Objective To observe the in vivo gene expression profile of the recombinant adenoassociated-2 virus mediated human GM-CSF, mouse GM-CSF (rAAV-2-hGM-CSF, rAAV-2-mGM-CSE )vector modified bone marrow mesenchymal stem cell (BMSC). Methods We transduced the BMSC by rAAV-2-hGM-CSF, rAAV-2-mGM-CSF at the condition which have acquired before respectively, then transfused the in vitro gene modified BMSC after 12 days proliferation in vitro to 6 weeks old nude mice through tail vein,while the BMSC transfused in control group hadn' t been gene modified. 2, 4, 6, 8 weeks after transfusion, count the total white blood cells and detect the hGM-CSF, mGM-CSF concentration in nude mice serum at that time point. Results Nude mice serum hGM-CSF levels were 23.77, 25.32, 19.77, 15.25 ng/L at 2, 4, 6, 8 weeks after transfusion compare to 36.25 ng/L, the in vitro level before transfusion; mGM-CSF levels were 34.96, 34.84, 35.50, 32.93 ng/L at 2, 4, 6, 8 weeks after transfusion compare to 25.14 ng/L, the in vitro level before transfusion; at the same time point the nude mice serum mGM-CSF levels were 17.34,17.44, 14.68, 16.85 ng/L in control group, rAAV-2-mGM-CSF transduced BMSC made the nude mice white blood cell count increased, but no changes in nude mice white blood cell count at rAAV-2-hGM-CSFtransduced BMSC and control group. Conclusion BMSC as a gene therapy vehicle, it can be gene modified in vitro, then the gene modified BMSC could let the therapeutic gene to have therapeutic effects in vivo.

Objective To investigate the changes of IL-8 in peripheral blood of patients with pustular psoriasis after acitretin treatment.Methods Dual antibody sandwich enzyme-linked immunosorbent assay (ELISA)was performed to measure the IL-8 levels in the peripheral blood of 30 patients with pustular psoriasis and 30 patients with psoriasis vulgaris before and after treatment,and in 30 normal human controls.The relationship between the IL-8 level and disease severity was assessed for patients with pustular psoriasis.Results After acitretin treatment,the condition was improved greatly in patients with pustular psoriasis,together with a significant decrease in the level of IL-8 in the peripheral blood(57.07 ± 12.02 pg/ml vs.96.84 ± 14.68 pg/ml,P ＜ 0.05).Conclusion IL-8 may be involved in the therapeutic mechanism of acitretin in pustular psoriasis.

BACKGROUND: Recombinant adeno-associated virus 2(rAAV-2) has attracted considerable attention due to its nonpathogenic nature in contrast to other viral vectors such as adenoviral and retroviral vectors in gene therapy attempts.OBJECTIVE: To explore rAAV-2 transduction to bone marrow mesenchymalstem cell(BMSC) in vitro and evaluate the possibility of using rAAV-2 as a vector for gene therapy of acute myelogenous leukemia(AML).DESIGN: An open experiment with cells as the observational subjects.SETTING: Department of Hematology, Nanfang Hospital, Southern Medical University.MATERIALS: The experiment was conducted in the Department of Hematology, Nanfang Hospital, Southern Medical University from February to July 2004. We used passages 3 to 5 BMSCs derived from six de novo AML patients and four healthy volunteers in this study.METHODS: BMSC was isolated from 6 to 10 mL of bone marrow aspirates obtained from the iliac crests of the patients who had been diagnosed as having de novo AML and from those of healthy volunteers. The acquired BMSC was infected by rAAV-2 which contained enhanced green fluorescent protein (rAAV-2-eGFP) at different multiplicity of infection(MOI) (MOI = 1 × 102,1 × 103, 1 × 104, 1 × 105, 1 × 106, 1 × 107) . Then we observed through phase contrast fluorescent microscope and flow cytometer to evaluate green fluorescent protein(GFP) expression 10 to 14 days after transduction. GFP expression was observed as the rAAV-2-eGFP transduced BMSC cultured in vitro. We also observed the in vitro gene expression profile of GFP in rAAV-2-eGFP transduced BMSC which was selected by neomycin ( G418). First, we confirmed GFP expression in BMSC through phase contrast fluorescent microscope, then on flow cytometer to detect the percentage of GFP expression.MAIN OUTCOME MEASURES: The efficiency of rAAV-2-eGFP transduction to BMSC. GFP expression was observed through phase contrast fluorescent microscope and flow cytometer at different time points after transduction.rAAV-2-eGFP to BMSC derived from normal volunteers and AML patients had no significant differences. GFP began to express 10 to 14 days after transduction, and the transduction efficiency ranged from 0. 3% to 1.4%. By changing infection condition, we could not make a higher transduction efficiency( P ＞ 0.05) . One round infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was ( 1. 030 ± 0. 034) %, 3 rounds of infection of BMSC by rAAV-2-eGFP at a MOI of 1 × 105 was (1. 140 ±0. 036)%, and coinfected by LipofectAMINE was (1. 380 ± 0. 054)%. However, 293 cell line which was the package cell of rAAV-2 could be efficiently transduced by AML patients transduced by rAAV-2-eGFP at MOI = 1 × 105: The percentage of GFP expression cell gradually decreased from 1.14% at day 12 after transduction to 0. 6% as cell passaged from 2 to 3, and maintained at a level of 0. 5% to 0. 6% later on till 61 days after transduction. After selected by neomycin(G418) 1 month later, rAAV-2-eGFP transduced BMSCs could maintain a long-term GFP expression at a level of 6.0% in vitro without significant decay within 100 days of observation period after transduction.CONCLUSION: The advantages of rAAV-2 mediated gene transduction lie in safety, no immune response to the host, and long-term expression maintained by the target gene. rAAV-2 and BMSC can be used for in vitro gene therapy, and as a systemic gene delivery system, it might be an alternative for systemic gene therapy in the future.

China ; Humans ; Meningococcal Infections ; Serogroup

OBJECTIVETo document morphological changes in hepatic microcirculation in liver tissue with hepatitis B and the pathogenesis of hepatic microcirculatory disturbances.

METHODSLiver tissue samples were obtained from patients with hepatitis B by liver biopsy. These samples were examined with a light microscope and transmission electron microscope.

RESULTSHepatic microcirculatory disturbances existed in patients with hepatitis B, including those with normal liver function, manifested by red blood cell aggregation in sinusoids seen under light microscope and sinusoidal capillarization seen under electron microscope. Weibel-Palade bodies in sinusoidal endothelial cells were seen in 26 out of 53 cases. Intimate contacts were found between lymphocyte/Kupffer cells and sinusoidal endothelial cells.

CONCLUSIONSHepatic microcirculatory disturbances exist in patients with hepatitis B. The appearance of Weibel-Palade bodies in sinusoidal endothelial cells may be a key step in the development of hepatic microcirculatory disturbances.

Adolescent ; Adult ; Aged ; Female ; Hepatitis B, Chronic ; pathology ; physiopathology ; Humans ; Liver ; blood supply ; pathology ; ultrastructure ; Liver Circulation ; Male ; Microcirculation ; pathology ; ultrastructure ; Microscopy, Electron ; Middle Aged

Objective: To evaluate the post-marketing safety of inactivated Enterovirus type 71 (EV-A71) vaccine (human diploid cell) . Methods: A total of 20 191 healthy children aged 6 to 59 months were invited to receive 2 doses of EV-A71 vaccine in Zhejiang Province from September 2016 to December 2017. Child caregivers were followed up on the 4^th or 5^th day after each EV-A71 vaccination, and the incidence of local, systemic, and other adverse events within 3 days after vaccination was recorded to assess vaccine safety. Describe the differences in adverse events among children with different characteristics. Results: A total of 32 230 doses were observed in this study, of which 20 191 and 12 039 were vaccinated for the first and the second dose, respectively; and the incidence of adverse events within 3 days was 2.045% (413 doses) and 1.611% (194 doses), respectively. After the first and the second dose, the number of systemic adverse events was the highest, 371 and 175 cases, respectively, with an incidence of 1.837% and 1.454%, respectively; the number of local adverse events was the lowest, 14 and 2 doses, respectively, with an incidence of 0.069% and 0.017%. Local adverse events occurred after vaccination were generally mild, and only 2 patients had level of 3; among the systemic adverse events, 39 patients had a fever level of 3 or higher, accounting for 8.2% of the total fever. Most of the symptoms in the local adverse events did not require treatment, only 3 cases of vaccination site rash and 2 cases of pruritus were self-purchased drugs or outpatient treatment; except for 5 cases of fever, the other symptoms were not hospitalized in the case of systemic adverse events. Conclusion The incidence of adverse events within 3 days after vaccination with EV-A71 vaccine was low in children, mainly systemic adverse events, and the prognosis was good.

Objective To study the spatial-temporal dynamical features of measles in Zhejiang province.Methods Data was from the China Disease Surveillance Information System and China Immunization Program Information Management System.Power-law method on spatial-temporal-multicomponent model was used to analyze the epidemic characteristics of measles in the districts of Zhejiang province.Results The incidence of measles in Zhejiang province was 2.72/ 100 000 (1 494 cases) in 2013.Compared to the first order adjacent matrix,Power-law method showed a lower value of Akaike information criterion.The follow-up impact from the previous measles epidemic was strong to the Keqiao,Xiaoshan and Yuecheng districts with the autoregressive component as 1.39,0.88 and 0.77,respectively.Local risk of measles seemed high in Keqiao,Qujiang and Xiaoshan districts with the endemic component as 4.06,3.74 and 3.55,respectively.Impact of the epidemic to the nearby districts was large in Keqiao,Shangyu districts and Jiande city with epidemic components as 3.08,2.54 and 2.21,respectively.Conclusion The spatial-temporal feature of measles in several districts of Zhejiang province appeared heterogeneous,suggesting the specific strategies should be taken to control the epidemics of measles.

Objective To evaluate the immunogenicity and safety of a booster dose of live attenua-ted measles-mumps-rubella (MMR) vaccine for 4-year-old children and to provide references for reasonable arrangement of MMR immunization schedule. Methods Children aged 4 years (54-60 months) old were recruited and divided into three groups as follows: Group 8 months MR [receiving live attenuated measles and rubella(MR) vaccination at 8 months and MMR vaccination at 18 months],Group 8 months MMR(re-ceiving MMR vaccination at both 8 and 18 months) and Group 12 months MMR(receiving MMR vaccination at both 12 and 22 months). Active follow-up was conducted for safety evaluation after immunization of all subjects with the booster dose of MMR vaccine. Blood samples were collected before and 35 days after vacci-nation and analyzed by ELISA to detect serum antibodies to measles,mumps and rubella. Results A total of 514 subjects were enrolled in this study of safety evaluation and 469 of them received serologic detection of antibodies twice. The rate of adverse reactions following vaccination was 17.12% (general reactions accoun-ted for 94.21%) and no severe adverse reactions were reported. No significant difference in the rates of ad-verse reactions was found among the three groups (χ2=4.82, P=0.090). Subjects who were seropositive for measles, mumps and rubella increased after immunization with MMR vaccine, accounting for 100%, 99.79% and 99.79%,respectively. Geometric mean concentrations (GMC) against measles, mumps and rubella in all subjects were 1.35,3.05 and 2.13 times higher than what they were before the immunization. Levels of antibodies to measles,mumps and rubella were all increased significantly in the three groups after immunization with the booster dose of MMR vaccine (Fisher Exact Test, P=0.000). Conclusion The booster dose of MMR vaccine increases the levels of serum antibodies in children aged 4 years old with high safety. It suggests that two doses of MMR vaccine should be encouraged in the immunization program in China.

Objective: To assess the 3-year antibody persistence after vaccination of domestic measles, mumps and rubella combined attenuated live vaccine (MMR) with different program. Methods: Children from three different vaccination strategies (Group 8 m MR: 8 months and 18 months vaccinated with measles-rubella combined attenuated live vaccine and domestic MMR,respectively; Group 8 m MMR: 8 months and 18 months both vaccinated with domestic MMR; Group 12 m MMR: 12 months and 22 months both vaccinated with domestic MMR ) were followed up in Zhejiang province in July 2015. There were 170 participants in Group 8 m MR, 171 participants in Group 8 m MMR and 173 participants in Group 12 m MMR selected by simple random sampling method .Blood samples (venous blood 2-3 ml) were collected 1 month after the first dose vaccination of MMR (only in Group 8 m MMR and Group 12 m MMR) and 3 years (36-38 months) after the last dose vaccination of MMR and tested for antibody IgG against Measles, Mumps and Rubella using ELISA. Seropostive rate and Geometric mean concentration (GMC) were calculated and compared among different groups by Chi-square test or Fisher exact test and Kruskal-Wallis H test. Results: A total of 514 participants (8 m MR: 170; 8 m MMR:171; 12 m MMR:173) were enrolled. The overall seropositivity rate of measles, mumps and rubella was 98.1% (504), 93.4% (480) and 88.1% (453), respectively, with corresponding GMC was 1 012.33 mU/ml, 502.87 U/ml and 50.53 U/ml respectively. There was no significant difference of seropositivity rate for measles among three groups (all groups were>97%). The highest seropositivity rate for mumps was found in the Group 12 m MMR with the rate of 98.8% (171/173), followed by Group 8 m MMR and Group 8 m MR with 93.0% (159/171) and 88.2%(150/170) respectively (Fisher exact test, P<0.001). The highest seropositivity rate for rubella was also found in the Group 12 m MMR with the rate of 94.8% (164/173), followed by Group 8 m MMR and Group 8 m MR with 86.6%(148/171) and 82.9%(141/170) respectively (Fisher exact test, P=0.002). The highest GMC of antibody against measles, mumps and rubella were all found in Group 12 m MMR, with 1 217.30 (1 119.35-1 323.82) mU/ml, 717.07 (643.83-798.65) U/ml and 62.54(56.21-69.58) U/ml respectively. The lowest GMC of antibody against measles and mumps were both in Group 12 m MR with 812.01 (734.52-897.67) mU/ml and 363.28 (305.42-432.11) U/ml respectively. The lowest GMC of antibody against rubella was in Group 8 m MMR with 44.10 (39.08-49.76) U/ml. These differences of GMCs among three groups were all reach significant means (P<0.05). Conclusion High level seropostive rates and GMCs were exist against measles and rubella after 3-year vaccination of domestic MMR among different program. Higher antibody level against mumps were found in those children with two doses vaccination of MMR.

Objective To study the effect of age-related white matter changes (ARWMC) on first symptomatic ischemic stroke events in the oldsters. Methods For the prospective study, a total of 368 eligible oldsters were enrolled in the study from January 2010 to August 2012. The degrees of ARWMC were assessed by ARWMC scale;according to the scores, they were divided into non ARWMC group, mild-moderate ARWMC group and severe ARWMC group. The patients were followed up once every 3 months. The clinical endpoint events and time (first symptomatic ischemic stroke, myocardial infarction and all-cause death) were recorded. Analyses of variance and Chi-square test were used to compare the differences of clinical data among the 3 groups. COX regression was used to assess the risk differences of first symptomatic ischemic stroke in the oldsters of three groups. Results After an average of follow-up for 48.7 months, 50 participants (13.6％) had first symptomatic ischemic stroke;25 (25.8％) were categorized as the severe ARWMC group, 22 (10.9％) were as the mild-medium group, and 3 (4.4％) were as the non ARWMC group. Among the three groups, the differences in age, history of hypertension, systolic blood pressure, incidence of clinical endpoint events and first symptomatic ischemic stroke, and follow-up time of endpoint events were statistically significant (P<0.05); patients from the severe ARWMC group were the oldest, and had the longest history of hypertension, the highest systolic blood pressure, the highest incidence of clinical end events and first symptomatic ischemic stroke, and the shortest follow-up period for clinical end events. COX regression analysis showed that the risk of first symptomatic ischemic stroke in the severe ARWMC group was about 8 times higher than that in the non ARWMC group (hazard ratio=9.012, 95％CI: 2.310-35.154, P=0.002). Conclusion In oldsters, severe ARWMC often accompany hypertension history and poor blood pressure controll, and it is an independent and serious risk factor for long-term first symptomatic ischemic stroke.