Can dida colonizait on associated with the humoral immune anamnestci respon se oft he dominant antigen in the early stage of invasive Candidosis / 医学研究生学报

Objective Rapid elevation of the IgG antibody against Candida Enolase ( Eno ) has been observed in patients with invasive candidosis in an early stage .The present study was to confirm the association of Candida colonization with humoral im-mune anamnestic response of the dominant antigen . Methods Twenty-four mice were randomized into group 1 treated by oral Candi-da colonization plus intraperitoneal infection ( immunocompetent , n=8) , 2 treated with immunosuppressant in addition to the treatment of group 1 ( immunocompromised , n=8) , 3 treated by oral Candida colonization only ( immunocompetent , n=4) and 4 treated by in-traperitoneal injection only( immunocompetent, n=4).The number of Eno-specific memory B-cells in the spleen and the levels of IgG , IgM and IgA antibodies were determined in the peripheral blood of the immunocompetent and immunocompromised invasive candidiasis mice . Results At 7 days after invasive infection , there were significantly more Eno-specific memory B-cells in the mice of groups1 ( 47.25 ± 13.81) and 2 (43.14±15.95) than in groups 3 (8.00±3.74) and 4(8.50±2.38) (P0.05).Eno-IgG antibodies were detected in the serum of the mice of the first two groups in the early stage of invasive infection and positively corre -lated with antigen-specific memory B-cells (r=0.737,P <00.1 ). Conclusion Rapid elevation of the Eno-IgG antibody level in the early stage of invasive infection after Candida colonization may be attributed to the rapid proliferation of humoral immune memory cells.

Detection of Candida albicans fructose bisphosphate aldolase antigen-specific T cells in healthy individuals by ELISPOT assay / 医学研究生学报

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.

Study on the Mechanism of Quinolone Resistance in Pseudomonas Aeruginosa / 现代检验医学杂志

Objective 30 Pseudomonasaeruginosa mechanism of resistance to quinolones.Methods For the determination of ciprofloxacin MIC by agar dilution method.Used PCR on DNA gyrase and topoisomerase Ⅳ,resistance genes gyrA,gyrB, parC and parE were amplified,and BLAST,to determine whether there was resistance to bits mutation point;using pulsed-field gel electrophoresis (PFGE)of these 30 strains homology analysis.Results The 28 bacterial strains gyrA gene ampli-fied fragment of 137 points were C→T mutation causes T83I;17 strains gyrB gene amplified fragment of 351 G→C lead to G466A;parC gene amplification 21 bacteria fragment 277 point increase with C→U mutation causes S87L change two differ-ent strains parE gene locus C→U mutation A425V and A473V cause change.PFGE results:30 Pseudomonas aeruginosa could be divided into six clones,Aclone 4,B clone 7,C clone 3,D clone 14,and two other single clones.Conclusion The tar-get mutant strains closely related to the epidemic clone type,the same changes in the same pop-type strains of drug targets, and proportional to the level of ciprofloxacin MICs value,the more the number of mutated genes,MICs value higher.GyrA gene most prone to mutation,the mutation was also the first to be discovered,more than any other target of the mutation mutations on binding of drugs and targets that would be the focus of concern.

Detection of Aspergillus fumigatus thioredoxin reductase GliT antigen-specific T cells by ELISPOT assay in healthy individuals and result evaluation / 医学研究生学报

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]) (P20/3 ×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 .

Development of an immunomagnetic bead assay for quantitative detection enolase of Candida albicans / 医学研究生学报

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 .

Detection and analysis of plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in carbapenem-resistant Escherichia coli / 中华检验医学杂志

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.

The state of non-culture-based laboratory diagnostic assays of invasive aspergillosis / 医学研究生学报

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.

Purification and properties of alkaline phosphatase from porcine kidney / 医学研究生学报

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.

Study on purification and properties of lactate dehydrogenase from human erythrocyte / 临床检验杂志

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.