Objective To evaluate the influential factors of iron acquisition during Francisella tularensis LVS infection of mouse macrophages.Methods F.tularensis LVS expressing green fluorescent protein was used to infect murine macrophage J774A.1 cells.Transferrin receptor 1(Tfr1)was detected with mono-antibody and visualized with a goat-anti mouse IgG conjugated to Alexa 594.The expression profile of 5 iron metabolism related genes of J774A.1 murine macrophages uninfected or infected with F.tularensis LVS was determined with real-time PCR.Immunoblot analysis was used to compare the Tfr1 expression of live Francisella infected macrophage with dead bacteria.Tfr1 knock-off in J774A.1 cells was performed with siRNA.The transfected cells were infected with Francisella for immunoblotting and microscopy and infection assay.Results It was revealed that the live vaccine strain of F.tularensis induced the expression of Tfr1 in host macrophages.Gene expression analysis indicated that F.tularensis LVS drove an active iron acquisition program with induction of Tfr1 and iron regulatory proteins(Irp1 and Irp2).It was shown by Western-blotting that the siRNA-Tfrc-1 could knock off about 75% of Tfr1 in J774A.1 cells.It was determined by infection assay that,Tfr1 was knocked off,the bacteria number at 1h infection with Francisella was not different from that of control(F=1.06,P=0.326 5),while it was decreased significantly after 24h of infection(F=24.12,P=0.000 6).Conclusions It is demonstrated that upregulation of the Tfr1 may be mediated by post-transcriptional regulation during early infection,but sustained later through increased expression of Irp 1 and Irp 2.Increased expression of Tfr1 expands the intracellular iron pool through transferrin-mediated delivery and may thus be readily available for uptaking by Francisella.Knocking off the expression of Tfr1 does not affect bacterial invasion.Francisella,however,may fail to proliferate in macrophages in which the expression of transferrin receptor has been suppressed.

Objective To assess the efficacy and safety of monoclonal antibody rituximab combined with cyclophosphamide (CTX) in the treatment of refractory and recurrent autoimmune hemolytic anemia.Methods Seven cases with refractory and recurrent autoimmune hemolytic anemia ( including 1 case of Evans syndrome) were recruited during January,2007 to December,2010.Treatment regimens were as follows:rituximab:375 mg/m2,1 time/week,2-6 courses; CTX:1 g,1/10 d,2-7 courses; combined with intravenous immunoglobulin (IVIG) 5 g,1 time/week,given 1 day after rituximab administration.The efficacy and safety of this regimen were assessed during follow-up.Results All the patients showed good responses (7/7).Six patients achieved complete remission (6/7) and one achieved partial remission ( 1/7 ).Average follow-up time for the patients was 27 months.All patients remained in remission during the 12-month follow-up visits.Two patients showed elevated indirect bilirubin and increased reticulocyte counts within 24 months.One patient achieved complete remission after additional rituximab therapy,and another patient remained partial remission after cyclosporine therapy.At the time of 36-month follow-up visit,the patient relapsed and was retreated with 3 courses of rituximab combined with CTX and eventually achieved partial remission.All patients tolerated the treatment well with few mild side effects.Conclusions Rituximab combined with CTX is effective and relatively safe in patients with refractory and recurrent autoimmune hemolytic anemia.Additional treatment to relapse patients about 12-24 months after drug withdrawal continues to be effective.

OBJECTIVETo establish fluorescent quantitative PCR method for detecting human herpes virus type 6 (HHV6).

METHODSAccording to the specific sequence of human herpes virus type 6 genes, the primers and the fluorescent probe (TaqMan) were designed and synthesized. The fragment generated from HHV 6 gene as template was cloned into the pMD18-T vector which was constructed from the pUC 18. And the positive recombinant plasmid was 1:10 diluted and used as standard quantitative template to make the standard curve for sample detection.

RESULTSThe standard curve indicated the linear relationship between Ct (cycle threshold) and template concentration. The clinical samples from 135 cases were detected by this system, 16 cases among 135 were positive.

CONCLUSIONThe fluorescent quantitative PCR method for the detection of human herpes virus type 6 is simple and accurate, and this method may be helpful to clinical diagnosis.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; DNA Primers ; DNA Probes ; DNA, Viral ; genetics ; Female ; Herpesvirus 6, Human ; genetics ; Humans ; Male ; Middle Aged ; Polymerase Chain Reaction ; methods ; Purpura, Thrombocytopenic, Idiopathic ; diagnosis ; virology ; Reproducibility of Results ; Roseolovirus Infections ; diagnosis ; virology ; Sensitivity and Specificity ; Young Adult

OBJECTIVETo compare the differences in the efficacy on distant version of naked eye in the patients of juvenile myopia between rotating manipulation and lifting-thrusting manipulation of acupuncture.

METHODSOne hundred and twenty cases (240 eyes) were randomized into a rotating manipulation group and a lifting-thrusting manipulation group, 60 cases (120 eyes) in each group. Additionally, a corrective lenses group, 60 cases (120 eyes), was set up as the control. In both manipulation groups, Cuanzhu (BL 2),Yuyao (EX-HN 4), Sizhukong (TE 23), Taiyang (EX-HN 5), Fengchi (GB 20), Zusanli (ST 36), Guangming (GB 37) and Sanyinjiao (SP 6) were punctured, but stimulated with rotating manipulation and lifting-thrusting manipulation respectively three times per week, 10 times as a treatment session and totally one session was required. In the corrective lenses group, the glasses were applied at daytime. The clinical efficacy and the changes in distant vision of naked eye before and after treatment were compared among the three groups.

RESULTSThe total effective rate was 87.5% (105/120) in the rotating manipulation group, which was better than 69.2% (83/120) in the lifting-thrusting manipulation group (P < 0.05). The distant vision of naked eye was improved apparently in the rotating manipulation group and the lifting-thrusting manipulation group after treatment (both P < 0.05). But it was not improved in the corrective lenses group (P > 0.05). The distant vision of naked eye was improved more apparently after treatment in the rotating manipulation group as compared with that in the lifting-thrusting manipulation group (0.75 +/- 0.23 vs 0.68 +/- 0.24, P < 0.05). For 96 cases (192 eyes) with acupuncture treatment, in 3-month follow-up, 87.0% (167/192) of the cases maintained the stable vision as the original level and 13.0% (25/192) of them were reduced in the vision In the acupuncture groups, it was found that the improvement of distant vision of naked eye was more obvious after treatment with younger age, better basic vision and shorter duration of sickness (all P < 0.05).

CONCLUSIONAcupuncture achieves the positive and sustainable clinical effect on juvenile myopia, and the results of rotating manipulation are superior to that of lifting-thrusting manipulation. Age, basic vision and duration of sickness impact the clinical efficacy.

Acupuncture Points ; Acupuncture Therapy ; instrumentation ; methods ; Adolescent ; Child ; Female ; Humans ; Myopia ; therapy ; Young Adult

BACKGROUNDThe goal of total knee arthroplasty (TKA) is to restore knee kinematics. Knee prosthesis design plays a very important role in successful restoration. Here, kinematics models of normal and prosthetic knees were created and validated using previously published data.

METHODSComputed tomography and magnetic resonance imaging scans of a healthy, anticorrosive female cadaver were used to establish a model of the entire lower limbs, including the femur, tibia, patella, fibula, distal femur cartilage, and medial and lateral menisci, as well as the anterior cruciate, posterior cruciate, medial collateral, and lateral collateral ligaments. The data from the three-dimensional models of the normal knee joint and a posterior-stabilized (PS) knee prosthesis were imported into finite element analysis software to create the final kinematic model of the TKA prosthesis, which was then validated by comparison with a previous study. The displacement of the medial/lateral femur and the internal rotation angle of the tibia were analyzed during 0-135° flexion.

RESULTSBoth the output data trends and the measured values derived from the normal knee's kinematics model were very close to the results reported in a previous in vivo study, suggesting that this model can be used for further analyses. The PS knee prosthesis underwent an abnormal forward displacement compared with the normal knee and has insufficient, or insufficiently aggressive, "rollback" compared with the lateral femur of the normal knee. In addition, a certain degree of reverse rotation occurs during flexion of the PS knee prosthesis.

CONCLUSIONSThere were still several differences between the kinematics of the PS knee prosthesis and a normal knee, suggesting room for improving the design of the PS knee prosthesis. The abnormal kinematics during early flexion shows that the design of the articular surface played a vital role in improving the kinematics of the PS knee prosthesis.

Adult ; Arthroplasty, Replacement, Knee ; methods ; Biomechanical Phenomena ; Female ; Humans ; Knee Prosthesis

OBJECTIVETo study the current situation and factors influencing the behavior of fruit and vegetable intake in adolescents, and to discuss health education strategy related to the behavior.

METHODS1677 students were selected from primary school and middle school in urban area of Hangzhou by a multistage sampling method. The behavior of fruit and vegetable intake was surveyed by questionnaires of the patient-centered assessment and counseling for exercise plus nutrition project (PACE+) for adolescent-scale of fruit and vegetable consumption, and analyzed by Transtheoretical Model on the servings of intake, the stage of change, process of change, decisional balance and the current status of self-efficacy.

RESULTSThe average intake of fruit and vegetable in adolescents was (3.21 +/- 1.50) servings per day ("one serving" means 100 g cooked vegetable or 100 g fruit). The behavior of consuming fruit and vegetable in most of the adolescents was in the contemplation stage, accounting for 36.91% (619/1677). The process of behavior change, decisional balance (Pros) and self-efficacy existed a positive correlation with the process of stage (r(process) = 0.38, r(decisional balance (Pros)) = 0.26, r(self-efficacy) = 0.33, t values were 16.78, 11.02 and 14.31, P < 0.05). The servings of fruit and vegetable intake existed a positive correlation with stage transition and self-efficacy (beta(stage transition) = 0.665, t = 35.07, P < 0.05; beta(self-efficacy) = 0.050, t = 2.63, P < 0.05), and existed a negative correlation with decisional balance (Cons) (beta(decisional balance (Cons)) = -0.051, t = -2.84, P < 0.05).

CONCLUSIONFruit and vegetable intake of these adolescents are under the recommended dietary intake. Along with behavior stage development and self-efficacy improvement, the intake of fruit and vegetable also increased correspondingly. Intervention strategies should aim at improving the awareness of adolescents on the health benefits of fruit and vegetable intake and promoting their confidence to reach the recommended servings.

Adolescent ; Adolescent Nutritional Physiological Phenomena ; Child ; China ; Eating ; Feeding Behavior ; Female ; Fruit ; Humans ; Male ; Models, Theoretical ; Students ; Surveys and Questionnaires ; Vegetables

Laser-induced breakdown spectroscopy (LIBS) was used for analysis of the distribution of S,Mn,Fe,Cr,Mo,Si,Al in a 34CrNiMo6 steel sample cut from a main shaft of wind driven generator.The MnS inclusion area in each ablation craters cover zone was extracted in the way of comparing the metallograph captured by optical microscopy before and after LIBS scanning ablation.The statistic relation between MnS inclusion area and signal intensity of S and Mn was analyzed.The result showed that the abnormal signal of S and Mn occurred at the same position with the existence of MnS inclusion,and their signal intensity showed linear relationship.The abnormal signal of S and Mn were triggered mainly by MnS inclusion.The statistic result also showed linear relationship between signal intensity and MnS inclusion area both for S and Mn.It was possible to determine the inclusion type,size and distribution by analyzing abnormal signal.A simplified ablation model was established to calculate the relation of S and Mn content to MnS inclusion area.The arithmetic result showed a linear relation between the content and MnS inclusion area both for S and Mn.The calculation confirmed the linear relationship between signal intensity and inclusion area observed in experiment.The linear relationship could be interfered by macro-segregation,micro-segregation,deviation in measuring inclusion area,and inclusion spatter in pre-ablation.

OBJECTIVETo explore in-vivo targeted imaging techniques for liver cancer detection using quantum dots (QDs) labeled probes in a nude mouse model of human hepatocellular carcinoma.

METHODSMercaptoacetic acid (MAA) modified QDs were linked to mouse-anti-human alpha-fetoprotein (AFP) monoclonal antibody to form water soluble QD-AFP-Ab probes, which were validated by spectra analyses and transmission electron microscope. The probes were firstly used to detect AFP antigen in human hepatocellular carcinoma cell line HCCLM6 in-vitro by one-step immunofluorescence method. In-vivo tumor xenografts and lung metastases models were then established by inoculation of HCCLM6 cells subcutaneously and into the tail vein of nude mice, respectively. QD-AFP-Ab probes were injected into the tail vein of the tumor bearing mice for live animal fluorescence imaging. Spectra of tumor and normal tissue were analyzed under illumination of Ti: sapphire laser. Serum levels of alanine amino transferase, aspartate amino transferase, blood urea nitrogen and creatinine were determined by conventional biochemical analysis. The liver, spleen, lungs, kidneys, heart and brain of the experimental nude mice were investigated for nonspecific uptake of the probes by confocal microscope.

RESULTSThe QD-AFP-Ab probes had broad excitation spectra and high fluorescence intensity. They could specifically and efficiently recognize AFP antigen in hepatocellular carcinoma cells. Tumor targeting imaging using these probes were successful without any acute toxicity to the experimental animals. Spectra analysis showed that the probes per field were lower in the centre than the periphery of the tumor. Non-specific uptake of QD-AFP-Ab probes occurred mainly in the liver, spleen and lungs.

CONCLUSIONSQD-AFP-Ab probes have good optical properties and biocompatibility for in-vivo targeted imaging of hepatocellular carcinoma. Such approach promises to be highly desirable for molecular targeted research of liver cancer.

Animals ; Antibodies, Monoclonal ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Diagnostic Imaging ; methods ; Fluorescent Antibody Technique ; methods ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Lung Neoplasms ; metabolism ; secondary ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microscopy, Fluorescence ; Molecular Probes ; metabolism ; pharmacokinetics ; toxicity ; Neoplasm Transplantation ; Quantum Dots ; Tissue Distribution ; alpha-Fetoproteins ; immunology ; metabolism

Objective To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD3+ T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity.Methods CD3+ T cells were sorted by magnetic activated cell-sorting system.The expressions of TET2 and DLK1 mRNA in bone marrow CD3+ T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR.Results The expression of TET2 mRNA in CD3+ T cells was down-regulated in the MDS patients by (0.16 ±0.15) fold compared with the controls ( P ＜ 0.05 ).The expression of TET2 mRNA in CD3+ T cells of MDS patients was positively correlated with serum complement C3 ( r =0.404,P ＜ 0.05 ).The expression of DLK1 mRNA in CD3+ T cells was up-regulated in the MDS patients by (1.61 ±0.88) folds compared with the controls (P＜0.05).Grouped by the chromosomes,the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [ ( 1.45 ± 0.44 ) folds,P ＜0.05 ].The expression of DLK1 mRNA in CD3+ T cells of MDS patients was positively correlated with the proportion of bone marrow blasts ( r =0.343,P ＜ 0.05 ).Conclusions The mRNA expression of TET2 in CD3+ T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased,which might decline the immune surveillance function.The findings would be useful for exploring the mechanism of immune tolerance.

Objective To investigate the effects and related factors of itraconazole in the treatment of invasive fungal infection (IFI) in the patients with blood diseases ( BD). Methods A total of 156 BD patients with IFI treated with itraconazole in General Hospital, Tianjin Medical University from 2005 to 2008, were retrospectively analyzed. Results Of these patients, 92 were with underlying malignant BD, and 64 with non-malignant BD; 77 possible IFI, and others proven IFI. A total of 94 (63. 5% ) patients were responded to itraconazole successfully, while 54 (36. 5% ) failed. The underlying malignant BD, post-chemotherapy, neutrophil count less than 0. 5 x 109/L, positive fungus culture, and bacteria infection were related with the response to itraconazole significantly, while patient's age, application of other antibiotics,positive C test, IFI localization, haemoglobin level and platelet counts were not Five patients was changed other anti-IFI therapy because of side effects, including gastrointestinal ill (3 cases with nausea or vomiting) and tachycardia (2 cases). Conclusions Itraconazole was effective and safe in the treatment of IFI in the patients with BD. Underlying malignant BD, agranulocytosis, bacteria infection, and delayed anti-IFI therapy might reduce itraconazole therapeutic effects.