Humans ; Infant ; Lymphohistiocytosis, Hemophagocytic ; complications ; Male ; Mucocutaneous Lymph Node Syndrome ; complications

Linoleic acid (C18∶2n-6) and ?-linolenic acid (C18∶3n-3) are found widely in fungi, plants and some lower animals. However, they can not be synthesized in mammals due to lack of △12 and ?-3 fatty acid desaturases. To enable endogenous production of essential fatty acids in mammalian cells, here the stable expression of a Caenorhabditis elegans gene FAT-2 encoding △12 fatty acid desaturase in CHO cells was reported. First, the FAT-2 coding sequence was cloned by RT-PCR. To facilitate high level synthesis of heterogeneous protein, the codon usage of the fatty acid desaturase genes was optimized according to the codon preference of mouse by site-directed mutagenesis, 2 synonymous mutations were introduced into FAT-2 gene by overlapping PCR. The codon-modified gene was finally fused to pBudCE4.1 vector (Invitrogen) under the control of CMV promoter. The expression vector pBudCE-FAT2 was linearized with NheⅠ, and then transfected CHO cells, the cells were under Zeocin selection for nine days and then propagated, then the transfected cells were harvested. The genome and total RNA were isolated for PCR and Norhern blot ananlysis. The results revealed that FAT-2 gene has been integrated into the genome of CHO cells and expressed properly. Fatty acids of total cellular lipids were analyzed by gas chromatography. The results indicate that the expression and function of △-12 fatty acid desaturase resulted in accumulation of linoleic acid. The levels of linoleic acid in transgenic cells were 2.4-fold higher than those in wild-type cells. The moderate linoleic acid in CHO cells was derived from cell culture media uptaken by cell membrane. The results demonstrate that a heterogenous desaturase gene can function well in mammalian cells and prove that transgenic approach is an efficient strategy for changing fatty acid composition of mammals.

Objective To investigate jugular vein morphological changes with three dimension phase contrast magnetic resonance venography(3D PC MRV),and to explore hemodynamic features using MR phase contrast cine (MR PC cine).Methods Sixty-five healthy volunteers performed 3D PC MRV and MR PC-cine sannings.MRV ranged from torcular herophili to brachiocephalic veins, and the raw data of PC-cine was acquired at cervical 2-3(C2-C3)level perpendicular to the Jugular veins(JVs)with the maximum encoding velocity of 50 cm/sec.Jugular vein showing absent or tip shape(cross-sectional area less than 12.5 mm2 )was considered abnormal,and flat,crescent,oval,round shapes were considered normal.Data of PC-cine was processed by computer to evaluate the hemodynamic features.Results Nine (13.85%)of 65 cases were abnormal that unilateral jugular vein showing needle-pointed narrow or absent,and 8 cases on the left,and one case on the right;Weak correlation was found between jugular veins pattern and the age.The right sided values in volunteers were higher than that of the left side.Conclusion The morphology and hemodynamics of jugular veins in volunteers showed significant difference between sides,and weak correlation is found between the morphology and aging.

Objective To evaluate the deep brain venous blood oxygen content changes in patients with multiple sclerosis(MS) using susceptibility weighted imaging (SWI), and to explore the ability of SWI in reflecting the clinical condition. Methods Forty-four MS patients were prospectively enrolled in the study. All the clinical-proved patients meeting the McDonald standards (2005 revised) underwent conventional MRI, SWI, and 12 cases of them underwent MRI review from 12 to 16 months interval. all the patients' clinical condition were quantified according to the expanded disability status scale(EDSS). The score was 0.5—6.5. Sixty-five age- and gender- matched healthy volunteers underwent conventional MRI and SWI. The blood oxygen content of the deep brain venous were estimated by the veins phase value, and differential phase values of blood vessels and surrounding tissues (Δφ) were processed with SPIN software. The blood vessels consist of bilateral BV, SMCV, ICV, STV and FMV, PMV, OMV. The difference of Δφvalue in different veins between MS patients and the controls was compared using independent sample t-test, and the Δφ value comparison of MS patients in different time were performed by using paired t test; The correlation ofΔφvalue between MS and EDSS was analyzed using Spearman correlation. Results TheΔφvalue of BV, SMCV, ICV, STV were 856.6 ± 246.4, 600.6 ± 155.2, 965.9 ± 205.4, 844.2 ± 149.7 in MS, and 767.6±145.1, 536.2±123.5, 892.8±156.3, 783.1±148.5 in controls, respectively. TheΔφvalue was higher in MS patients than the controls (t=2.157, 2.303, 2.005, 2.103,P<0.05). The twelve patients'Δφvalues of BV, ICV,STV were 729.4±275.1, 906.1±219.2, 737.2±159.1 in the first time, and 923.2±211.6, 1017.3±211.1, 919.3 ± 165.9 in the second time, and all the values increased in the review of the interval of 12 to 16 months (t=-3.092,-6.420,-3.972,P<0.05). The phase value of PMV and OMV had significant positive correlation with EDSS scores(r=0.638, 0.642,P<0.01). Conclusions The state of hypoxia of the brain parenchyma appears in MS patients, and hypoxia may become worse with the extension of course. The extent of hypoxia can reflect the disability of the patients.

Objective To investigate the current status of secondary prevention of cerebral infarction in Longquanyi District of Chengdu. Methods Prospective Investigation on the secondary prevention was conducted in 94 pa-tients with cerebral infarction at 3, 12 and 24 months following stroke. Results The percentages of patients receiving anti-platelet therapy, antihypertensive treatment, antidiabetic drug treatment and statin therapy were 96.8%, 92.1%, 91.7%and 63.8%at hospital discharge, 79.8%, 88.9%, 91.7%and 55.3%at 3 months, 76.1%, 75.0%and 41.4%, 63.8%at 12 months and 33.0%, 71.4%;58.3%and 22.3%at 24 months, respectively. None of stroke patients with atrial fibrillation re-ceived anticoagulation therapy. Conclusion There is a large gap between the current practice of secondary prevention and guideline in patients with cerebral infarction in Longquanyi District of Chengdu.

Objective To establish a rabbit model of restenosis and analyze the expressions of VEGFmRNA and TGF-?_1mRNA during the intimal proliferation.We also explored the relationship between VEGFmRNA,TGF-?_1mRNA and restenosis.Methods 40 healthy male New Zealand white rabbits were evenly divided into three injury groups and one control group.Right carotid arteries were injured with PCI balloon in the injury groups.10 rabbits of each injury group were sacrificed on weeks 1,2 and 4 after the injury.VEGFmRNA and TGF-?_1mRNA were examined by in situ hybridization.All the samples were analyzed using a computerized imaging analysis system.Results In the injury groups,neointimal areas were significantly larger than those in control group(P

OBJECTIVEThis study examined the levels of exhaled nitric oxide (eNO) and peripheral blood eosinophils (EOS) as well as the correlation between the two markers in children with bronchial asthma (AS),AS complicated by allergic rhinitis (AS/AR) and chronic cough variant asthma (CVA), in order to explore the value of eNOS detection in children with AS.

METHODSThe eNO level was measured using light-emitting electrochemical photometry in 12 children with AS, 29 children with AS/AR and 10 children with CVA. Peripheral blood EOS was counted by blood cell counter (Coulter JT). Forced expiratory volume in one second (FEV1) was assessed by lung function measurement. Thirty children without atopic disease and acute respiratory infection as well as without a family history of atopic diseasea served as the control group.

RESULTSThe levels of eNO and blood EOS in the AS, the AS/AR and the CVA groups were significantly higher than those in the control group (p<0.01). The AS/AR group showed increased levels of eNO (50.3 + or - 6.7 ppb) and EOS (5.9 + or -4.2 x 109 ) compared with the AS (30.5 + or - 8.8 ppb and 4.2 + or - 3.2 x 109 respectively) and the CVA groups (26.0 + or - 3.2 ppb and 3.7 + or - 6.9 x 109 respectively) (p<0.05). There were no significant differences in eNO and EOS levels between the AS and the CVA groups. The eNO level was positively correlated with the EOS level (r=0.51, p<0.05), but not with FEV1 (r=0.144, p>0.05) in the AS group.

CONCLUSIONSNO is highly expressed in children with symptoms of atopy and can reflect the levels of eosinophilic airway inflammation in children with AS.

Adolescent ; Asthma ; blood ; physiopathology ; Breath Tests ; Child ; Child, Preschool ; Eosinophils ; physiology ; Female ; Forced Expiratory Volume ; Humans ; Male ; Nitric Oxide ; metabolism

OBJECTIVETo establish a transgenic cell line with stable expression of CD14.

METHODSTotal RNA extracted from peripheral blood mononuclear cells was treated with RNAase-free DNAase, the human CD14 gene was cloned and sequenced through the RT-PCR, T-A clone techniques and ABI PRISM377 machine. Eukaryotic expression vector pcDNA3.1(+)/CD14 was constructed by cleaving with double restriction endonucleases EcoR I/Xba I and ligating with T4 ligase. The human cervical cancer cell line Hela was transfected with the positive recombinant plasmid pcDNA3.1(+)/CD14 using superfect transfection reagent. Positive clones were selected by G418 at a concentration of 0.5 μg/μl and the expression of human CD14 on the transfected Hela cells was confirmed by quantitative PCR and immunofluorescent assay.

RESULTSThere was significantly difference om expression of CD14 mRNA between the blank pcDNA3.1(+) transfected cells and pcDNA3.1(+)/CD14 transfected cells (P<0.01). The fluorescence was significantly stronger on the stable cell line Hela-CD14 than that on the transiently transfected Hela cells,and no visible fluorescence was observed in blank vector transfected cells.

CONCLUSIONThe transfectant cell line Hela-CD14 with stable expression of human CD14 has been successfully established, which can be used to study human CD14 molecular and CD14-associated monocyte/macrophage cell diseases.

Female ; Gene Expression ; Genetic Vectors ; HeLa Cells ; Humans ; Lipopolysaccharide Receptors ; genetics ; metabolism ; Plasmids ; genetics ; Transfection

In order to provide the evidences for CD19 as a better antibody targeting molecule for B lineage acute leukemias than CD20 through the multi-parameter flow-cytometry analysis of leukemia cells, the samples from 321 patients with acute leukemia (AL) were immunophenotyped by multi-color flow cytometry and CD45/SSC gating strategy followed by the analysis of CD19 and CD20 expression. The results showed that the positive rate of CD19 (115/116, 99.1%) in 116 cases with B lineage acute lymphoblastic leukemia (B lineage ALL) was significantly higher than that of CD20 (33/116, 28.4%) (P < 0.01); in 17 patients with B lineage/Myeloid (B/My) acute mixed lineage leukemia (AMLL), the former positive rate (17/17, 100%) was also higher than the latter (5/17, 29.4%) (P < 0.01). Both of the two antigens were negative in 29 patients with acute T lymphoblastic leukemia and 7 patients with T/My AMLL. The positive rates of CD19 and CD20 in 152 patients with acute myeloid leukemia (AML) were 7.2% and 2.0%, respectively. The difference of the fluorescence intensity between the two antigens on the cells from each patient with B lineage ALL or B/My AMLL was statistically significant (t = 20.68, P < 0.001). The specificity of CD19 and CD20 in B lymphocytic lineage was 92.3% (132/143) and 92.7% (38/41), respectively, while the sensitivity was 99.2% (132/133) and 28.6% (38/133), respectively, the former sensitivity was significantly higher than the latter (chi(2) = 144.018, P = 0.001). It is concluded that CD19 continuously and steadily express on almost all subtypes of B lineage leukemic cells with homogeneous pattern while only a small number of leukemias express CD20. Both the specificity and sensitivity of CD19 were very high with a much broader reaction pattern than that of CD20 on this group of diseases. These indicate that CD19 may be a better antibody targeting molecule than CD20 for patients with B-lineage acute leukemia.
Acute Disease ; Adolescent ; Adult ; Antigens, CD19 ; biosynthesis ; Antigens, CD20 ; biosynthesis ; Bone Marrow Cells ; immunology ; metabolism ; pathology ; Cell Lineage ; Child ; Child, Preschool ; Female ; Flow Cytometry ; Humans ; Immunophenotyping ; Infant ; Leukemia ; blood ; immunology ; pathology ; Leukocytes, Mononuclear ; immunology ; metabolism ; pathology ; Male ; Middle Aged ; Tumor Cells, Cultured

OBJECTIVETo construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.

METHODSPrimers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.

RESULTThe cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.

CONCLUSIONThe ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Antibodies, Monoclonal ; Biotechnology ; methods ; Cells, Cultured ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Lipopolysaccharide Receptors ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics