Objective To determine the modality of Leptospira interrogans invading human and murine mononuclear-macrophages and diversity of leptospiral phagocytotic vesicle formation. Methods Transmission electron microscopy was applied to observe the invasion of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai into murine mononuclear-macrophage-like cell line J774A. 1 and PMA-activated human monocyte line THP-1 and the formation of leptospiral phagocytotic vesicles. By using immunofluorescence plus either laser confocal microscopy or fluorescence spectrophotometry, the changes of intracellular leptospiral numbers in J774A. 1 and PMA-activated THP-1 cells before and after block with endocytosis inhibitors monodansylcadaverin (MDC), phenylarsine oxide (PAO) and clathrin antibody were investigated. Results The leptospires in J774A. 1 cells were located in phagocytotic vesicles while the leptospires in THP-1 cells had no package with phagocytotic vesicle membrane. Both MDC and PAO presented the effect inhibiting endocytosis of L. interrogans into J774A. 1 and THP-1 cells in dose-dependent manner. The numbers of leptospires in J774A. 1 and THP-1 cells that pre-blocked with 10 μmol/L or above MDC and 1 μmol/L or above PAO were significantly less than that in the two cells untreated with MDC and PAO (P＜0.05＝. After J774A. 1 and THP-1 cells were blocked with clathrin antibody, the numbers of intracellular leptospires were also remarkbly decreased ( P<0.05 ).Conclusion Leptospira interrogans can invade into both human and murine mononuclear-macrophages through the way of clathrin-dependent endocytosis. There is an opposite diversity of leptospiral phagocytotic vesicle formations in human and murine mononuclear-macrophages, which may result in the difference of pathogenesis in human and mice after infected with L. interrogans.

Objective To investigate the dynamic changes of lymphocyte subsets (lymphocyte count, CD3 +T,CD4 +T,CD8 +T,CD19 +B,CD56 +NK)in children with septic shock.Methods Peripheral blood lymphocyte subsets were analysed in 25 cases with severe septic shock and 24 cases with mild septic shock on day 1 ,3,8,and compared with those of 25 healthy volunteers.Children with severe septic shock were divided in-to survival group and dying group according to the outcome.The detection data were compared.Results In the first day of admission,peripheral blood lymphocyte subsets in children with septic shock decreased significantly compared with the normal children,and there was significant difference in the three groups(P <0.05 ).In the third day of admission,children with mild septic shock had statistical increase of CD4 +T,CD3 +T and lympho-cyte count(P <0.05),and children with severe septic shock had statistical decrease of lymphocyte subsets ex-cept CD56 +NK.In the eighth day of admission,children with mild septic shock had no significant difference in CD4 +T lymphocyte count compared with control group(P >0.05),but children with severe septic shock had statistical increase of CD4 +T,CD3 +T and lymphocyte count(P <0.05).There were 5 cases died who had se-vere septic shock in day 2 to day 5 after admission.The count of lymphocyte subsets were detected significantly lower in dying group than survival group in the third day of admission(P <0.05).Conclusion The continuous low level of lymphocyte subsets indicates bad outcome.The patientˊs condition improved with increase of CD4 +T,CD3 +T,lymphocyte count.CD4 +T is the most sensitive.Dynamic detection of lymphocyte subsets in the pe-ripheral blood of children with septic shock is of great clinical significance in judging the severity of disease and cura-tive effect.

Objective To determine the change of expression level of Leptospira interrogans sph2 gene, and hemolytic and cell apoptosis-inducing activities of sphingomyelinase hemolysin Sph2. Methods Entire sph2 gene fragment was amplified by PCR from genomic DNA of L. Interrogans serovar serogroup Icterohaemorrhagiae serovar Lai strain Lai, and sequenced after T-A cloning. Subsequently, a prokaryotic expression system of sph2 gene was constructed. The expression of target recombinant Sph2( rSph2 ) was examined by SDS-PAGE and the expressed rSph2 was extracted by Ni-NTA affinity chromatogaphy. The hemolytic activity of rSph2 was measured by hemolytic test in sheep blood agar plate and spectrophotometry-based hemoglobin measurement, and the apoptosis-inducing activity of rSph2 to murine mononuclear-macrophagelike cell line(J774A. 1) and hepatic cell line(IAR20) was determined by flow cytometry. A real-time fluorescence quantitative RT-PCR was applied to detect the change of sph2 mRNA levels before and after L. Interrogans strain Lai infecting J774A. 1 and IAR20 cells. Results The cloned sph2 gene had 100% sequence identity to the corresponding gene in GenBank. The constructed prokaryotic expression system was able to efficiently express rSph2. The rSph2 could lyse sheep erythrocytes in concentration-dependent pattern. 10μg/ml rSph2 could induce the apoptosis of J774A. 1 cells and IAR20 cells, and the peak apoptotic rates were 23.96% and 32.92%, respectively. The mRNA level of sph2 gene was significantly elevated within 0.5-2 h of L. Interrogans strain Lai infecting either J774A. 1 or IAR20 cells, and then the mRNA level was quickly descended. Conclusion The sph2 gene of L. Interrogans strain Lai has a transient expression when the microbe contacts host cells. rSph2 possesses activities of sheep erythrocyte lysis and inducing macrophage and hepatocyte apoptosis, indicating Sph2 as an important virulence factor during pathogenic process of Leptospira.

Objective To evaluate the apphcation value of peripheral blood neutrophil CD64 expression in early differential diagnosis of etiology in children with community-acquired pneumonia.Methods From June 2014 to June 2015 in our hospital,total of 99 cases of different pathogen infection were divided into three groups,bacterial pneumonia group (n =41),non-bacterial pneumonia group(n =38),healthy children group (n =20);and the non-bacterial pneumonia group was divided into two subgroups,viral pneumonia group (n =22) and mycoplasma pneumoniae pneumonia group(n =16).Flow cytometry was used to test the median fluorescence intensity (MFI) of peripheral blood neutrophil CD64.The levels of peripheral blood CRP,WBC and neutrophil percentage were detected.Results The levels of CD64 expression,CRP and WBC in bacterial pneumonia group were statistically higher than those of non-bacterial pneumonia group and healthy children group respectively (both P ＜ 0.01).The optimal cutoff value of CD64 was 6 519 MFI when using ROC curve,and the sensitivity and specificity of the diagnosis of bacterial pneumonia were 87.8％ and 89.7％ respectively.The levels of CD64 expression in bacterial pneumonia group were significantly higher than those in viral pneumonia group,mycoplasma pneumoniae pneumonia group and healthy children group (all P ＜ 0.01).Conclusion The expression of peripheral blood neutrophil CD64 increases in children with community-acquired pneumonia of bacteria infection.It can be used to guide early diagnosis of children with bacterial pneumonia and the using of antibiotics.

Objective To investigate the clinical efficacy ofβ-lactam combined with macrolides antibiotics in treatment of severe community-acquired pneumonia (CAP) in children. Methods Children with severe CAP on admission between 2012 February and 2012 April were divided into treatment group and control group. With the same symptom specific supportive treat-ment, the patients in the treatment group were treated with both cefmetazole and azithromycin, while the patients in the control group were treated with cefmetazole alone. The total effective rate, number of days of symptoms and signs disappeared and num-ber of days of hospitalization were observed. Results The total effective rate was 87.8%in the trearment group and 61.3%in the control group with significant difference (P<0.05). Compared with control group, the recovery time of temperature, time of pulmonary rale disappearing and cough retraction were reduced (P<0.05). As well as the number of days of hospitalization was decreased (P<0.05). Conclusions The treatment of severe CAP in children with combination of azithromycin and cefmetazole results in better curative effect. A combined medication ofβ-lactam and macrolides antibiotics may be rational and effective.

Adult ; Drug Resistance, Microbial ; Female ; Humans ; Male ; Middle Aged ; Pneumonia ; complications ; microbiology ; Silicotuberculosis ; complications ; microbiology

Objective To improve the understanding of pulmonary mucormycosis by analyzing the clinical manifestations,imaging features,diagnosis,treatment and prognosis of this disease.Methods The clinical data of eight patients diagnosed as pulmonary mucormycosis by histopathologic examination were retrospectively analyzed.Results Eight patients included six males and two females with age from 36 days to 66 years.Underlying conditions covered diabetes (n =4),renal transplantation (n =3),premature (n =1) and long-term corticosteroid treatment in two cases.Imaging manifestations revealed multiple irregular lumps or nodules in three cases,multiple cavities with thick wall in three cases,diffuse lung infiltrate in one case and lung opacities in one case.The diagnoses of seven patients were confirmed by percutaneous needle lung biopsy and the remaining one was diagnosed with fiberoptic bronchoscopy biopsy.Surgery combined with amphotericin B liposome(60 mg/d for three weeks)was applied to one patient who was cured with no recurrence after a 22 month follow-up.Three cases were given amphotericin B liposome (a newborn with 7mg/d for 62 days,the other two 60 mg/d for 31 days and 70 mg/d for 71 days respectively).All had achieved marked response with follow up from 8 to 29 months,but one patient relapsed and died of recurrent lung mucormycosis.The other three patients were treated with itraconazole 400-200 mg/d from 21 days to 1 year with duration of follow up from 1 month to 20 months.One patient was not evaluable due to missing.Two patients relapsed and one died.Conclusion Pulmonary mucormycosis is difficult to diagnose and treat with a high mortality.Percutaneous tranthoracic lung biopsy is a useful diagnostic method.Amphotericin B liposome or itraconazole may be active against mucus.Early control of causes is essential to improve the prognosis and reduce the recurrence in patients with pulmonary mucormycosis.

Objective To establish a quick electrochemical biosensor for the detection of nucleic acid of Ebola virus . Methods The DNA tetrahedral nanostructure was self-assembled on gold surface by strong Au-S chemical bonds , leaving the target probe at the top .A biotinylated-ssDNA was introduced as the detection probe by specific binding of the captured target sequence , before avidin-horseradish peroxidase ( HRP) was used as a signal amplifier to transduce amperometric sig-nal through interactions with TMB substrate .Results The results indicated that the nucleotide sequence of Ebola virus could be recognized and detected by the sensor .The linear range for the detection of target DNA was from 1.0 ×10 -9 to 5.0 ×10 -6 mol/L,and the detection limit was 5.2 ×10 -10 mol/L.Conclusion The fabricated sensor is demonstrated to be sensitive and specific for the detection of Ebola virus nucleotide .

Objective To investigate the status of clinical nutrition management in patients in PICU,and to provide data for promoting the improvement and development of clinical nutrition of pediatric critically ill patients.Methods A questionnaire survey was conducted on PICU specialists.The nutrition assessment,nutrition intervention and nutrition management of critically ill children in PICU were investigated.The results were summarized and analyzed.Results A total of 39 PICU specialists were involved in this survey.The nutritional assessment methods and guidelines in domestic PICU were not unified.Twenty-five respondents (64.1%) believed that both clinical performance and the scales as the basis can decide whether the patients should be fed or not;all respondents believed that nutritional assessment and intervention time need to be determined by the needs of the patients;23 respondents(58.9%) used weight only as their nutritional monitoring indicators.Twenty-eight respondents(71.8%) considered that gastric tube was the first choice way to feeding for the critically ill children;20 respondents(51.3%) believed that critically ill children should be fed within 24 hours.Twelve respondents (33.3%) believed that critically ill children should be fed between 24 to 48 hours.Thirty-three respondents(84.6%) advocated early enteral nutrition;36 respondents (92.3%) considered that the main reasons of fasting in critically ill children were vomiting or abdominal distension or gastrointestinal bleeding.Twenty-eight respondents(71.8%) believed that according to the results of gastrointestinal function evaluation,they made decisions whether the patient to fast or not.Twenty-three respondents(59.0%) considered that specialists in PICU were the decision maker of the clinical nutrition in critically ill children.Twenty-four (61.5%) of the respondents believed that we needed to establish our own routines in management of nutrition in PICU.Conclusion At present in China,a lot of achievements have been made in the nutritional assessment,monitoring,early enteral nutrition intervention and management in critically ill children,but it is not enough.We need to make more effort to enhance the critically nutrition level in PICU,and we have a lot of research to do about nutrition assessment and nutrition intervention mode.It is recommended to establish Chinese guidelines or consensus to enhance the level of nutritional treatment of critically ill children.