OBJECTIVETo investigate the effects of survivin antisense oligonucleotide (ODN) on cell proliferation and apoptosis of HL-60 cells.

METHODSSynthetic ODN was completely phosphorothioate-modified. Cationic lipid-mediated antisense ODN was transferred into HL-60 cells. The expression of survivin mRNA and protein was detected by RT-PCR and Western Blot. The incorporation of MTT was used as the measurement of HL-60 proliferation. The cell-cycle and apoptosis were analyzed by flow cytometry.

RESULTSHL-60 cells spontaneously expressed survivin mRNA and protein. Both mRNA and protein expression of survivin decreased significantly in the antisense ODN transfected cells in comparison to that in the original cells and cells transfected with sense ODN. Survivin antisense ODN significantly inhibited cell proliferation and induced apoptosis in a dose-dependent manner. The cell-cycle in the antisense ODN-transfected cells stopped at the G2/M phase.

CONCLUSIONSAntisense ODN targeting at survivin mRNA can inhibit HL-60 cell proliferation and induce G2/M stop and apoptosis.

Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; HL-60 Cells ; drug effects ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; antagonists & inhibitors ; genetics ; Neoplasm Proteins ; antagonists & inhibitors ; genetics ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis

BACKGROUNDSubcutaneous specific immunotherapy has been demonstrated to be capable of inducing T-cell regulatory response. Interleukin-10 (IL-10) plays a crucial role in inducing allergen-specific tolerance. However the reports of the changes of IL-10 in house dust mite (HDM)-specific immunotherapy were varied. The aim of this study was to evaluate the function of IL-10-secreting regulatory T cells in asthma children successfully treated with HDM immunotherapy.

METHODSPeripheral blood mononuclear cells (PBMCs) were isolated from 27 patients following 1.5 - 2 years of HDM-specific immunotherapy (SIT, SIT group) and from 27 matched treated asthmatic children allergic to HDM (asthma group). After 48 hours of in vitro stimulation with HDM extracts, IL-10-secreting regulatory T cells were measured by four colour flow cytometry. Sera were tested for allergen-specific IgG(4) and IgE using the Immuno CAP 100 assay.

RESULTSPBMCs from children undergoing immunotherapy following HDM extracts stimuli produced significantly more IL-10 compared with the asthma group. The frequency of iTreg cells and aTreg cells increased in SIT group after HDM stimulation, while it was not affected in the asthma group. Among the iTreg cells and aTreg cells, the frequency of CD4(+)CD25(-)Foxp3(-)IL-10(+) Treg cells increased the most which was 2 times higher than that in unstimulated cultures in SIT group. The levels of HDM-specific IgG(4) of SIT group was significiently higher compared with asthma group, but there was no correlation of the levels of HDM-specific IgG(4) and IL-10 secreting Treg cells.

CONCLUSIONSHDM-specific immunotherapy can successfully upregulate the frequency of IL-10-secreting Treg cells. CD4(+)CD25(-)Foxp3(-)IL-10(+) Treg cells may play a key role in inducing the immune tolerance in HDM-specific immunotherapy.

Animals ; Asthma ; immunology ; therapy ; Child ; Female ; Flow Cytometry ; Humans ; Immunotherapy ; Interleukin-10 ; secretion ; Male ; Pyroglyphidae ; immunology ; T-Lymphocytes, Regulatory ; immunology

Objective To study the effect of low-frequency suprathreshold repetitive transcranial magnetic stimulation (rTMS) of the unaffected hemisphere on recovery of motor function in patients with acute stroke. Methods A total of 26 patients with middle cerebral artery territory infarction were randomly assigned to unaffected hemisphere stimulation group and control group (not receiving any stimulation,n=13).The patients in the stimulation group were treated with rTMS 3 to 5 d after the onset of symptoms with the frequency of 1 Hz and 70％ of the intensity (about 2.1T actual output) and the 1200 pulses per day for 10 consecutive d.The motor evoked potential (MEP) latency,central motor conduction time (CMCT),scores of National Institutes of Health Stroke Scale (NIHSS) and modified Barthel index (MBI) of the affected brain region were recorded on the 1 st of experiment (before the treatment),10 and 40 d after treatment. Results The scores of clinical futction scale and neuroelectrophysiologic parameters before treatment had no statistical significance between the 2 groups (P＞0.05).The scores of clinical function scale after the treatment in the 2 groups were obviously higher than those before treatment (P＜0.05). And the improvement of motor function in the unaffected hemisphere stimulation group was statistically obvious as compared with that in the control group (P＜0.05):the score of NIHSS and the MBI in the stimulation group were obviously higher than those in the control group (P＜0.05).The neuroelectricity physiological indexs in the 2 groups after treatment gained improvement in comparision to those before treatment:the MEP latency on the 40th d of treatment and CMCT on the 10th and 40th d of treatment in the unaffected hemisphere stimulation group was significantly different as compared with those before treatment (P＜0.05); the CMCT on the 10th and 40th d of treatment in the unaffected hemisphere stimulation group was shorter as compared with that in the control group. Conclusion The frequency of 1 Hz and 70％ of the intensity (about 2.1T actual output) in rTMS of the unaffected hemisphere can shorten CMCT and improve the motor function in patients with acute stroke.

E2 gene of classical swine fever virus (CSFV) was cloned into secretory pPIC9K Pichia pastoris expression vector. After being linearized by digestion, the vector was transformed into Pichia pastoris by electroporation to integrate with the genome, the transformants with high copies were screened by G418 and were induced to express with methonal. The results of SDS-PAGE and Western blot demonstrated that the supernatant of the induced P. pastoris culture contained protein E2. The results of the study on the immunological activity indicated that the protein E2 expressed in P. pastoris can elicit animal bodies to produce antibodies against protein E2.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Classical swine fever virus ; genetics ; immunology ; Cloning, Molecular ; Gene Expression ; Pichia ; Rabbits ; Swine ; Viral Envelope Proteins ; genetics ; immunology

The major antigen region of E2 gene of Hog Cholera Prevalent Strain (Guangxi Yuling Strain) and Chinese Hog Cholera Lapinised Virus (C-strain) derived from hog and rabbit spleen tissue, was amplified by reverse transcription polymerase chain reaction(RT-PCR) and the nested Polymerase Chain Reaction (nPCR). After the amplified fragments were cloned into the expression vector pPROEX-HTb, the recombinant plasmids pPROEX-GXYL and pPROEX-C were obtained. The insert position, the size and the reading frame were right by PCR, restriction digestion and the sequence analysis. SDS-PAGE indicated that both of the reciepient germs transducted and induced by the recombinant plasmids pPROEX-GXYL and pPROEX-C could express the major antigen region of E2 gene. Western-blot indicated that the expressed antigen protein could be recognized by the positive serum of CSFV.
Blotting, Western ; Cloning, Molecular ; Escherichia coli ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo explore the location of telocytes in pulmonary tissues.

METHODSElectron microscopy, immunohistochemistry, primary cell culture and vital cell staining were used to identify the distribution of telocytes in mouse bronchial and pulmonary tissues.

RESULTSTelocytes were identified in the interstitial space between bronchial cartilage (cricoid) and smooth muscle by scanning and transmission electron microscope in mouse. By transmission electron micrscope and immunohistochemistry, telocytes were found in the interstitial spaces of lung parenchyma in connection with capillaries and bronchia. Telocytes expressed CD34, c-kit and vimetin by immunohistochemistry. After isolation, cultured telocytes demonstrated typical morphological feature, i.e. cells with telopode, which was seen as luminal structures with alternating thin and thick segments under electron microscope.

CONCLUSIONTelocytes are present in the interstitial space between cricoid cartilage and smooth muscle.

Animals ; Antigens, CD34 ; metabolism ; Bronchi ; cytology ; metabolism ; ultrastructure ; Cell Culture Techniques ; Immunohistochemistry ; Interstitial Cells of Cajal ; cytology ; metabolism ; ultrastructure ; Lung ; cytology ; metabolism ; ultrastructure ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Proto-Oncogene Proteins c-kit ; metabolism ; Vimentin ; metabolism

OBJECTIVETo summarize the experience of diagnosis and treatment of esophageal leiomyoma.

METHODSClinical data of 52 patients with esophageal leiomyoma were analyzed from 1993 to 2002.

RESULTSAbout 54% patients in this group had difficulty of food intake. The diagnostic accuracy of gastrointestinal barium meal series, computed tomography, gastric endoscope and endoscopic ultrasonography (EUS) for esophageal leiomyoma was 64% 44% 27% and 90% respectively. All patients received operation, resection of esophageal leiomyoma by videothoracoscopy (VAS) and endoscope were performed in 6, 9 patients respectively. The remaining 37 patients received regular open operation,in whom 32 cases received enucleation of esophageal leiomyoma, 5 cases received partial esophageal resection and esophageal-gastric anastomosis. No serious complications occurred except only one case needed operation again because of bleeding.

CONCLUSIONEUS is an effective method for diagnosing esophageal leiomyoma. VAS and endoscopic treatment should be considered for suitable cases in order to reduce the trauma.

Esophageal Neoplasms ; diagnostic imaging ; surgery ; Female ; Follow-Up Studies ; Humans ; Leiomyoma ; diagnostic imaging ; surgery ; Male ; Middle Aged ; Thoracoscopy ; Ultrasonography

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.
Genetic Markers ; Green Fluorescent Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Plasmids ; Recombination, Genetic ; Tobacco ; genetics

OBJECTIVETo investigate the incidence of HEV infection in population of non-remunerate blood donors in Shaoxing.

METHODSBlood specimens from 3701 non-remunerate blood donors were collected, ELISA were used to study anti-HEV IgG and IgM antibodies, RT-PCR were further used to study HEV RNA in samples from donors whose anti-HEV IgM was positive.

RESULTSAnti-HEV IgG positive rate was 29.19% (1107/3701), anti-HEV IgM positive rate was 1.35% (50/3701) among non-remunerate blood donors in Shaoxin. Six cases were positive for HEV RNA. The positive rate was 0.16% (6/3701), and all the 6 cases belonged to HEV genotype 1. Different seasons showed no interference on the positive rate of IgG and IgM.

CONCLUSIONThe detecting and studying of HEV infection among donors was important to ensure the safety of blood products and blood transfusion.

Adolescent ; Adult ; Age Factors ; Blood Donors ; China ; epidemiology ; Enzyme-Linked Immunosorbent Assay ; Female ; Hepatitis E ; blood ; epidemiology ; virology ; Hepatitis E virus ; classification ; genetics ; immunology ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood ; Male ; Middle Aged ; Phylogeny ; RNA, Viral ; blood ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

OBJECTIVETo assess the quality control for the maximal expiratory flow-volume (MEFV) curve in school-age children.

METHODSEight hundred and sixty-two children who had two or more MEFV manoeuvres were classified into ≥6-year-old (n=379), ≥8-year-old (n=210), ≥10-year-old (n=64), and 12-17-year-old groups (n=109). The parameters of quality control and concordance with quality control criteria for MEFV were compared between the two groups. In addition, patients who were diagnosed with asthma were classified into two groups, one with normal pulmonary function (n=155) and the other with abnormal pulmonary function (n=62), based on the results of spirometry. Differences in the parameters of quality control for spirometry were compared between the two groups.

RESULTSEight hundred and sixty-two children underwent 2 367 MEFV manoeuvres, 97.8% of which met the start of test criterion for backward extrapolated volume (VBE) of less than 0.15 L, with the highest concordance in the &ge;6-year-old group and the lowest concordance in the 12-17-year-old group. Three hundred and eighty-one children (44.2%) met the end of test criterion for forced expiratory time (FET) and the concordance in children over 10 years of age was lower than that in children under 10 years of age (P<0.05). Differences in two best forced expiratory volume in first second (FEV1) and forced vital capacity (FVC) manoeuvres were within 150 mL in 91.9% and 84.8%, respectively, of the children. The parameters of quality control for spirometry were better for asthmatic children with abnormal pulmonary function compared with asthmatic children with normal pulmonary function (P<0.05).

CONCLUSIONSConcordance with the start of test criteria and the manoeuvre repeatability criteria is high, whereas the concordance with the end of test criteria is low. It is suggested that the concordance with the FET criteria should be improved.

Adolescent ; Age Factors ; Child ; Female ; Forced Expiratory Volume ; Humans ; Male ; Maximal Expiratory Flow-Volume Curves ; Quality Control