Migration and spread of Bacillus cereus in mice with endophthalmitis and influence on inflammation / 中华微生物学和免疫学杂志

Objective:To investigate the causes of strong pathogenicity of Bacillus cereus ( B. cereus) in a mouse model of B. cereus endophthalmitis and the factors that might be related to the prognosis of the disease. Methods:C57BL/6J mice aged 6-8 weeks were injected with 1 μl PBS solution containing 100 CFU B. cereus into the vitreous cavity to construct traumatic endophthalmitis model, and a control group was set up by injecting the contralateral eyeball with 1 μl sterile PBS. A mouse model of Staphylococcus epidermidis ( S. epidermidis) endophthalmitis was constructed in the same way as disease control group. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the levels of inflammatory cytokines at different time points. Histology, electroretinogram and transmission electron microscopy were used to detect the progression of endophthalmitis and retinal function at different time points. Results:B. cereus grew significantly faster than S. epidermidis in the eyes of C57BL/6 mice and gradually moved to the cornea 12 h after infection. The results of transmission electron microscopy showed that many more B. cereus were found in the iris with sparse pigment particles, while S. epidermidis could not be detected in the anterior segment after infection. The electroretinogram results showed that the amplitude of A wave and B wave of mice with B. cereus endophthalmitis decreased significantly 6 h after infection, and the B wave could not be detected 12 h after infection. Moreover, the amplitude reduction at different time points was significantly larger than that in the S. epidermidis endophthalmitis group. Histological examination found that compared with the S. epidermidis endophthalmitis group, the mice with B. cereus endophthalmitis had significantly increased inflammatory cells in the anterior chamber and vitreous cavity with a higher degree of infiltration, which was more destructive to the tissue structure. ELISA results showed that the activity of myeloperoxidase (MPO) was significantly stronger in the B. cereus endophthalmitis group than in the S. epidermidis endophthalmitis group, suggesting that a much more severe inflammation was induced. The expression of IL-6, TNF-α and IL-1β at the transcription and protein levels in the mouse model of B. cereus endophthalmitis were significantly higher than those in the mice with S. epidermidis endophthalmitis. Conclusions:B. cereus could induce severe endophthalmitis and tissue destruction in the eye due to its rapid growth and migration ability, which was an important factor leading to vision loss.

Diagnosis value of peripheral blood TEMs percentage in the AFP negative early hepatocellular carcinoma / 中华检验医学杂志

Objective To evaluate the diagnosis value of the percentage of Tie 2-expressing monocytes(TEMs)in CD14+CD16+monocytes of peripheral blood from hepatocellular carcinoma(HCC) patients with negative AFP and tumor size≤3 cm.Methods Flow Cytometry(FCM)was used to determine the percentage of TEMs in CD14+CD16+monocytes of peripheral blood from patients with HCC(n=82), liver cirrhosis(n=29), chronic hepatitis B(n=28), and healthy controls(n=31).Abbott i2000 microparticle chemiluminescence immunoassay analyzer was used to determine the plasma alpha -fetoprotein (AFP)levels.The difference among multi groups was analyzed by the Kruskal-Wallis H test.Two independent groups were analyzed by the Mann-Whitney U test.The chi-square test was used in the rate comparison.The correlation between TEMs and AFP was analyzed by Spearman rank correlation analysis. Morever, the areas under the receiver operating characteristic curves(ROC-AUC), sensitivity and specificity of TEMs or AFP in differentiating HCC, HCC with AFP negative or tumor size≤3 cm were analyzed.Results The percentage of TEMs in CD14 +CD16 +monocytes of peripheral blood from HCC or HCC with negative AFP or HCC with tumor size≤3 cm was significantly higher than that in patients with liver cirrhosis,chronic hepatitis B and healthy controls(P<0.05).ROC-AUC of TEMs and AFP in the diagnosis of HCC were 0.701(95% CI 0.626-0.768)and 0.712(95% CI 0.638-0.779) respectively.When the cut-off values of TEMs and AFP were 4.95%and 20 μg/L,the sensitivities of TEMs and AFP were 71.95%and 45.12%,and the specificities of TEMs and AFP were 70.45%and 85.23%. The sensitivity of TEMs in the diagnosis of HCC was significantly higher than that of AFP(χ2=12.16,P=0.000).The specificity of AFP was significantly higher than that of TEMs(χ2=5.57,P=0.018).There was a highest sensitivity(89.02%)in TEMs/AFP method,and there was a highest specificity(93.18%) in TEMs+AFP method in the diagnosis of HCC.There was no significant difference between the ROC-AUC for the TEMs and the AFP in the diagnosis of 26 patients with tumor size≤3 cm HCC(0.776 vs 0.645,Z=1.805,P=0.071),TEMs/AFP had the highest sensitivity(84.62%),while TEMs+AFP had the highest specificity(93.18%)in the diagnosis of tumor size≤3cm HCC.The ROC-AUC for the TEMs in the diagnosis of 45 patients with AFP negative HCC was 0.739(95%CI 0.648-0.829).The sensitivity and specificity of TEMs were 80.0% and 70.45% respectively.There was no correlation between the level of plasma AFP and the percentage of TEMs(r=-0.169, P=0.129)determined by Spearmans rank correlation coefficient.Conclusions TEMs is valuable in the diagnosis of HCC with negative AFP and tumor size≤3cm,and the two tests of TEMs and AFP can complement each other in the diagnosis of patients with HCC.

Prognostic significance of proteins expression by immunohistochemical method in diffuse large B cell lymphoma / 中华血液学杂志

Objective@#To analyze the prognostic significance of TP53, Bcl-2, Bcl-6, Myc proteins expression by immunohistochemical method (IHC) in diffuse large B cell Lymphoma (DLBCL) .@*Methods@#Clinical and pathologic data of 223 patients with DLBCL hospitalized in Zhejiang First Hospital from March 2009 to June 2015 were retrospectively analyzed.@*Results@#The 223 cases, a median age of 56 years old with a male predominance, had shown a 39.0% of TP53 positive expression, 38.6% of Myc, 69.1% of Bcl-2, 56.5% of Bcl-6, and 22.7% of Myc/Bcl-2 double expression. According to Hans’ classification, 27.4% were GCB and 72.6% were non-GCB. With a median follow-up of 38 (2-97) months, the 3 and 5 years survival rates were 70% and 66% , respectively. By multivariate analysis, TP53 over-expression and Myc/Bcl-2 double expression were independently associated with poor outcomes. 3-year and 5-year overall survival were 59% and 57% for patients with TP53 positive, 77% and 71% for patients with TP53 negative expression. Patients with non-GCB subtype receiving chemotherapy combined with rituximab had a higher OS than those without rituximab. But rituximab did not improve the prognosis of patients with TP53 positive.@*Conclusion@#Myc/Bcl-2 double expression and TP53 over-expression are poor prognosis for DLBCL patients. Patients with Myc/Bcl-2 double expression have shorter OS. Patients with non-GCB subtype who received chemotherapy combined with rituximab have a better OS than those without rituximab. But rituximab does not improve the prognosis of patients with TP53 positive.

Diagnosis Value of Detecting Plasma Dickkopf-1 Patients with Hepatocellular Carcinoma / 现代检验医学杂志

Objective To evaluate the diagnosis value of plasma Dickkopf-1(DKK1)in hepatocellular carcinoma (HCC)pa-tients.Methods Selected 48 patients with HCC,20 patients with liver cirrhosis (LC),20 patients with chronic hepatitis B (CHB),and all of them were clinically diagnosed in the Third People’s Hospital of Nantong City,chose 20 cases of health examination as healthy controls (HC),that were ruled out other chronic disease.Enzyme-linked immunosorbent assay (ELISA)and Abbott i2000 microparticle chemiluminescence immunoassay analyzer were used to determine the plasma DKK1 and alpha-fetoprotein (AFP)levels.At the same time,analysesed and compared the receiver operating characteristic (ROC)curve and correlation of the DKK1 and AFP results.Results The plasma level of DKK1 in patients with HCC was significantly higher than that in patients with chronic hepatitis B,cirrhosis and healthy controls (Z=-4.132~-5.828,P<0.001).The area under ROC curve (AUC)of plasma DKK1 in the diagnosis of HCC was 0.889 and 95% confidence in-terval was 0.831~0.947,when the Cut-off value of DKK1 was 565 ng/L,the sensitivity was 93.8% and specificity was 70% in diagnosing HCC.The areaunder ROC curve of AFP in the diagnosis of HCC was 0.759 and 95% confidence interval was 0.667~0.850.The AUC of DKK1 was significantly higher than that of AFP (Z=2.28,P=0.022).DKK1 and AFP in diagnosing HCC were no significantly correlated (r=0.148,P=0.316).21 patients with AFP under 20μg/L showed higer DKK1 above 565 ng/L in 48 patients with HCC.Conclusion Detection of DKK1 in plasma can be used as a complement of AFP in the diagnosis of HCC,Especially DKK1 early diagnostic value of AFP negative HCC patients is remarkable.

Efficacy of HAA regimen in the treatment of 64 patients with refractory/relapsed acute myeloid leukemia / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of the HAA regimen (homoharringtonine,cytarabine and aclarubicin)as salvage chemotherapy in the treatment of refractory/relapsed acute myeloid leukemia (AML).</p><p><b>METHODS</b>We retrospectively analyzed 64 patients with refractory/relapsed AML who received the HAA regimen as salvage chemotherapy. The complete remission (CR)rate was analyzed. Kaplan-Meier method was used to estimate overall survival (OS) and relapse free survival (RFS), and the differences were compared by Log-rank test.</p><p><b>RESULTS</b>The overall CR rate was 70.1%, and 67.1% of the patients attained CR after the first induction course. The early death rate was 0. The median follow-up time was 61 (range:6-120) months. The estimated 3-year OS rate was 46.8% and the estimated 3-year RFS rate was 42.8%. The CR rates of patients with favorable/intermediate and unfavorable cytogenetics were 76.4% and 33.3%, respectively. The 3-year OS of favorable/intermediate and unfavorable group were 53.7% and 10.0%, respectively. The median survival time of unfavorable group was only 8 months. The side effects associated with the HAA regimen were tolerable, in which the most common toxicities were myelosuppression and infection.</p><p><b>CONCLUSION</b>HAA regimen is associated with a higher rate of CR and longer-term survival and its toxicity can be tolerated. The regimen is suitable for refractory/relapsed AML patients with favorable or intermediate risk .</p>

Comparing and analysis of the early-onset and late-onset neonatal sepsis / 中国综合临床

Objective To analyse the pathway of infection,risk factors,clinical characteristics and pathogenic bacteria distribution of neonatal sepsis.Methods Eighty-eight neonatal with sepsis were enrooled from January 2006 to December 2011 in the First Affiliated Hospital of Lanzhou University.According to disease stage,they were divided into early-onset sepsis group and late-onset sepsis groups.Results Respiratory infections in two groups was the majority (44.7％ vs.46.0％),and there was no significant difference (P =0.906).That premature birth,low birth weight infant,amniotic fluid pollution and asphyxia were risk factors for early-onset sepsis.The most common clinical characteristics in early-onset sepsis were eating less milk(57.9％,22/38) and had fever of the late-onset sepsis(42.0％,21/50).Forty-one cases were with positive blood culture and the rate was 46.6％ (41/88),the blood culture specimens were mainly Gram-positive bacteria in two group (75.0％ (15/20),90.4％ (19/21)).The most common pathogenic bacteria were Staphylococcus aureus and Coagulase-negative staphylococci.Conclusion Premature birth,low birth weight,meconium,asphyxia are risk factors of early onset neonatal sepsis.Early-onset sepsis often shows poor feeding,and late-onset sepsis in children prone to be fever.The common pathogenic germ of early-onset and late-onset sepsis are Grampositivebacteria,Staphylococcus aureus and coagulase-negative staphylococci common.As for neonate with highrisk factors,clinical features of early pathologic examination should be performed in order to further clarify the diagnosis and taking clinical therapy.

Identification of hemolysin BL from Bacillus cereus of patients with endophthalmitis and its biological activity detection in vitro / 中华微生物学和免疫学杂志

Objective To identify the hemolysin BL(HBL) gene from Bacillus cereus of patients with endophthalmitis comfirmed by API bacterial identification test strip,and detect its biological activity in vitro.Methods Three pairs of specific primers were designed according to the gene sequence of HBL(B,L1 and L2 components),then the PCR assay were established through condition optimization,and to further detect five Bacillus cereus strains isolated from clinical patients with endophthalmitis.HBL with biological activity was extracted by salt fractionation from a randomly selected strain,and a series of different concentrations of HBL were prepared and acted on sheep red blood cells(SRBC),Vero cells and Hela cells; virulence of HBL was also assessed through observating lethal effect of which on mice with intraperitoneal injection.Results Three genes of HBL were detected in all B.cereus strains from clinical patients; Strong hemolytic activity of HBL showed obvious time-and dose-dependent.In the study,we found the morphological changes of Vero and Hela cells caused by HBL were different,but cell death were the same result with contents released; Within 48 h after infection,lethality of HBL for mice showed 100％ with the concentration of more than 2.0 HU/ml,and was also in a time-and dose-dependent manner.Conclusion HBL isolated from B.Cereus had high hemolysis activity and low concentration.The expression of all BL genes provided a strong basis for the clinical feature of B.Cereus infection,which was developed rapidly and with a poor prognosis.It also provided a new method for rapid diagnosis and molecular epidemiology investigation in clinical.

The efficacy and safety of HAA regimen as induction chemotherapy in 150 newly diagnosed acute myeloid leukemia / 中华内科杂志

Objective To explore the efficacy and safety of HAA regimen (homoharringtonine,cytarabine and aclarubicin) in the treatment of 150 newly diagnosed adult acute myeloid leukemia (AML).Methods All patients entered the study from May 1999 to June 2008 were treated with HAA regimen. Coxsurvival analysis was used to estimate the survival rate and differences between M1/M2 and M4/M5 were compared with 2-sided log-rank test. Results Out of the 150 patients, 121 (81%) achieved complete remission (CR). After the first course, CR rate was 68%. The CR rates of 97%, 84% and 38% were achieved in patients with favorable, intermediate and unfavorable cytogenetics, respectively. For the patients with CR, the median follow-up time was 16.5 ( 1.5-100.5 ) months, and the estimated 3-year survival rate was 45%. The estimated 3-year relapse free survival rate was 52% for the 121 patients with CR.Conclusions HAA regimen may be an efficacious and safe regimen with a good toleration in the induction therapy for newly diagnosed AML, and a high CR rate could be achieved with only one or two courses.

Expression and significance of transcription factor EBF3 in tissues of human primary hepatocellular cancer / 临床检验杂志

Objective To investigate the expression differences of mRNA and protein of early B-cell factor 3(EBF3) in the tissues between hepatocellular carcinoma(HCC) and distant noncancerous tissues and the clinical significance.Methods The expression levels of EBF3 mRNA in 18 pairs surgical specimens of HCC and their distant noncancerous tissues were detected by real-time fluorescence quantitative polymerase chain reaction(FQ-RT-PCR).Western blotting was used to detect the expression levels of EBF3 protein in 5 pairs surgical specimens of HCC and distant noncancerous tissues.Results The ratios of EBF3 mRNA tobeta2 microglobulin mRNA in eighteen liver cancerous tissues(0.52?0.17) were significantly higher than those in distant liver noncancerous tissues(0.28?0.23,t=3.56,P=0.0011).The average gray scale level of EBF3 in five liver cancerous tissues(26.35?14.06)were significantly higher than those in distant noncancerous tissues(7.86?8.47,t=2.52,P=0.036).Conclusion Theexpression levels of EBF3 mRNA and protein up-regulated in primary hepatocellular carcinoma,suggestting that EBF3 may contribute to occurrence of primary hepatocellular carcinoma.

The effect of early B cell factor 3 on HepG2 cell cycling / 中国免疫学杂志

Objective:To clone the encoding sequence of human EBF3 gene,construct recombinant eukaryotic expression plasmid vector pEGFP/EBF3,and study the effect of EBF3 on HepG2 cell cycling.Methods:Total RNA was isolated from placental tissue.Full-length human EBF3 cDNA was amplified by RT-PCR,cloned into eukaryotic expression plasmid vector pEGFP-N1 and sequenced.The expression and sub-cellular localization of the fusion protein EBF3-EGFP in HepG2 cells were analyzed by Western blot.Cell cycles were analyzed with flow cytometry analysis.Results:Obtained full encoding sequence of early B cell factor 3 was identical with that included in GeneBank,and the eukaryotic expression plasmid vector pEGFP/EBF3 was constructed correctly.24 h after transfected by pEGFP/EBF3,the fusion protein EBF3-EGFP was observed mainly in the cellular nucleus under the inverted fluorescence microscope.Western blot analysis confirmed that the EBF3-EGFP fusion proteins of Mr 87 000 were detected in both cytoplasmic and nuclear protein of the HepG2 transfected by pEGFP/EBF3 for 24 h or 48 h.Flow cytometry analysis revealed that the percentage of cells in the S phase was markedly increased in HepG2 cells transfected by pEGFP/EBF3 as compared with that in pEGFP-N1 transfected cells.These findings suggested that transfection of EBF3 gene into HepG2 induced cell proliferation by increasing the number of cells from G1 phase to G2 phase.Conclusion:The recombinant eukaryotic expression plasmid vector pEGFP/EBF3 is successfully established.The percentage of cells in the S phase is markedly increased in pEGFP/EBF3 transfected cells as opposed to pEGFP-N1 transfected cells.It is likely that EBF3 promotes HepG2 cells proliferation through DNA replication.