OBJECTIVETo investigate the expression profile of interleuki-1β (IL-1β) in rat myocardium at different time points during hypoxia/reoxygenation(H/R)transition.

METHODSThe isolated Langendorff perfused rat heart model was established.Forty SD rats were randomly divided into sham group (A group) and hypoxia/reoxygenation group (H/R group). The H/R group rats were subdivided into H/R 0.5 h group(B group), H/R 1 h group(C group), H/R 2 h group(D group)according to reoxygenation time. The left ventricular development pressure(LVDP), maximal rates of increase/decrease of the left ventricular pressure(±dp/dtmax) were continuously recorded. The concentration of interleukin-1β(IL-lβ) and creatine kinase-MB (CK-MB) in myocardium was measured by ELISA. The mRNA expression of IL-lβ in myocardium was determined by RT-PCR. Microstructure of myocardium was observed under light microscopy.

RESULTSThe value of LVDP and ±dp/dtmax in hypoxia/reoxygenation group rat were significantly lower than that in sham group(P < 0.05). The expression of IL-lβ and CK-MB at protein level and the expression of IL-1β at mRNA level in hypoxia /reoxygenation group were higher than that in sham group(P < 0. 05). There were significant differences of the above parameters among H/R 0.5 h, 1 h, 2 h group(P <0.05). The concentration of IL-1β and CK-MB, the mRNA expression of IL-1β were higher in H/R 2 h group than that of other groups(P < 0.05).

CONCLUSIONThe high expression of IL-Iβ in myocardium after myocardial hypoxia /reoxygenation in rats might lead to. ischemia/reperfusion injury.

Animals ; Creatine Kinase, MB Form ; metabolism ; Disease Models, Animal ; Hypoxia ; metabolism ; pathology ; Interleukin-1beta ; metabolism ; Myocardial Ischemia ; metabolism ; Myocardium ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo establish the controls for allele detection of blood groups s and Ok(a). A multiplex PCR method for the detection of three blood group antigens Fy(a), s and Ok(a) was developed and used to investigate the distribution of these blood groups in Chinese random blood donors.

METHODSPolymerase chain reaction (PCR)-based, gene site-directed mutagenesis (SDM) technique were used to make site-directed mutagenesis for the single nucleotide polymorphism (SNP) sites of the blood group alleles (the 153 C/T point mutation of the GYPB gene, and the 274 G/A point mutation of the BSG gene) as controls for allele detection. Sequence specific primers were designed according to the SNP sites of alleles of blood group antigens Fy(a), s and Ok(a). A multiplex PCR system was developed and 438 random donor samples were screened for the blood group antigens Fy(a), s and Ok(a).

RESULTSThe controls for alleles in blood groups s and Ok(a) were successfully made with the SDM technique, a multiplex PCR system was set up and successfully used to analyze the genotypes of three blood group antigens Fy(a), s and Ok(a). Two Fy(a-) samples were detected in the 438 samples, no s- and Ok(a-) sample was found.

CONCLUSIONThe PCR-based SDM technique can be used to obtain the unavailable controls in blood group genotyping. The multiplex PCR technique established in this study is an efficient genotyping method for blood groups Fy(a), s and Ok(a).

Alleles ; Base Sequence ; Blood Donors ; Blood Group Antigens ; genetics ; Blood Grouping and Crossmatching ; standards ; Genotype ; Humans ; Mutagenesis, Site-Directed ; Polymerase Chain Reaction ; standards ; Polymorphism, Single Nucleotide ; Reference Standards

BACKGROUNDRapamycin (RAPA) is a relatively new immunosuppressant drug that functions as a serine/threonine kinase inhibitor to prevent rejection in organ transplantation. RAPA blocks activation of T-effector (Teff) cells by inhibiting the response to interleukin-2. Recently, RAPA was also shown to selectively expand the T-regulator (Treg) cell population. To date, no studies have examined the mechanism by which RAPA converts Teff cells to Treg cells.

METHODSPeripheral CD4(+)CD25(-) naive T cells were cultivated with RAPA and B cells as antigen-presenting cells (APCs) in vitro. CD4(+)CD25(-) T cells were harvested after 6 days and analyzed for expression of forkhead box protein 3 (Foxp3) using flow cytometry. CD4(+)CD25(+)CD127(-) subsets as the converted Tregs were isolated from the mixed lymphocyte reactions (MLR) with CD127 negative selection, followed by CD4 and CD25 positive selection using microbeads and magnetic separation column (MSC). Moreover, mRNA was extracted from converted Tregs and C57BL/6 naive CD4(+)CD25(+) T cells and Foxp3 levels were examined by quantitative real-time polymerase chain reaction (rt-PCR). A total of 1 x 10(5) carboxyfluorescein succinimidyl ester (CFSE)-labeled naive CD4(+)CD25(-) T cells/well from C57BL/6 mice were cocultured with DBA/2 or C3H maturation of dendritic cells (mDCs) (0.25 x 10(5)/well) in 96-well round-bottom plates for 6 days. Then 1 x 10(5) or 0.25 x 10(5) converted Treg cells were added to every well as regulatory cells. Cells were harvested after 6 days of culture and analyzed for proliferation of CFSE-labeled naive CD4(+)CD25(-) T cells using flow cytometry. Data were analyzed using CellQuest software.

RESULTSWe found that RAPA can convert peripheral CD4(+)CD25(-) naive T Cells to CD4(+)Foxp3(+) Treg cells using B cells as APCs, and this subtype of Treg can potently suppress Teff proliferation and maintain antigenic specificity.

CONCLUSIONOur findings provide evidence that RAPA induces Treg cell conversion from Teff cells and uncovers an additional mechanism for tolerance induction by RAPA.

Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigen-Presenting Cells ; drug effects ; immunology ; metabolism ; B-Lymphocytes ; drug effects ; immunology ; metabolism ; CD4-Positive T-Lymphocytes ; drug effects ; immunology ; metabolism ; Cell Proliferation ; drug effects ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Forkhead Transcription Factors ; metabolism ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Interleukin-7 Receptor alpha Subunit ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mitomycin ; pharmacology ; Polymerase Chain Reaction ; Sirolimus ; pharmacology ; T-Lymphocytes, Regulatory ; drug effects ; immunology ; metabolism

OBJECTIVETo elucidate the molecular background of Del phenotype in the Chinese population and explore new Del alleles.

METHODSFive hundred and fifteen RhD negative blood samples was tested by Rh typing test, indirect antiglobulin test and adsorption and elution assay to screen the Del phenotype. DNA of all the Del samples was analysed by multiplex polymerase chain reaction (MPX PCR) for the presence of RHD and by sequence-specific primer polymerase chain reaction (PCR-SSP) for Del alleles: RHD 1227A and RHD 885T. Samples which showed the negative result by PCR-SSP, were additionally analysed by genomic DNA sequencing and cDNA sequencing.

RESULTSSeventy-nine Del samples were found by adsorption and elution assay. All these samples had RHD exons 3, 4, 5, 6, 7 and 9. Except 4 Del samples, other 75 Del samples carried the RHD 1227A allele. None of the samples had the RHD 885T allele. Four novel RHD alleles were found in these four Del sample. There were RHD 3G-->A (GenBank DQ310735), RHD 28C-->T, RHD 53T-->C (GenBank DQ451877,DQ451878), RHD 251T-->C (GenBank DQ310734).

CONCLUSIONfnRh blood group system is very complex. New D variation phenotypes and new RHD alleles may be discovered ceaselessly.

Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; Gene Frequency ; Genetics, Population ; methods ; Genotype ; Humans ; Molecular Sequence Data ; Mutation ; Phenotype ; Polymerase Chain Reaction ; Rh-Hr Blood-Group System ; genetics ; Sequence Analysis, DNA

OBJECTIVETo investigate class I integron of Shigella flexneri, its prevalence in children, and its relation to bacterial resistance to antimicrobial agents.

METHODSTotally 51 strains of Shigella flexneri were isolated from fecal samples of children suffering from bacterial diarrhea seen between June 2004 and November 2004 at Children's Hospital of Fudan University. Polymerase chain reaction (PCR) was employed to amplify various integron markers, including intI1, gene cassette region and 3' conserved region of class I intrgron; susceptibility of Shigella flexneri strains to 7 antimicrobial agents was determined by K-B (Kriby-Bauer) method.

RESULTSForty-six strains of Shigella flexneri had intI gene with a positive rate of 90.2% (46/51); 24 strains of Shigella flexneri were positive for qacEDelta1-sul1, the positive rate was 47.1% (24/51); proportion of the isolates positive for all the three regions of class I integron was 43.1% (22/51); 46 strains of intI positive Shigella flexneri were all positive for ant (3'')-I. Among 46 strains of intI positive isolates, proportions of the isolates positive and negative for qacEDelta1-sul1 were 47.8% (22/46) and 52.2% (24/46), respectively. In the class I integron positive Shigella flexneri, the resistance rates of ampicillin (chi(2) = 10.13, P < 0.01) and chloramphenicol (chi(2) = 19.97, P < 0.01) were significantly higher than those in the class I integron-negative group.

CONCLUSIONSClass I integron was detected in 90.2% of Shigella flexneri in children; carriage of class I integron is related to antimicrobial resistance of Shigella flexneri.

Anti-Bacterial Agents ; pharmacology ; Child ; DNA, Bacterial ; genetics ; Diarrhea ; microbiology ; Drug Resistance, Bacterial ; genetics ; Dysentery, Bacillary ; drug therapy ; microbiology ; Feces ; microbiology ; Humans ; Integrons ; drug effects ; genetics ; Polymerase Chain Reaction ; Retrospective Studies ; Shigella flexneri ; drug effects ; genetics ; isolation & purification

Objective:To evaluate the biological characters of C57-TgN(HBV adr2.0)SMMU transgenic mice. Methods: Integration,expression,replication and histology change of hepatitis B virus gene in F6 transgenic mice were estimated by ge-nomic DNA PCR,Western blotting,ELISA,immunohistochemistry,serum DNA PCR,transmission electron microscopy and H-E staining. Results: Hepatitis B virus gene was integrated into F6 C57-TgN(HBV adr2. 0)SMMU transgenic mice and expressed HBsAg,HBcAg and X protein in liver tissue. HBsAg and HBeAg were expressed in serum of 19. 54% and 3. 39% F6 transgenic mice. Hepatitis B virus were replicated in serum and liver tissue of transgenic mice. Long-term integration,expression and replication of hepatitis B virus gene induced pathological lesion of transgenic mice liver and lung. Conclusion: C57-TgNCHBV adr2. 0)SMMU transgenic mice line has the biological characters including integration of hepatitis B virus gene into genomic DNA,expression and replication of hepatitis B virus gene in serum and liver, and histological change in liver and lung. It is a valuable animal system to study pathogenesis, treatment and prevention of hepatitis B virus.

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OBJECTIVEImprovement in lung function was reported after acupuncture treatment of chronic obstructive pulmonary disease (COPD), but little is known about the underlying mechanisms. Because an immune response imbalance could be seen in COPD, we hypothesize that electroacupuncture (EA) may play a role in regulating inflammatory cytokines and contribute to lung protection in a rat model of smoke-induced COPD.

METHODSA COPD model using male Sprague-Dawley rats exposed to cigarette smoke was established. The rats were randomly divided into four groups (control, sham, COPD, and COPD plus EA), and COPD model was evaluated by measuring pulmonary pathological changes and lung function. EA was applied to the acupuncture point Zusanli (ST36) for 30 min/d for 14 d in sham and COPD rats. Bronchoalveolar lavage fluid (BALF) was used to measure levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and malonaldehyde (MDA).

RESULTSCompared with the control rats, COPD rats had significant changes in lung resistance (RL) and lung compliance (CL) (both P<0.01), bronchi and bronchiole airway obstruction (P<0.01), and levels of MDA, TNF-α, and IL-1β (P<0.01). There were no significant differences between the control and the sham groups. Compared with the COPD rats, the COPD plus EA rats had decreased RL and increased CL (both P<0.05), and reduced bronchi and bronchiole airway obstruction (P<0.05, P<0.01, respectively), while levels of TNF-α, IL-1β, and MDA in BALF were lowered (P<0.05 and P<0.01, respectively). However, TNF-α and IL-1β levels of the EA group rats remained higher than those of the control group (P<0.05).

CONCLUSIONEA at ST36 can reduce lung injury in a COPD rat model, and beneficial effects may be related to down-regulation of inflammatory cytokines. The anti-inflammatory and antioxidant effects may prolong the clinical benefit of EA.

Acupuncture Points ; Animals ; Bronchoalveolar Lavage Fluid ; immunology ; Disease Models, Animal ; Electroacupuncture ; Humans ; Interleukin-1beta ; immunology ; Male ; Pulmonary Disease, Chronic Obstructive ; etiology ; immunology ; therapy ; Rats ; Rats, Sprague-Dawley ; Smoking ; adverse effects ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVEA reliable method for genotyping blood group antigens Dib, k, Jsb1910 and Jsb2019 was developed. Through screening for rare blood types, the National Rare Blood Bank of China may be enriched.

METHODSThe controls for allele detection of blood groups Dib, k, Jsb1910 and Jsb2019 were prepared via polymerase chain reaction (PCR)-mediated gene site-directed mutagenesis (SDM) technique. Sequence-specific primers were designed according to known single nucleotide polymorphism (SNP) sites of alleles of blood groups antigens Dib, k, Jsb1910 and Jsb2019, a multiplex PCR system was developed by optimizing PCR reaction system. And 4190 random healthy donors samples were screened for the blood group antigens.

RESULTSUsing SDM technique, controls for alleles in blood group Dib, k, Jsb1910 and Jsb2019 were successfully generated. And a multiplex PCR system for genotyping above blood groups was developed. After verification, the system has performed with good stability and reproducibility. Two Di (b-) samples have been discovered from 4190 samples, no k- and Js(b-) sample was found.

CONCLUSIONMultiplex PCR features rapid detection, high throughput and low cost, and can be used for screening for donors of rare blood types. Information of donors may be registered in a database, which in turn can help those with rare blood types or require long-term blood transfusion to obtain matched blood, thereby reduce the adverse reactions of blood transfusion.

Blood Group Antigens ; genetics ; Erythrocytes ; immunology ; Genotyping Techniques ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Mutagenesis, Site-Directed ; Polymorphism, Single Nucleotide

OBJECTIVETo study the effect of rapamycin in inducing naïve murine effector T cell (Teff) convert to regulatory T cell (Treg) in vitro.

METHODSThe forkhead box protein 3 (FoxP3) negative Teff were isolated and purified from the spleen and lymph node of C57 BL/6 murines aged 6-8 weeks, then Teff were cultured in three groups with mature dendritic cells (mDC), B cells, and plate coated Anti-CD3. In addition, the control wells and the test wells were prepared in each group, rapamycin were not added in the control wells but added in the test wells with concentrations of 1, 10, 50, and 100 nmol/L. Percentages of FoxP3 positive Treg were examined by flow cytometry after 4 days in Anti-CD3 group and after 6 days in the other two groups.

RESULTSAs shown by the flow cytometry, the percentages of FoxP3 positive Treg were as follows in three group: in the mDC group, it was 0.01% in the control well and 0.39%, 0.47%, 0.34%, and 0.26% in test wells; in B cell group, it was 0.01% in the control wells and 5.56%, 5.89%, 7.15%, and 4.72% in the test wells; in Anti-CD3 group, it was 0.93% in the control wells and 1.35%, 1.07%, 1.02%, and 1.19% in test wells. No significant difference was found between the test wells and control wells in the mDC group and Anti-CD3 group; however, the percentages of FoxP3 positive Treg was significantly different between the test wells and control wells in the B cell group (P < 0.01).

CONCLUSIONWhen B cell is acted as the antigen-presenting cell, rapamycin can effectively induce Teff convert to Treg in vitro.

Animals ; B-Lymphocytes ; cytology ; drug effects ; immunology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; immunology ; Flow Cytometry ; Forkhead Transcription Factors ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Precursor Cells, T-Lymphoid ; cytology ; drug effects ; immunology ; Sirolimus ; pharmacology ; T-Lymphocyte Subsets ; cytology ; drug effects ; immunology ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; immunology