In high-risk patients,such as patients with hematologic malignancies,or patients after allogeneic stem-cell or solid-organ transplantation,early diagnosis of invasive aspergillosis(IA) is essential,as missing or delayed diagnosis of IA results in increasing rates of mortality.However,diagnosis of IA is difficult because classic tests have low sensitivity and specificity.The limited sensitivity and specificity of conventional assays for the detection of IA and the growing number of IA patients have led to the development of new assays.These methods include antigen detection systems,such as galactomannan(GM) assay,and different molecular methods(PCR assays).The GM assay is commercially available.However,it still need to be evaluated in large patient cohorts,especially children.A range of different PCR assays have been developed,targeting different gene regions,including a variety of amplicon detection methods.These molecular assays provide high potential in terms of sensitivity and specificity,but vary widely in their feasibility and up to now have not been standardised.Taken together,new non-culture-based diagnostic assays are noninvasive,simple,rapid and highly sensitive.Thus,they might be valuable tools to make early diagonosis,to reduce empirical antifungal therapy and to monitor the therapy.

Objective To preparing the reference material(RM) of lactate dehydrogenase(LD),we purification of LD from human erythrocyte(RBC)and studied its properties.Methods Using a modified procedure which including complete hemolysis of the RBC,hydroxyapatite treatment,(NH4)2 SO 4 Precipitation,CM- Sephadex C 50,Sephadex G100 and 5'- AMP- Sepharose 4B affinity chromatography.Results The purified LD has a sepecific activity of 163.0 kU/g protein.It is almost free of contaminating enzymes.Two corresponding bands were observed on both PAG plates stained for either protein or LD activity after electrophresis had been done.The apparent Michaelis constants were of 1.000 and 0.179 mmol/L for L-lactate and NAD and of 0.119 mmol/L 0.062 mmol/L for pyruvate and NADH respectively.Conclusion The final purified LD was found to be very similar to that in human serum in catalytic properties.It is intended to be a fundament to prepare a RM for the measurement of LD.

Objective: For preparing the reference material of alkaline phosphatase(ALP), the purification and properties of ALP from porcine kidney were studied. Methods: The ALP purification procedure included isolation microvillus of porcine kidney cortices by solubilization the membrance-binding enzymes with 1-butanol, precipitation with ammonium sulfate, chromatography on DEAE-Sephacel,ConA-Sepharose,Sephadex G-200 and a immuno-affinity column of anti-GGT antibodies. Results: The purified enzyme had a specific activity of 402 kU/g protein at 37℃and was almost free of contaminating enzymes. The apparent Michaelis constants was 1.35 mmol/L, and the optimum pH was 10.40. Conclusion: The kinetic properties of the preparation were very close to those of the enzyme present in the human serum, The product was suitable as enzyme preparation for producing the enzyme reference material.

Hypermethylation of the promoter region is an important mean for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferase are considered to be the main cause of promoter hypermethylation. In order to further explore the epigenetic mechanism of tumor suppressor gene RASSF1A inactivation, 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA methyltransferase inhibitor, was used to treat the human biliary tract carcinoma cell line QBC-939 at the concentration of 5 micromol/L for 24 h in this study. After the chemical intervention with 5-Aza-CdR, the methylation status in the promoter region of RASSF1A gene was detected by methylation specific PCR (MS-PCR), and the expression alteration of RASSF1A mRNA and protein were observed by RT-PCR and Western Blot respectively. Following the treatment with 5-Aza-CdR, methylation status in the promoter region of RASSF1A gene was reversed from methylation to unmethylation. A 280 bp DNA band which represented RASS1FA expression at transcriptional level and a 40 kDa (1 kDa=0.9921 ku) protein band which represented RASSF1A expression at protein level were detected by RT-PCR and Western Blot respectively in the experimental group cells and there were no corresponding bands in the control group cells. The experimental results suggest that 5-Aza-CdR can induce demethylation in the promoter region of RASSF1A. It can also reverse epigenetic transcriptional silencing caused by DNA methylation and induce the re-expression of RASSF1A in QBC-939. This study also suggest that the mechanism of RASSF1A inactivation is very closely related to the methylation of the promoter region, which may provide a new epigenetic understanding for tumor related gene inactivation and the pathogenesis of biliary tract carcinoma.

Objective The positron generated at the dose deposition site by using high-energy carbon ions to hit the material annihilate with the negative electron in the material to release the gamma photon.The positron-emitting isotope (PEI) distributions in the target volume are activated significantly by carbon ions.Therefore, the mean values of positron emission tomography (PET) activity could be related to the delivered doses to the clinical target volume from carbon ion.This specialty can be used for the image registration fusion of the carbon ion treatment planning computed tomography (CT) and treatment verification PET-CT.After radiation in the almost same decay period, the relationship between the different target volume and the PET-CT SUV of different every single fraction dose can be found, then the range of SUV for the radiation target could be decided.So this PET-CT standardized uptake value (SUV) range can also provide a reference for the correlation and consistency in planning target dose verification and evaluation for the clinical trial.Methods The head phantom was used as a simulation of the real human body, the 1 cc, 4 cc, and 10 cc cube volume target contouring were done in the TPS, the 90 degree fixed carbon ion beams were delivered in different single fraction effective dose of 2.5 GyE, 5 GyE, and 8 GyE.After the beam delivery, later the PET-CT scanning was performed and parameters of scanning followed the trial regulation.The MIM Maestro software was used for the image processing and fusion to determine the maximum, minimum, average, and total values of SUV in the virtual clinical target volumes for the different single fraction dose.Results The results showed that for the same target volume, the SUV range of target had an approximate linear correlation with effective dose of target (P=0.000).The same effective dose for the different target volumes got the same SUV range (P>0.05).Conclusions For the carbon ion treatment plan, the SUV range from image registration and fusion of planning CT and PET-CT after treatment can be used to make an evaluation for accuracy of the dose distribution.And this method also could be used in the hyper-fraction treatment plan.In the SUV range research of different decay periods, the similar method can be performed for the exploration.

Objective To study molecular epidemiology and carbapenem-resistance mechanism of four Escherichia coli strains isolated from general surgery wards. Methods Antibiotic susceptibility was carried out by K-B gar diffusion and agar dilution methods. Carbapenemases were screened by three dimensional test and EDTA-Na_2-disk synergy test. Pulsed-field gel electropboresis (PFGE) was performed to analyze molecular epidemiology of isolates. Plasmid was extracted by using an alkalinelysis technique. Conjunction experiment, transformation assay, specific PCR and DNA sequencing were performed to confirm carbapenemase genotype and its transmission mechanism Results Four Escherichia coli isolates were resistant to most antimicrobials including carbapenem. PFGE showed that the four isolates belong to four different clonal strains. Specific PCR and DNA sequence analysis identified that carbapenem resistance in four clinical isolates was mediated by KPC-2 encoded on an approximately 56 000 bp plasmid, and this plasmid did not harbor aminoglycosides and fluorquinolones resistant genes. Conclusion Four Escherichia coli isolates with carbapenem resistance are obtained from our hospital, and KPC-2 plasmid is main cause of carbapenem resistance in these isolates.

Objective The effects of candesartan,an angiotensin Ⅱ type 1 receptor blocker (ARB) were investigated on advanced glycation end-products accumulation and the receptor for AGE (RAGE) expression in type 2 diabetic KK/Ta mouse kidneys.MethodsKK/Ta mice(n=72)were random divided into three groups(n=24) and it was treated with candesartan [4 mg/(kg·d)] or vehicle from 6 or 12 to 28 weeks of age.BALB/c mice(n=24) treated with vehicle were used as controls.Body weight,blood pressure,blood glucose,urinary microalbumin,urinary creatinine and serum creatinine were measured every four weeks.At 28 weeks,renal expressions of carboxymethyllysine and RAGE were evaluated by immunohistochemistry and/or competitive RT-PCR.Results KK/Ta mice developed high body weight,high blood glucose,and high urinary microalbumin/creatinine ratio in KK/Ta mice at 28 weeks of age,and it was significantly higher than that of BALB/c mice [(427.49±89.37)mg/g vs (9.54±3.25)mg/g,P＜0.01 ].Protein and mRNA expressions of RAGE were upregulated in KK/Ta kidneys with increased immunostaining intensities of carboxymethyllysine.Candesartan treatment has markedly reduced urinary microalbumin/creatinine ratio [Early treatment group (32.18±9.41)mg/g,Late treatment group (53.20±7.26)mg/g,P＜0.01 ].Treatment with candesartan down-regulated the protein and mRNA expressions of RAGE and reduced the accumulation of carboxymethyllysine.There were no significant differences between the two treatment groups (from 6 or 12 weeks).ConclusionsThe results suggest that candesartan,an ARB,reduces advanced glycation end-products accumulation and subsequent albuminuria by down-regulating RAGE expression in type 2 diabetic KK/Ta mouse kidneys.

Objective To develop an immunomagnetic bead ( IMB) assay for quantitative detection enolase ( Eno) of Candi-da albicans, and to improve the diagonosis of invasive candidiasis . Methods The immunomagnetic bead was prepared by conjuga-ting with Anti Eno of Candida albicans monoclonal antibody .HRP-conjugated goat polyclonal antibody against Candida albicans Eno was employed as detecting antibody .The performance parameters of the IMB assay including precision , specificity, linear range and limit of detection were verified by using recombinant Candida albicans Eno.Then the developed assay was applied to determine Eno levels in supernatant of pathogenic fungi cultures . Results The intra and inter-coefficient of variation was 4.54%, 5.87% and 5.26%, 8.82%at the concentration of 25 ng/mL and 5 ng/mL, respectively.The limit of detection was 0.5 ng/mL.The linear range was(0.5-50) ng/mL.The level of Eno in Candida albicans culture after incubated in 37℃for 24 h was 3.89 ng/mL and gradually in-creased to 37.89 ng/mL at 120 h.There was a positive correlation between the level of Eno and growth hyphae of Candiad albicans. There was weak cross reaction with Candida parapsilosis and no cross reaction with Candida tropicalis, Candida guilliermondii, Candida glabrata, Cryptococcus neoformans and Saccharomyces cerevisiae. Conclusion An IMB assay for quantitative detection Eno of Candida albicans was developed , which was more sensitive , rapid and reliable than previous qualitative ELISA .The IMB assay has the potential to be applied to the research in invasive candidasis .

Objective The purpose of this study was to evaluate the protective potential of the Aspergillus fumigatus thiore -doxin reductase GliT ( TR) antigen by establishing and optimizing ELISPOT assay for TR antigen-specific T cells ( TR/AST) secreting IFN-γand IL-4 in peripheral blood mononuclear cells ( PBMCs ) and explore the role of TR/AST in invasive aspergillosis ( IA ) . Methods We optimized the reaction conditions of ELISPOT by preliminary checkerboard titration and determined the frequencies of positive spot-forming cells ( SFCs) specifically secreting IFN-γand IL-4 in the PBMCs of 20 healthy individuals with TR as specific stimulant and with PHA and PMA as positive controls ,. Results Checkerboard titration demonstrated the best result of ELISPOT with the TR antigen at the final concentration of 10μg/well and PBMCs at 3 ×105/well.The median frequency of IFN-γSFCs was sig-nificantly higher (15 [3.5, 59.5]) than that of IL-4 SFCs (0 [0, 0]) (P<0.001).TR induced IFN-γresponses in all the 20 healthy donors, including 9 cases of strong IFN-γresponse (SFCs>20/3 ×105 PBMCs), accounting for 45%, but failed to induce IL-4 response in 19 of the healthy individuals . Conclusion The Aspergillus fumigatus TR antigen could induce an immunodominant Th1 response , and therefore might be a potential protective antigen .

Objective Invasive candidiasis is associated with a significant mortality clinically.The purpose of this study was to observe the T cells immune response to fructose bisphosphate aldolase (Fba), an immunodominant antigen of Candida albicans, and determine whether the antigen has the possibility of priming cellular immune protection in invasive candiasis.Methods Using ELISPOT assay, we determined the frequencies of positive spot-forming cells (SFCs) of Fba antigen-specific T cells secreting IFN-γ, IL-4 and IL-17A in the peripheral blood mononuclear cells (PBMCs) of 26 healthy individuals.Results After Fba stimulation, the frequencies of positive SFCs of IFN-γ, IL-4 and IL-17A in the 26 healthy subjects were 23 (9.75, 42.50), 0 (0, 0.25) and 1.5 (0.75, 8.25), respectively, with statistically significant differences among the three (P<0.01).The response rates of IFN-γ (100% [26/26]) and IL-17A (76.92% [20/26]) were significantly higher than that of IL-4 (15.38% [4/26]) (P<0.01).Fba-induced strong response (SCFs ≥20) for IFN-γ was observed in 57.69% (15/26) of the healthy individuals, that for IL-17A in only 1, while that for IL-4 in none.Responses of both Th1 and Th17 cells to Fba were found in 65.38% (17/26) of the subjects, that of Th1 cells in 19.23% (5/26), but that of Th2 cells in none.Conclusion Fba of Candida albicans can induce immunodominant responses of Th1 and Th17 cells and is a potential vaccine against invasive candiasis.