences of site-specific DNA demethylation on transcription activity of MMP9 promoter. Results Compared with PBS-treated control, TNF-α significantly increased the expression of MMP9 in HaCaT cells for indicated culture duration ( P < 0. 05 ). Real time PCR, Western blot, and ELISA analysis demonstrated that the mRNA and protein levels of MMP9 were increased initially, followed by a decline with prolonged incubation time. After TNF-α treatment, varied degrees of DNA demethylation occurred at 10 CpG sites in the promoter of MMP9, and the changes at the -36 bp site were statistically significant (P<0. 05). The demethylation at the -36 bp site greatly increased the transcription activity of MMP9. Conclusion TNF-α promotes MMP9 expression in HaCaT cells through inducing -36 bp site DNA demethylation on the promoter of MMP9.

Objective To evaluate the role of Restriction fragment length polymorphism (RFLP) analysis in detection of the fragment of GEF1α/a gene which are both located at ct and a mating type loci in identification of species, varieties, genotypes and mating types of Cryptococcus neoformans species complex(Cryptococcus neoformans and Cryptococcus gattii). Methods The GEF1α/a gene was selected from 20 genes which both located at α and a mating type loci for RFLP analysis, according to the requirements of sequence similarities and primer design in PCR-RFLP analysis. Primer pair was designed from the conserved regions of GEF1α/a genes of distinct genotypes and mating types of reference strains to amplify a fragment of GEF1α/a gene from Cryptococcus neoformans and Cryptococcns gattii strains tested. Sequence alignment,restriction maps analysis, endonucleases selection and electrophoresis stimulation were conducted by using DNAMAN and Vector NTI software. EeoT14 Ⅰ and Hap Ⅱ endonucleases were selected for RFLP analysis of the GEF1α/a fragments amplified from 125 isolates of Cryptococcns neoformans and Cryptococcus gattii. Results An approximate 1 300 bp fragment was amplified from total 82 Cryptococcus neoformans and 43 Cryptoceccus gattii isolates. However, negative PCR results were found in the reference strains of Cryptococcus laurentii, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krnsei,Candida glabrata, Trichosporon asahii, Aspergillus fumigatns and Aspergillus flavus. RFLP analysis successfully identified the species, varieties, genotypes and mating types of total 125 isolates of Cryptococcus neoformans and Cryptococcns gattii tested in this study. Condusion PCR-RFLP analysis of the GEF1α/a fragment has the potential value in identification of species, varieties, genotypes and mating types of Cryptococeus neoformans species complex simultaneously and rapidly, and may be a useful tool in molecular epidemiological analysis.

Objective To identify the AD hybrid strains and its hybrid types within Cryptococcus neoformans.Methods Difierent hybrid types of AD strains were analyzed by PCR 0f STE20 and MF genes within MAT locus and CIA4 and GPal genes out of MAT locus.The PCR-RFLP analysis of g6341 gene was also performed.Results The mating types of 18 AD strains were precisely identified by PCR of STE20 gene,whereas those of H strain were not identified.CL44 gene was better than the GPal gene in PCR identification of the AD hybrids.In the RFLP analysis of g6341 gene,AD strains were grouped into 2 distinct RFLP patterns based on the mating type on serotype A allele.The mating types of AD strains were not identified by the molecular analyses based on the CL44,GPal and g6341 genes.Conclusion It is necessary to use multi-locus analyses of genes within and out of the MAT locus in precise identification of the AD strains and their hybrid types of Cryptococcus neoformans.

Objective To assess the subgenotypes of pathogenic Cryptococcus gattii isolates from China and to elucidate the epidemiological links between these domestic isolates and those from other parts of the world. Methods DNA was extracted from 9 clinical isolates of Ctyptococcus gattii from China. The partially variable regions of the three unlinked loci, namely IGS1, PLB1 and GEF1, were amplified and sequenced, and the bioinformation at these loci was obtained from GenBank for multi-locus sequences alignment and phylogenetic analysis. Results Of these 9 clinical isolates, 8 were genotype VG Ⅰ and mating type α with the same sequences at the tested regions as the reference strain WM276, which was a representative isolate of an independent subgenotype; 1 was of genotype VG Ⅱ and mating type α, which was the first report in China, with the tested sequences consistent with those of the referrence strain R272. Sequencing and phylogenetic analysis of GEF1 gene, which was located at mating type locus, successfully identified the genotypes and mating types of all the Cryptococcus gattii isolates involved here. Conclusions Multi-locus sequence analysis shows that causative Cryptococcus gattii isolates of genotype VG Ⅰ in China carry similar sequences at the tested loci in IGS1, PLB1 and GEF1 genes, to a widely distributed subgenotype in the world, and the sequences of the first VG Ⅱ genotype isolate from China resemble the less virulent subgenotype VG Ⅱ b found in Vancouver islands.

Objective To investigate the molecular epidemiology of clinical cryptococcal isolates from China by analyzing the constituents and distributions of the varieties,genotypes and mating types (MAT)of them.Methods (1)PCR fingerprinting and PCR amplification were performed by using the minisatellite-specific core sequence of wild-type phage M13 as single primer.Genotypes of the 110 cryptococcal isolates from China were assigned by comparison with the reference strains of the 8 major molecular types loaded on gel.(2)Identification of the varieties and mating types was carried out by PCR using the specific primers of the varieties and mating types.Results Of the 110 clinical cryptococcal isolates,strains of Cryptococcus neoformans var.grubii with genetype VNⅠ and mating type MATα were the most representative ones(89.1%)followed by strains of C.neoformans var.gattii(8.2%)including isolates of genotype VG I,mating type MATα(7.3%)and genotype VGⅡ,mating type MATα(0.9%);AD hybrids with the genotype VNⅢ,mating type MAT-/α and genotype VN Ⅲ,mating type MATα/-(1.8%);and isolate of C.neoformans var.neoformans with the genotype VNⅣ and mating type MATa(0.9%).Conclusion Of the clinical isolates from China,all three varieties and AD hybrids are found.The vast majority(>99%) of strains possess the α allele in MAT locus and most of them are C.neoformans vat.grubii with the genotype VN I,which accord with the data of most studies of clinical molecular epidemiology in other geographic areas.However.no genotype of VNⅡ.VGⅢ and VGⅣ isolates are found in this study.

Objective To assess the outcome of percutaneous vertebroplasty for symptomatic vertebral hemangiomas. Methods Five cases with 7 symptomatic vertebral hemangiomas were treated with percutaneous vertebroplasty. Aggressive lesions were treated with absolute alcohol injection in addition. Patients were followed-up and clinical manifestations were observed and CT, MRI and X-ray plain film were compared between before and after vertebroplasty. Results Procedures were successful without complications. Most of the symptoms resolved within 24 hours after vertebraplasty. All patients were followed-up for 12～50 months and free of neurological deficits and symptoms. Imaging follow-up showed no vertebral collapse, nor recurrance of hemangiomas. Conclusion With effective long-term follow-up and quick elimination of symptoms, precutaneous vertebroplasty, added with absolute alcohol injection in aggressive cases, proves to be a safe and effective treatment for symptomatic vertebral hemangiomas.

Objective To determine in vitro drug susceptibility to five antifungal agents of clinical Cryptococcus neoformans strains isolated from different areas of China in recent ten years. Methods Eighty clinical isolates of Cryptococcus neoformans were isolated from Shanghai, Guangdong, Fujian, Beijing and some other areas of China from 1998 to 2007. The minimal inhibitory concentrations (MIC) of the isolates to five antifungal agents, including amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole, were determined using broth microdilution procedure (document M27-A2) recommended by the Clinical and Laboratory Standards Institute (CLSI). Kruskal-Wallis rank sum test was employed for the statistical analysis. Results The MIC50 of the Cryptococcus neoforrnans isolates tested for amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole were 0.5, 4, 2, 0.25 and ≤0.031 3 mg/L, respectively; and the MIC<,90> of the isolates tested for the above antifungal agents were 1, 8, 4, 0.5 and 0.062 5 mg/L, respectively. Among the tested isolates, 3 (3.8 %) were resistant to flucytosine, 4 (5.0 %) were resistant to itraconazole. All isolates were susceptible to amphotericin B and voriconazole. There was no significant difference in MIC of the strains isolated from any particular years to the five agents (χ2=0.500,2.687,2.211, 2.660,0.677,P>0.05). Conclusions The Cryptococcusneoformans isolates are highly susceptible to the five antifungal agents, while a few strains are resistant to flucytosine or itraconazole. The drug susceptibilities of the strains isolated from particular years are similar.

Objective To analyze the composition and distribution of pathogens from 1366 superficial candidiasis cases in Shanghai.Methods Candida species identification was carried out for 1366 adults or children with superficial candidiasis by using CHROMagar Candida plates,API20C AUX system,etc.Pal's agar,Xylose assimilation and the test for growth at 45 ℃ were utilized to differentiate Candida dubliniensis.Newly identified pathogenic Candida species including Candida orthopsilosis,Candida metapsilosis,Candida fermentati,Candida nivariensis and Candida bracarensis were differentiated by molecular biological methods.Finally,the composition and distribution of pathogens in superficial candidiasis cases were statistically analyzed.Results A total of 1366 Candida strains,included 2 Candida orthopsilosis strains and 4 Candida metapsilosis strains,were isolated from these cases.Among these isolates,Candida albicans predominated(79.0％),followed by Candida parapsilosis(9.5％),Candida tropicalis(2.9％)and Candida guilliermondii(1.9％).The composition of Candida species was significantly different between child and adult patients(x2 =196.46,P ＜ 0.01),with the isolation rate of non-albicans Candida species being 14.4％ and 45.8％ respectively in child and adult patients.Conclusions Candida albicans is still the dominant pathogen of superficial candidiasis.Candida orthopsilosis and Candida metapsilosis can cause superficial candidiasis.The isolation rote of non-albicans Candida species is higher in adult patients than in child patients.

Objective To establish a multiplex PCR targeting the intergenic spacer regions (IGS) for the identification of Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii.Methods Primers were designed by using the software ClustalW2 and Oligo 6 based on the sequence of IGS1 region,which shows high sequence variability in the genome of Cryptococcus neoformans and Cryptococcus gattii.,for the multiplex PCR.Then,the developed multiplex PCR was performed to identify 51 Cryptococcus neoformans strains representing genotypes VNI-VNIV and VNB as well as 41 Cryptococcus gattii strains representing genotypes VGI-VGIV.The identification results were compared with those from common PCR by using primers GPA1A,CLA4D and SODlgattii specific to Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii,respectively,as well as with those from the canavanine-glycine-bromothymol blue (CGB) medium-based culture.Results The developed multiplex PCR successfully identified the 92 Cryptococcus neoformans and Cryptococcus gattii.strains,and yielded negative results from the other tested pathogenic yeasts,which revealed a high specificity of the designed primers.False positive results were observed in the identification of two Cryptococcus gattii strains with GPA1A primer-based PCR,one Cryptococcus gattii strain with CLA4D primer-based PCR,one var.grubii strain and one var.neoformans strain with CGB culture,while no false negative results were observed in the detection of these Cryptococcus strains by any of these methods.Conclusions The developed multiplex PCR in this study can rapidly and accurately identify Cryptococcus neoformans var.grubii,var.neoformans,AD hybrid,and Cryptococcus gattii,with superior performance in comparison with common PCR and CGB medium-based culture.

Objective To investigate the effects of Mycobacterium vaccae vaccine on immunological function and clinical character in elderly patients with stable chronic obstructive pulmonary disease (COPD). MethodsA total of 100 elderly patients with stable COPD were randomly divided into immunotherapy group (group A, n= 50) and non-immunotherapy group (group B, n= 50), and normal control group (group C, n = 50). The levels of peripheral blood T-lymphocyte subsets (CD3+ , CD4+, CD8+ , CD4+/CD8+ ratio), natural killer cells (NK cells), immunoglobulins (IgG, IgA, IgM) and cytokines (IL-6, IL-8, TNF-a) were measured respectively before and after therapy. Group A and B were followed up for 1 year, then the times of acute outbreak and hospitalization of patients in the two groups were also compared. Results The levels of CD4 + ,CD4+/CD8+ ratio and NK cells in group A, B were significantly lower before therapy (P＜0. 05～0. 01＝, and the levels of IL-6, IL -8, TNF-a and IgA were significantly higher than in group C (P＜0. 01＝. After treatment with Mycobacterium vaccae vaccine in group A, the levels of CD4+ , CD4+/CD8+ ratio and NK cells were significantly higher (P＜0. 05-0. 01＝ and IL-6, IL-8, TNF-a and IgA were significantly lower than before treatment (all P＜0. 01＝. These levels showed no significant changes in group B after treatment (P＞0. 05). After 1-year follow-up, the times of acute outbreak and hospitalization on patients were statistically lower in group A than in group B (P＜ 0. 01 ).ConclusionsMycobacterium vaccae vaccine can improve cellular immunity function and reduce the times of acute outbreak and hospitalization in patients with stable COPD, so it has a higher clinical application value.