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AIM:To investigate the role of damaged mitochondria in dendritic cell ( DC) apoptosis induced by Vibrio vulnificus (Vv) and its possible mechanism.METHODS: DC2.4 cells were co-cultured with Vv 1.1758 strain. Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect reactive oxygen species ( ROS) and intracellular Ca 2+concentration in the invaded cells , respectively .The cellular apoptotic rates and mitochondrial membrane potential (Δψm ) were measured by flow cytometry.The expression of nuclear factor-kappa B p65 (NF-κB p65) and tumor necrosis factor-al-pha (TNF-α) was detected by Western blotting.RESULTS:Vv 1.1758 induced DC2.4 cell apoptosis.Vv 1.1758 bacte-ria invaded into the DC2.4 cells by binding with cellular membrane though the end of the body .In the invaded DC2.4 cells, the visible mitochondrial damage, elevated ROS and intracellular Ca2+levels, and declinedΔψm were presented.Af-ter 1 h of co-culture, NF-κB p65 began to rise and reached the peak at 5 h, and then slightly decreased at 6 h.The TNF-αlevel increased after 2 h of co-culture and reached the peak at 6 h.CONCLUSION:The damaged mitochondria play an important role in DC apoptosis induced by Vv , and its possible mechanism may associate with the elevation of ROS and in-tracellular Ca2+level, and the declined Δψm.Meanwhile, NF-κB p65 and TNF-αare potential critical signaling molecules in the process of apoptosis .
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Objective To observe the process of Vibrio vulnificus inducing dendritic cell strain apoptosis.Methods We established the mixed culture model of mouse dendritic cell ( DC 2.4 strain) and Vibrio vulnificus( Vv1.1758 strain ),analyzed morphological characteristics of cell apoptosis by DAPI fluorescence staining,detect DNA fragmentation level of apoptosis cells by DNA Ladder assay,analyze DC2.4 apoptosis rate by Annexin V FITC/PI staining,determine activities of caspase-3 and caspase-8 by means of spectrophotometric method and detect changes of mitochondrial transmembrane potential ( △ Ψm ) by JC-1 fluorescence labeling.Results After Vv1.1758 strain and DC2.4 cell were mixed and cultured for 4 h,DAPI fluorescence staining showed typical apoptosis characteristics-chromatin condensation and marginalization; DNA agarose gel electrophoresis showed apoptosis band; apoptosis rates at 2,4 and 6 h were respectively (37.8±9.8) ％,(54.3 ± 12.7 ) ％ and ( 68.2± 14.6 ) ％ ; Mitochondrial transmembrane potentials (△Ψm) at 1 h,2 h and 4 h reduced by 7.1％,16.1％ and 46.7％ respectively; caspase-8 activity increased at 1.5 h and reached the peak at 2 h [ (2.48±0.19) U/μg],while caspase-3 activity started to increase at 3h and reached the peak at 4 h [ ( 1.91 ±0.16) U/μg ].Conclusion Vibrio vulnificus could induce dendritic cells by two pathways: reducing mitochondrial transmembrane potential and activating caspase-8 promoter and finally activate effector caspase-3 to promote apoptosis.
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Objective To investigate the invasion,internalization and the organelles damage of the cultured dendritic cells ( DC2.4 strain) during Vibrio vulnificus (Vv) infection.Methods The study model was the cultured DCs infected by Vv 1.1758 strain.Electron microscopy was used to observe the localization of bacteria in different time point of infection,cell morphology and the process of organelles changes.The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined by the fluorescence microscope.Results The Vv were pinocytosed into the DC cells through double-sides,and localized at 1-2 μm of the inner side membrane.It cost 1.27,1.87,3.43 hours reaching the infection ratio of 25％,50％,75％,respectively.Using electron microscopy,the DCs had been observed the phagosome formation within 1h,chromatin activation within 2 h,chromatin aggregation 4 h,and the significant cytoskeleton structure disruption within 6 h.Endoplasmic reticulum,mitochondria and lysosomes became swollen.In DCs,the protruding filaments gradually reduced,and their shape changed from the point-like to the linearlike aggregation at the inner side of the plasma membrane,extended microtubules disappeared,the microtubules at the outside nuclear membrane striking rearranged.Conclusion After DC was infected by Vv,the bacteria were pinocytosed into the inner side of DC membrane,and the microfilaments were observed to move from the cytoplasm to cell membrane.In addition,the microtubules moved from the synapse and the cell membrane to the nuclear membrane.The high lethality of Vv could provoke to the DCs cytoskeleton rearrangements.
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Objective To study the uncertainty and traceability of HBV DNA assays and discuss the comparability of results among different detection systems. Methods Different detecting systems were used to detect HBV DNA using the national standard substance as "quality control substance". The uncertainty of the results was evaluated referring "Guidelines for estimating and reporting measurement uncerTAinty of chemical test results" of NATA The results were traced back to the national standard substance. According to the CLSI document EP9-A2, the results were analyzed and subjected to bias estimation with the t(0.05sv) √u2b1+ u2b2 as the criterion clinically accepted to investigate the comparability of different detecting systems. Results The means (-y) measured by 3 HBV DNA assay systems were 6.15,5.88,and 6.31 lg(kIU/L) respectively. Except system A,both the biases of system B and C had statistical significance (all P ＜ 0. 05) and expanded uncertainty of three detection systems was varied, but the difference was within the maximum acceptable range (± 0. 5) of the external quality assessment by National Center for Clinical Laboratory. Being traceable to national standard substance, the results of HBV DNA of the three detecting systems were (5.45 ± 1.23), (5.55 ± 1.32) and (5.42 ± 1.25) lg(kIU/L), respectively.There was significant difference among three systems (F = 5.63, P ＜ 0. 05). Comparing system A and B,there was significant difference in statistic (q = 5. 12, P ＜ 0. 05) and the difference between system B and C also had statistically significant (q = 6. 85, P ＜ 0. 05), but the results between system A and C had no statistical difference (q = 1.85,P ＞ 0. 05). Among these three systems, the difference of any two detection systems had no statistical significance (all P ＞ 0. 05). It showed that system bias was acceptable in clinical application and the results between different systems were comparable. Conclusions It is necessary to estimate the uncertainty and traceability when comparing the HBV DNA assay among the different labs. It also needs to estimate the bias of different systems and evaluate the clinical acceptability to ensure the accuracy and comparability of the results.
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OBJECTIVE To evaluate the detect method of neopterin(Npt) by enzyme-linked immunosorbent assay(ELISA) and discuss the application of Npt in the viral encephalitis.METHODS The Npt was detected by ELISA.The methodology was investigated by sensitivity,precision,rate of recovery,interference and reference value.The npt was detected by ELISA assay in peripheral blood of 50 healthy people,30 cases of cerebrospinal fluid in children with viral encephalitis and 12 cases of cerebrospinal fluid in the control group.RESULTS In this method,within-run CVs were 4.94% and 5.55%;between-run CVs were 5.99% and 6.57%.The sensitivity was 1.08 nmol/L;the rate of recovery was 95.8-107.8%.Various indexes of the methodology coincided with the requirements of clinical laboratory.The reference value of serum Npt was 0-7.84 nmol/L.The Npt of cerebrospinal fluid was(34.09 ? 36.34) nmol/L in the viral encephalitis group,and(4.55 ? 2.89)nmol/L in the control group,and the Npt of cerebrospinal fluid in patient was significantly higher than that in control group(P
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OBJECTIVE To analyze the pathogenic results and drug resistance of bacteria isolated from serous effusion specimens from Jan 2006 to Feb 2008 so as to provide evidence for clinical pathogenic analysis and selection of antibiotic. METHODS The bacteria identification and the antimicrobial susceptibility were assayed by routine methods. RESULTS The positive rate of pathogens was 6.80% from 721 serous effusion specimens.There were 49 strains of pathogens which included 15 of Gram-positive bacteria and 28 of Gram-negative bacteria and 6 fungi strains.The most frequently isolated pathogens were Enterobacteriaceae(32.65%) followed by Enterococcus(18.37%),and Pseudomonas aeruginosa(12.25%).The most active compounds against Enterococcus were vanconmycin and linezolid(100.00%),nitrofurantoin and tetracycline(66.67%);the most active compounds against Staphylococcus were vancomycin,linezolid and nitrofurantoin(100.00%),tetracycline and rifampin(75.00%);The most active compounds against Gram-negative bacilli were piperacillin/tazobactam(67.86%),imipenem(64.29%),tobramycin(60.71%),gentamicin(53.57%) and levofloxacin(46.43%). CONCLUSIONS The prevailing pathogens in serous effusion are Gram-negative bacilli,especially Enterobacteriaceae.Data collected in present study provide a valuable information for prophylactic and empirical antibiotic use for serous infection.
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Objective To investigate the cultivation, biochemical features and drug susceptibilities of Vibrio vulnificus. Methods Three strains of Vibrio vulnificus were isolated from fatal patients in the Second Hospital of Jiaxing, Zhejiang province. Cultivation, identification and antibacterial susceptibility test were performed. Results Vibrio vulnificus grew on blood agar as dull-gray, opaque colonies with β-hemolysis. The organism presented positive in lactose, cellobiose fermentation and O/129 (10 μg) tests, but lack of inositol and rhamnus. The antibacterial susceptibility tests showed that Vibrio vulnificus strains were sensitive to ciprofloxacin, levofloxacin, imipenem, cefepime, ceftazidime, ceftriaxone, compound sulfamethoxazole and nitrofurantoin, however, resistant to gentamicin, tobramycin, aztreonam and cefazolin. Conclusions Vibrio vulnificus can be isolated from blood, bubbles fluid, and stool. Rapid identification, early diagnosis, and prompt empirical antibacterial therapy are important for reducing the mortality.
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0.05), the IFN ?R1 in advanced cases without splenectomy was low ( P
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Objective To explore the occurrence and development of schistosomiasis japonica hepatic fibrosis, and the treatment countermeasure. Methods Three groups of patients,including advanced schistosomiasis (splenectomy involved), non-advanced schistosomiasis splenomegaly and advanced schistosomiasis with hepatitis, were followed up. Biochemical tests including hepatic fibrosis indexes, liver ultrasonography,lymphocyte subcluster, and membrane CD35 were examined. Immunohistochemical tests of liver cells were performed in some patients. Results A total of 94.06% of 1212 patients took various anti-schistosome treatments. The difference was significant for positive antigen and antibody to schistosome, LN and HA ,and the ultrasonic indexes of liver fibrosis among the three groups. The degree of liver substance pathohistological examinations showed inflammation had a positive correlation with hepatic fibrosis. The contents of C-Ⅰ, C-Ⅲ in portal regions, central vein and hepatic sinusoid were increased in advanced schistosomiasis hepatic fibrosis. In hepatic fibrosis, hepatic sinusoid showed two pathological changes of dilation and stricture. Conclusions In schistosomiasis japonica, hepatic sinusoid changes play a great role in hepatic fibrosis.Hepatic fibrosis would still develop even after the thorough anti-schistosome therapy. So, anti-fibrosis therapy is necessary.