Search

During the thermite welding of in-service oil pipelines, empirical values are often used for the dosage of aluminum powder welding flux, and the uncertainty of temperature and residual stress generated during the welding poses a serious threat to the construction of pipeline and its safe operation. In response to this problem, the thermite welding process of pipelines in different wall thickness and with different dosages of welding flux was analyzed with the numerical simulation software ANSYS.Specifically, a finite element model of thermite welding was established, and thus the change law of the temperature field and residual stress field with different dosages of aluminum powder welding flux and different pipeline wall thicknesses was obtained,which was also verified by test. As indicated by the results, the maximum temperature in the welding area of pipelines with same wall thickness rises with the increasing dosage of welding flux. The maximum temperature in the welding area decreases with the increasing of pipeline wall thickness in the case that equivalent welding flux is used. Besides, with the increasing of pipeline wall thickness, the maximum residue stress at the weld is up to 150 MPa, lower than the yield limit of material, so that no burn-through and crack will occur during thermite welding, and the simulation results are consistent with the test results. Moreover, it is safe and reliable to conduct thermite welding in 6 mm to 8 mm pipeline in service with 10 g to 20 g of aluminum powder. Generally, the numerical simulation method could provide reference for the safe operation of thermite welding of in-service pipelines, thus reducing the construction risks of pipelines in service.

Objective*To study the regulation of transcription factor T-bet in antibody isotype switching via the optimized CRISPR/Cas9-mediated gene editing system in primary B cells. Methods*In order to demonstrate the regulation of T-bet in antibody isotype switching, the transcriptome data of in vitro-derived B cells from public database GEO was analyzed. The retroviral small guide RNA (sgRNA) vector was constructed to deliver sgRNA targeting Tbx21 (T-box 21, encoding T-bet), and retroviral packaging cell line Plat-E was used for retrovirus production. The retroviruses with sg-Tbx21 plasmids were harvested and concentrated. Primary B cells isolated from Cas9 transgenic mice were activated in vitro by synergistic stimulation of anti-B cell receptor antibody, anti-CD40 antibody and a Toll-like receptor agonist, and then infected with concentrated retroviruses with sg-Tbx21. The genome DNA was extracted from the infected cells isolated by flow cytometry, and then PCR amplification was performed for target gene sequence. ICE CRISPR analysis tool was used to calculate the gene knockout efficiency. The edited B cells were induced to plasma cells in vitro and the antibody isotypes of IgM, IgG2c, and IgG3 production in the culture supernatant were assessed by ELISA. Independent sample t-test was used to analyze the difference of antibody levels in the supernatant between the sg-NC editing cells and sg-Tbx21 editing cells. Results*(1) According to the analysis of GEO database, interferon-γ induced the expression of Tbx21 in B cells, and T-bet expression promoted antibody isotype switching-related pathways. (2) The retroviral infection efficiency was improved in primary B cells after in vitro activation, which were infected by the concentrated high titer viruses. (3) The sg-Tbx21 plasmids were delivered to Cas9 transgenic B cells by retroviruses, which led to T-bet knockout efficiency of about 59%. (4) Cultured in the plasma cells-polarizing condition, the Tbx21 edited B cells secreted less IgG2c into the culture supernatant (P=0.000), while the levels of IgM and IgG3 did not significantly change. Conclusion*The efficiency of sgRNA delivery can be improved by concentrating retroviruses and activating primary B cells through synergistic stimulation, and Tbx21 gene can be knocked out in primary B cells of Cas9 transgenic mice; it is proved that T-bet knockout hinders the production of antibody isotype IgG2c through this system. [ABSTRACT FROM AUTHOR]

Objective·To take the systemic lupus erythematosus (SLE) -associated single nucleotide polymorphism (SNP) rs1978421 reported in the genome-wide association study (GWAS) as an example, and establish the research strategy for the functional study of disease-associated genetic variants located in the non-coding region through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/ Cas9) system. Methods·Single guide RNA (sgRNA) was designed for SNP rs1978421. T7 endonuclease I (T7E I) experiment was used to verify the cleavage efficiency of sgRNA in HEK293T cells. The homologous recombinant template and expression plasmid of Cas9-green fluorescent protein (GFP)/ sgRNA were transfected into U-937 cells by electroporation transfection method. GFP positive U-937 single cells were sorted by flow cytometry, and the cells were cultured for the following 14 days. Then the genotype identification was carried out. After the homologous recombinant cell clones were screened, quantitative real-time PCR (qPCR) was used to quantify the upstream and downstream genes of rs1978421 within 2 Mb and miR-146a, and the effects of different alleles of rs1978421 on the expression of miR-146a and its surrounding genes were analyzed. Results·T7E I assay showed that about 24% of the total rs1978421 sites in HEK293T were cleaved. In homologous recombination experiment of the U-937 cells, a total of 141 cell lines were obtained and only six were successful, while the ratio of homologous recombination was 4.3%. qPCR showed that there was no significant difference in the expression of miR-146a and the detected genes between wild type TT and homologous recombinant CC in the U-937 cells (all P>0.05). Conclusion·SNP rs1978421 has no regulatory effect on the surrounding genes within 2 Mb and miR-146a. However, this study proves the feasibility of CRISPR-mediated homologous recombination technology for the functional study of SNP sites in the non-coding regions. [ABSTRACT FROM AUTHOR]

Objective · To construct retroviral small guide RNA (sgRNA) expression vector for studying gene function in mouse T cells. Methods · The short hairpin RNA (shRNA)-expressing retroviral vector MSCV-LTR-miR30-PIG (LMP) was digested with Xho1 and Sal1 to obtain the backbone of the retroviral vector. The lentiviral sgRNA vector was used as template for PCR amplification to obtain the U6-sgRNA-PGK-Puro-BFP fragment. The PCR fragment was then assembled into the retroviral vector backbone by homologous recombination. To verify the effectiveness of the system, sgRNA sequences targeting Il17a, Rorc and Irf4 were designed and cloned. CD4 T cells isolated from Cas9 transgenic mice were infected with individual sgRNA retrovirus and differentiated into Th17 cells. After cytokine intracellular staining, the percentage of Il17a positive population was detected by flow cytometry. Unpaired t test was used to compare the data of independent samples between the two groups. Results · The sgRNA retroviral vector was successfully constructed. The retroviral vector was used to successfully deliver sgRNA to CD4 T cells, which mutated Il17a gene. The percentage of Il17a positive population was significantly reduced in the Rorc-sgRNA and Irf4-sgRNA positive populations (P<0.05). Conclusion · The sgRNA retroviral vector is successfully used to deliver sgRNA to target cells. In addition, the results of Rorc and Irf4 gene knockout experiments prove that the system can be used for studying gene function in mouse T cells. [ABSTRACT FROM AUTHOR]

Objective * To investigate the intrinsically regulatory role of transcription factor STAT3 on the activation and proliferation of CD8+ T cells in immunodeficient Rag1-/- mice. Methods * The CD8+CD44-CD62L+Naïve T cells, from CD45.1 wild type (WT) mice and CD45.2-CD8 (ΔStat3) mice, were sorted by flow cytometry cell sorter, equally mixed and labelled with Carboxyl Fluorescein diacetate Succinimidyl Ester (CFSE), and then transferred (i.v.) to the Rag1-/- mice. Ten days after CD8+ T cells transfer, the proportion and proliferation of CD8+ T cells in the spleen, mesenteric lymph nodes (mLNs) and popliteal lymph nodes (pLNs) were determined by flow cytometry. TNF-α, IFN-γ, FasL and granzyme B (GraB) produced by the CD8+ T cells were measured with flow cytometry after PMA and ionomycin stimulation. The statistical significance of activation and effectors expressed between WT and ΔStat3 CD8+ T cell was analyzed by t test. Results * The percentages of WT CD8+ T cells in spleen, mLNs and pLNs were significantly higher than that in ΔStat3 CD8+ T cells (76.2% vs 23.4%, 82.1% vs 17.4%, 64.5% vs 32.3%, with all P=0.008). The percentage and cell number of activated CD44+CD8+ T cells in WT CD8+ T cells were much higher than those in ΔStat3 CD8+ T cells in pLNs (all P=0.008). In spleen, pLNs and mLNs, the levels of TNF-α, IFN-γ, GraB and CD107a expressed in WT CD8+ T cells were higher than those in ΔStat3 CD8+ T cells (all P<0.05). Conclusion · Rag1-/- mice can work as an ideal model to evaluate the survival, proliferation, activation and function of CD8+ T cells in vivo. STAT3 intrinsically regulates the proliferation, activation and function of CD8+ T cells in vivo. [ABSTRACT FROM AUTHOR]

Objective: To diagnose and explore the genetic etiology of a neonate with Hereditary epidermolysis bullosa., Methods: A neonate who was admitted to Suqian Hospital Affiliated to Xuzhou Medical University on July 10, 2021 was selected as the study subject. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA. And target gene capture and next-generation sequencing were carried out. Candidate variants were verified by Sanger sequencing and pathogenicity analysis., Results: The child was found to harbor compound heterozygous variants of the COL17A1 gene, namely c.997C>T (p.Q333X) and c.3481dupT (p.Y1161fs*2), which were respectively inherited from his father and mother. Both variants were predicted to be pathogenic., Conclusion: The child was diagnosed with Generalized atrophic benign epidermolysis bullosa due to the compound heterozygous variants of the COL17A1 gene.

A series of strict control measures were imposed in the Beijing-Tianjin-Hebei region in early 2020 to control the spread of COVID-19. These measures have led to a reduction of anthropogenic air pollutants, providing an opportunity to observe the contribution of human activities to local air pollution. In this study, the control period was divided into four stages:the before, early, middle, and later stages. Based on a variety of data including meteorological, traffic, and industrial manufacturing datasets, statistical methods were combined with spatial analysis to evaluate changes in air pollution and associated human impacts during each stage. In addition, suggestions are made for further regional air pollution control in the Beijing-Tianjin-Hebei area. Key results are as follows:① Overall, the AQI and the concentrations of six air pollutants, especially SO 2 , PM 10 , and NO 2 , were lower during control period than during the equivalent period in 2019 (reductions of 26.5%, 24.3%, and 16.9%, respectively). From the before to later stages, pollutants (except O 3 ) showed a downward trend while O 3 increased significantly during the before stage (by 76.2%) and the growth rate slowed during the middle and later stages; ②During the prior stage, Beijing experienced two periods with heavy air pollution days as a result of the local accumulation of pollutants, secondary transformation, and regional transport. The concentration of PM 2.5 in February was nearly 60% lower than in February 2014 under similar meteorological conditions in Beijing; ③ Following an increase in traffic volume and industrial activity, changes in air pollutants tended to be stable or slightly increase during the middle and later stages of the control period. The grey relation coefficients between thermal radiation intensity anomalies and the main pollutants in heavy industrial cities were greater than 0.6, which means that the control of industrial emissions remains key to controlling air pollution.

Objective: To explore the genetic etiology of a child with lymphangiectasia and lymphedema., Methods: DNA sample of the patient was extracted and subjected to whole exome sequencing. Suspected variants were verified by Sanger sequencing., Results: The patient was found to carry compound heterozygote variants (c.521G>A and c.472C>T) of the CCBE1 gene, which were respectively inherited from his parents., Conclusion: The compound heterozygote variants of the CCBE1 gene probably underlie the disease in this child.

Objective: To identify potential mutations of the CLS gene in a Chinese pedigree affected with Coffin-Lowry syndrome., Methods: Whole exome sequencing was applied to detect potential mutation in the proband, and the result was verified by Sanger sequencing., Results: The proband was found to carry a c.966&#95;967delAA (p.Arg323Thr fs*11) deletional mutation in the RPS6KA3 gene. The same mutation was also found in his mother., Conclusion: The c.966&#95;967delAA (p.Arg323Thr fs*11) deletional mutation of the RPS6KA3 gene probably underlies the disorder in this pedigree.

Objective: To correlate genotype with clinical phenotype of a child featuring multiple congenital malformations., Methods: Clinical examination of the patient was carried out. Chromosome microarray analysis (CMA) was employed to detect genomic copy number variations (CNVs), and quantitative PCR (qPCR) was used for verifying the result., Results: The child had congenital heart disease (ventricular septal defect, atrial septal defect, pulmonary arterial hypertension, and tricuspid regurgitation), psychomotor retardation, agenesis of corpus callosum, hypospadias and scoliosis. CMA has detected a 1.8 Mb deletion at 7p22.3, a 1.8 Mb duplication at 7p22.3p22.2 and a 23.5 Mb duplication at 7q33q36.3 in the fetus, all of which were de novo in origin., Conclusion: CMA can precisely detect microdeletion/duplications and facilitate the genotype-phenotype correlation analysis.

Objective: To explore the genetic basis of a child with congenital heart disease (CHD)., Methods: Clinical examination of the child was carried out. Chromosomal microarray analysis (CMA) and quantitative PCR were carried out to detect copy number variations., Results: The major features of the child included CHD (ventricular septal defect, severe pulmonary hypertension, tricuspid regurgitation, patent ductus arteriosus, and patent foramen ovale), severe pneumonia and liver failure. A de novo 3.2 Mb deletion encompassing 25 genes in 13q34 and a paternal 2.2 Mb duplication in 19p13.3 were revealed by CMA and qPCR., Conclusion: The 13q34 region probably contains susceptibility genes for CHD.

Objective: To explore the clinical and genetic features of a child with primary ciliary dyskinesia., Methods: Genomic DNA of the child and her parents was extracted and subjected to targeted gene capture and next generation sequencing. Suspected mutation was verified by Sanger sequencing, with its nature and impact predicted by Bioinformatic analysis., Results: Clinical manifestations of the child mainly included severe pneumonia, bronchiectasia, nasosinusitis and pneumothorax. DNA sequencing showed that she has carried compound heterozygous mutations of the CCNO gene, namely c.848T>C (p.L283P) and c.262&#95;263 insGGCCCGGCCC (p.Q88Rfs*51), which were respectively inherited from her mother and father., Conclusion: The child was diagnosed with primary ciliary dyskinesia caused by the compound heterozygous mutations of the CCNO gene.

To investigate the effect of flavonoids from Sophora flavescens in aging mice induced by D-galactose and its mechanism. Totally 60 mice were randomly divided into six groups: the control group, the model group, the piracetam group (positive control group) and flavonoids from S. flavescens low, medium and high doses groups. Except for the control group, all of the rest groups were subcutaneously injected with D-galactose (160 mg x kg(-1)) for successively 30 days to establish the sub-acute senescent model. Meanwhile, flavonoids from S. flavescens low, medium and high doses groups were respectively administered with 150, 300 and 600 mg xkg-('1)of flavonoids from S. flavescens for 30 days. The learning and memory abilities of mice were determined by avoiding darkness ex-eriment and jumping stair experiment. The contents of malondialdehyde (MDA) tumor necrosis factor-aα NF-aα the activities of superoxide dismutase (SOD) monoamine oxidase-B (MAO-B) Na'(+)K'(+)-ATPase and Ca2(+ )-ATPase in the brain of mice were deter-ined respectively after the behavioral experiments. The activity of lactic dehydrogenase ( DH) in blood serum was also determined and analyzed by microscope after HE staining to observe the changes in hippocampal organizational structure. Compared with the model group, flavonoids from S. favescens medium and high doses groups showed significantly increases in the latency of avoiding darkness and jumping stair experiments; flavonoids from S. fllvescens low, medium and high doses groups and the piracetam group showed de-reases in the numbers of errors in avoiding darkness experiment; the flavonoids from S. flavescens high dose group and the piracetam group showed reduction- n the number of errors in jumping stair experiment (P <0 . 5 or P <0 . 1). Flavonoids from S. flavescens me-ium and high doses groups and the piracetam group showed improvements in the activities of SOD, Na'(+)K'(+)ATPase in the brain of mice and declines in the contents of MDA and TNF-aα the activity of MAO-B in the brain of mice, the activity of LDH in blood serum (P <0 . 5 or P <0 . 1). Flavonoids from S. flavescens low, medium and high doses groups and the piracetam group also showed im-rovement in the activity of Ca2(+ )ATPase, with statistical difference from the model group (P <0 . 5 or P <0 . 1). The pathological result showed decreases in the number of cells of hippocampal dentate gyrus area, sparse cell arrangement, incomplete cellular mor-hology, scarce cytoplasm, blurred boundary between nucleus and cytoplasm, nuclei anachromasis, irregular pyknosis and unconspicu-us nucleoli in the model group. Compared with the model group, flavonoids from S. flavescens low, medium and high doses groups and the piracetam group showed improvements in hippocampal organization tissues. Flavonoids from S. favescens can improve the learning and memory ability of senescent mice induced by D-galactose. Its mechanism may be correlated with the enhancement of anti-oxidative actions by lowering TNF-aαcontent, which results in the stability of cell membrane and the reduction in MAO-B activity.

Objective: To investigate the cardiac injury effect of different intensities microwave radiation on rabbits and its possible mechanism., Methods: Rabbits were radiated by intensity of 50, 100, 150 and 200 mW/cm2 2450 MHz microwave for 20 min. 6 h after microwave radiation, the heart tissue was taken. ATP and mitochondria complex IV and V were measured in myocardial cells. The changes of myocardial tissue were observed by light microscopic. The expression of Caspase-3 and HSP 70 were detected by western blotting., Results: The activity of ATP and mitochondria complex IV and V decreased significantly compared with normal control in cardiac tissue. 100, 150 and 200 mW/cm2 microwave radiation group vs. control group (P <0. 05). The HE staining result showed that myocardial cell appears edema, muscle fiber malalignment, cells appeared obvious injury. Results of western blotting showed that the expression of Caspase-3 and HSP 70 protein increased significantly in different dosage radiation group (P <0. 05)., Conclusion: Microwave radiation has injury effect on rabbit heart. The possible mechanism may be related with inducing cell apoptosis by changing of stress level in myocardial cell.

This study is designed to explore the possible effects of Hemerocallis citrina baroni flavonids (HCBF) on liver fibrosis induced by CCl4 in rats. The liver fibrosis model was induced by CCl4, and HCBF were administered by gastric perfusion at 25 and 50 mg x kg(-1) qd for 50 days, while the contents of alanine transaminase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), superoxide dismutase (SOD), maleic dialdehyde (MDA) and transforming growth factor-β1 (TGF-β1) were measured and the contents of PINP were measured in liver tissue, and the expression of TGF-β1 were observed by immunohistochemisty and Western blot. The pathological changes of liver tissue were examined by HE. The results showed that HCBF (25, 50 mg x kg(-1)) improved the liver function significantly through reducing the level of ALT, AST, GGT and ALP (P < 0.05 or P < 0.01), and increasing the content of SOD (P < 0.01), while reducing the content of MDA (P < 0.05 or P < 0.01), the expression of TGF-β1 (P < 0.05) and the content of PINP (P < 0.05). The results suggest that HCBF (25, 50 mg x kg(-1)) may inhibit the liver injury induced by CCl4 by decreasing the oxidative stress.

Objective: To investigate the effect of Inonotus obliquus polysaccharides on testicular injury induced by exposure to high power microwave (HPM) in rats., Methods: A total of 30 male Wistar rats were randomly divided into 5 groups, i.e., the normal control group, the microwave radiation model group, the treatment group, the new microwave radiation model group, and the prevention group, 6 in each group. All rats, except those in the normal control group, were exposed to microwave at an average power density of 200 mW/cm2 for 6 min. Rats in the control group and the model group were administered with normal saline by gastrogavage, once a day. Rats in the treatment group and the prevention group were given with Inonotus obliquus polysaccharides by gastrogavage, 2 mL each time (400 mg/kg body weight), once a day. All rats were sacrificed on the 11th day.The sperm density and the rate of sperm deformity were determined. Pathological changes of testis were observed by light microscope and transmission electron microscope., Results: Short-term HPM irradiation could significantly reduce the sperm density and increase the sperm deformity rate (P < 0.05). Meanwhile, obvious pathological changes of testes occurred. Compared with the two model groups, the sperm density increased and the sperm deformity rate decreased in the treatment group and the prevention group (P < 0.05). Under the light microscope, injuries of spermatogenic cells and stromal cells, as well as vascular dilatation and congestion were obviously alleviated in the treatment group and the prevention group. Mitochondrial swelling and endoplasmic reticulum expansion shown by ultrastructural observation were also significantly alleviated. Of them, injuries of spermatogenic cells and inflammation response were milder in the treatment group than in the prevention group., Conclusions: Inonotus obliquus polysaccharides had significant protective effect on microwave radiation induced testicular injury. Better effect was obtained by therapeutic medication than preventive medication.

Objective: To explore the efficacy of Schizandra Chinensis polysaccharide (SCP) on cyclophosphamide (CTX) induced dyszoospermia of rats and its effects on reproductive hormones., Methods: SCP was extracted by ethanol-alkali solution. Fifty male Wistar rats were randomly divided into 5 groups, i.e., the normal control group, the model group, the low dose SCP group (100 mg/kg), the middle dose SCP group (200 mg/kg), and the high dose SCP group (400 mg/kg). Except the normal control group, the dyezoospermia rat model was established by peritoneal injection of CTX at the daily dose of 80 mg/kg, once daily for 5 successive days. After modeling, SCP was intragastrically administered at corresponding dose to the three SCP groups. Equal volume of normal saline was given to rats in the normal control group and the model group by gastrogavage. All the medication was performed once daily for 60 successive days. The blood serum and testis were withdrawal 24 h after the last intragastric administration. The levels of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone (T) in the testis homogenate were determined by enzyme linked immunosorbent assay. The sperm count, the motility rate, and the teratospermia rate were compared. The morphology of the testis was observed using HE staining., Results: Compared with the normal control group, the sperm count and the motility rate decreased, the teratospermia rate increased, the serum levels of FSH and LH increased, the T content in the testis homogenate decreased in the model group, showing statistical difference (P <0.01). Compared with the model group, the sperm count and the motility rate increased, the teratospermia rate decreased, the serum levels of FSH and LH decreased, the T content in the testis homogenate increased in the model group, showing statistical difference (P <0.01, P <0.05). All the indices showed dose-dependent manner in the SCP groups. The histological results showed the pathological injury in the testicular tissue was improved in all SCP groups., Conclusion: SCP showed obvious therapeutical effects on CTX induced dyszoospermia in rats, and its mechanisms might be correlate with recovering the regulation function of hypothalamus-hypophysis-gonad axis.

Hydrophilicity/hydrophobicity, the same as chemical signals and mechanical stimuli, is an important characteristic of material surface, induces a cascade events of intercelluar proteins and genes, and determines cells biologic behaviors in vitro eventually. In this review, we summarize the available reports to review the methods of hydrophilic/hydrophobic surface modification, and its effects on protein adsorption and cells biologic behaviors.

Aim: To develop a flow cytometry using (5-carboxyfluorescein diacetate succinmidyl ester, CFSE) to detect the proliferation of specific T lymphocytes from equine infectious anemia virus (EIAV)., Methods: Peripheral blood mononuclear cells (PBMC) were isolated, stained with CFSE and incubated with EIAV for 5 days. After interacted with either CD4(+) or CD8(+) antibody, the cells were detected for proliferated population, which contained serially 2-fold reduced CFSE in CD4(+) and CD8(+) T lymphocytes., Results: The concentration of CFSE, and the type, concentration and reaction time of EIAV-specific antigens were tested and optimized to improve the sensitivity and specificity of this flow cytometry-based assay. PBMCs isolated from the horses infected with either virulent or vaccine strains of EIAV and health controls were analyzed to evaluate this technique. The proliferation rates of CD4(+) and CD8(+) T lymphocytes were observed in the cells from differently treated horses., Conclusion: The CFSE-based flow cytometry is a reliable tool to analyze antigen-specific proliferation of lymphocytes, which plays an important role in evaluating the immune responses induced by EIAV and other viruses.

Objective: The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-gamma expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-gamma expression in different subtypes of T lymphocytes in EIAV-infected horses., Methods: Peripheral blood mononuclear cells (PBMC) were prepared from horses inoculated with EIAV vaccine stain FDDV (fetal donkey dermal-attenuated virus vaccine), virulent strain LV or saline (health control), were stimulated in vitro with FDDV or PMA + Inomycin. Brefeldin A was added into the cell culture to block protein secretions. Stimulated cells were then labeled with monoclonal antibodies specific for equine CD4 and CD8 surface markers, as well as an IFN-gammaspecific monoclonal antibody. Flow cytometry was applied to detect CD4+ and CD8+ lymphocytes expressing IFN-gamma., Results: IFN-gamma positive cell population isolated from FDDV-immunized horses was 1.7 +/- 0.9% in CD4+ T cells and 6.1 +/- 1.2% in CD8+ T cells (n=4). In contrast, only 0.6 +/- 0.1% of IFN-gamma positive cells in CD4+ subset and 2.4 +/- 0.9% of IFN-gamma positive cells in CD8+ subset of T cells were detected for PBMC isolated from LV-infected horses (n=4)., Conclusion: The multi-fluorescence cell flowmetry detecting both the IFN-gamma expressing cells and subsets of T lymphocytes simultaneously, is specific, stable and reproducible in evaluating antigen-specific response of IFN-gamma-producing lymphocytes. This is an essential approach to study the protective immunity induced by EIAV vaccine.

The purpose of this investigation was to evaluate the effect of transforming growth factor-beta (TGF-beta) on the efficacy of Schwann cell (SC) and on the repair of peripheral nerve defect. 50 ng x ml(-1) TGF-beta was shown to promote the proliferation of SC by MTT and flow cytometry (FCM) assay, and NGF synthesis in SC culture media was noted to be of significantly higher concentration by ELISA method (P<0.05). SCs mixed with bovine acellular matrix (BAM), fetal bovine serum and media based on definite ratio were injected into polylactideco-glycolide acid (PLGA)guide. 30 SD rats, each had a man-made sciatic nerve defect 15 mm long, were randomly divided into 3 groups: experiment group (PLGA conduit+SC+TGF-beta), control group(PLGA conduit+SC), and autograft group. After 16 weeks, it was demonstrated that the effect of the test group was not significantly different from that of the autograft group, but it was better than that of the control group by means of electrophysiological test and sciatic nerve function index (SFI). TGF-beta can promote not only the proliferation, but also the NGF synthesis of SC obviously. The use of exogenous TGF-beta in the repair of peripheral nerve defect may produce better curative effect.

Objective: To observe whether single nucleotide polymorphisms (SNPs) within the OLF1/EBF-associated zinc finger protein (OAZ) gene are associated with lupus nephritis (LN) susceptibility in Chinese population., Methods: Eight SNPs located around the positive microsatellite marker D16S517 within OAZ gene with relatively high heterozygosity were chosen for genotyping on 184 systemic lupus erythromatosus (SLE) patients, including 101 non-LN patients and 83 LN patients, and 286 normal controls using TaqMan MGB allelic discrimination method. Data were collected by SDS 2.0 software. Haplotypes and their frequencies were constructed and estimated, and linkage disequilibrium analysis between pairs of SNPs was evaluated by calculating the D Prime using Helixtree program. Case-control study was performed between the SLE, LN, and non-LN groups and normal control group., Results: (1) The frequency of SNP rs1344531 T allele was 47.0% in the SLE patients, significantly higher than that in the controls [38.1%,; chi(2) = 7.300, P = 0.008, OR (95% CI) = 1.441 (1.105 - 1.878)], which showed that the frequency of SNP rs1344531 T allele is associated with SLE susceptibility. The genotypic distribution of SNP rs1344531(CC/CT/TT) differed significantly between the SLE patients (25.5%/54.9%/19.6%) and normal controls (38.1%/47.6%/14.3%) (chi(2) = 8.394, P = 0.015). The CC genotype frequency of the SLE patients was 25.5%, significantly lower than that of the normal controls [38.1%; chi(2) = 7.976, P = 0.005, OR (95% CI) = 0.557 (0.370 - 0.838)] (2) The SNP rs1344531 T allele frequency of the SLE patients was 53.0%, significantly higher than that of the normal controls [38.1%; chi(2) = 11.769, P = 0.001, OR (95% CI) = 1.832 (1.293 - 2.596)], which showed an associated between SNP rs1344531 T allele frequency and LN susceptibility. The genotypic distribution of SNP rs1344531 (CC/CT/TT) differed significantly between the LN patients (22.9%/48.2%/28.9%) and the normal controls (38.1%/47.6%/14.3%) (chi(2) = 12.065, P = 0.002). The CC genotype frequency of the LN patients was 22.9%, significantly lower than that of the normal controls (38.1%) [chi(2) = 6.578, P = 0.013, OR (95% CI) = 0.481 (0.274 - 0.848)]. The TT genotype frequency of the LN patients was 28.9%, significantly higher than that of the normal controls (14.3%) [chi(2) = 9.423, P = 0.003, OR (95% CI) = 2.431 (1.363 - 4.334)]. No statistical significance was observed between the non-LN patients and normal controls in TT genotype frequency. (3) The frequencies of haplotypes containing rs1344531:rs1420676-rs1344531(C-T) [chi(2) = 11.731, P = 0.001, OR (95% CI) = 1.867 (1.302 - 2.676)], rs3803665-rs1420676-rs1344531(C-C-T) [chi(2) = 8.876, P = 0.004, OR (95% CI) = 1.772 (1.213 - 2.589)], and rs2292155-rs3803665-rs1420676-rs1344531(C-C-C-T) [chi(2) = 9.962, P = 0.002, OR (95% CI) = 1.915 (1.274 - 2.880)] were all significantly higher in the LN patients in comparison with the normal control group (41.0% versus 27.1%; 33.3% versus 21.8%; and 27.0% versus 16.2%); while the frequencies of other haplotypes: rs1344531-rs2080353(C-A) [chi(2) = 8.06, P = 0.005, OR (95% CI) = 0.603 (0.424 - 0.856)], rs1344531-rs2080353-rs933564 (C-A-G) [chi(2) = 7.929, P = 0.006, OR (95% CI) = 0.602 (0.422 - 0.859)] were significantly lower than those of the normal control group (39.5% versus 52.2%, and 36.6% versus 49.2%), which produced additional support for such association., Conclusion: SNP rs1344531 and some haplotypes containing SNP rs1344531 within OAZ are significantly associated with LN susceptibility. Genetic variants of the OAZ gene are involved in the pathogenesis of LN.

Objective: To investigate the expression levels of interferon (IFN)-alpha, -beta, -omega and -gamma genes and proteins in the peripheral blood of patients with systemic lupus erythematosus (SLE), and to evaluate any possible connections between these expression levels with clinical features., Methods: 144 SLE patients, 27 non-SLE patients with rheumatisms and 59 normal controls were recruited for the research, and all subjects were drawn blood to isolate plasma and elute total RNA. SYBR Green Dye based real-time quantitative PCR method was used to compare the expression levels of 4 IFNs in patients with SLE and those in the controls. The significance for the correlation of these expression levels and disease activity and specificity was studied., Results: (1) mRNA expression levels of all 4 IFNs in SLE patients were remarkably lower than those observed in normal controls (P < 0.01 in all); IFN-alpha expression levels in SLE patients were increased than those observed in non-SLE group (P < 0.01). (2) The expression levels of all 4 IFNs in active SLE patients were similar to those observed in inactive SLE patients (P > 0.05 in all), and expression levels of all 4 IFNs in patients with SLE were not correlated with involvements of kidney, lung, brain, blood. (3) IFN score in SLE patients was remarkably lower than that observed in normal controls (P < 0.01). (4) The expression levels of all 4 IFNs in both SLE patients and normal controls were positively correlated with each other (P < 0.01 in all). (5) Protein levels of IFN-alpha, IFN-beta and IFN-omega in patients with SLE were similar to those observed in normal group (P > 0.05 in all), protein level of IFN-alpha in active SLE group was apparently elevated than in inactive SLE group (P < 0.01), and protein level of IFN-gamma has a trend to increase in SLE group than in normal group., Conclusions: Decreased expression levels of IFN-alpha, -beta, -omega and -gamma have implicated significance in the determination of SLE disease specificity, of which IFN-alpha is the best. Lower expression level of IFN score has also significance in the determination of SLE disease specificity. Higher protein level of IFN-alpha is helpful to judging SLE disease activity.

Objective: To observe the association between systemic lupus erythematosus (SLE) and gene polymorphisms of OLF-1/EBF associated zinc finger protein(OAZ)., Methods: Verified single nucleotide polymorphisms (SNPs) with relatively high heterozygosity were chosen for allelic discrimination in 244 Chinese SLE pedigrees. Then transmissions of single SNP, and haplotypes were calculated by Genehunter software..OAZ mRNA level was also measured for comparing gene expression in patients of different haplotypes., Results: Genotyping of five SNPs within OAZ gene introns indicated there was no preferential transmission of single SNP, and haplotype T-A-G-G for rs1344531-rs2080353-rs933564-rs1345431 showed only weak linkage with the disease (P=0.04). However, haplotypes combining SNPs and the SLE-associated D16S517 allele showed significant association with SLE susceptibility (for rs933564-d16s517 G-271bp t:non-t=93:29 P<0.000001, for rs2080353-rs933564-d16s517 A-G-271bp t:non-t=88:35 P=0.000002). The haplotype A-G-271bp-G of Rs2080353-rs933564-D16s517-rs1345431 was also transmitted to patients preferentially (P=0.0084) and it showed a tendency to affect gene expression., Conclusion: Special polymorphism haplotype of OAZ gene is associated with Chinese SLE. OAZ may suggest a new pathway for lupus.

Objective: To investigate the expression levels of lymphocyte antigen 6 complex, locus E (LY6E) and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) genes in the peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE), and to evaluate the relations between these gene expression levels and disease activity., Methods: The clinical data of 144 SLE patients, 27 non-SLE patients with rheumatic diseases, and 59 normal controls were collected. The SLE patients were further divided into 2 subgroups: active SLE group (n = 87) and non-active SLE group (n = 57) according to SLEDAI scores. Specimens of peripheral blood were drawn; total RNA was extracted and transcribed into cDNA. Sybr green dye based real-time quantitative PCR method was used to compare the expression levels (indicated as-DeltaDeltaCT value) of LY6E and IFIT1 in patients with SLE and those in the controls., Results: (1) The-DeltaDeltaCT value of LY6E expression level of the SLE patients was 5.4760 +/- 1.9806, significantly higher than those of the non-SLE patients (3.4323 +/- 1.7456) and normal controls (4.5198 +/- 1.6359, both P = 0.001). (2) The-DeltaDeltaCT value of LY6E and IFIT1 mRNA expression of the active SLE patients were 6.1960 +/- 1.7729 and 6.4997 +/- 2.6297 respectively, significantly higher than those observed in the inactive SLE patients (4.3770 +/- 1.7764 and 4.1327 +/- 2.6044 respectively, both P = 0.000). The-DeltaDeltaCT values of LY6E and IFIT1 mRNA of the SLE patients were correlated with the SLEDAI scores, and with the numbers of matched criteria used in the diagnosis of SLE (P < 0.001)., Conclusion: As IFN-induced genes, elevated expression of LY6E mRNA is specific to diagnosis of SLE. The real time expression levels of LY6E and IFIT1 genes are associated with SLE disease activity. To inhibit the expression of LY6E and IFIT1 may become a novel therapeutic approach for SLE.

Objective: To observe whether deoxyribonuclease I (DNASE1) gene expression and its DNASE1 mRNA expression was detected by real-time polymerase chain reaction and its alternatively spliced transcripts were performed by capillary electrophoresis. An analysis was also made to disclose whether specific single nucleotide polymorphisms (SNPs) haplotype had effects onDNASE1 gene expression and its alternatively spliced transcripts., Results: DNASE1 gene expression was higher in SLE patients than in normal controls (P<0.001), and in patients it was found to be of no relationship with SLE disease activity index score. However, it was increased in female patients. Capillary electrophoresis revealed that the pattern of alternatively spliced transcripts in patients was not the same as that in normal controls. Moreover, it seemed that different SNPs haplotype combination might show different transcript pattern in SLE patients., Conclusion: In SLE patients, DNASE1 gene expression is abnormal and there are alternatively spliced transcripts different from those in normal controls. DNASE1 gene is a critical factor in the pathogenesis of SLE.

Objective: To investigate the protein-to-protein interaction of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), a newly discovered systemic lupus erythematosus (SLE) related up-regulated gene, and its possible function., Methods: Peripheral blood of 40 SLE patients was obtained to extract total RNA and synthesized cDNA. Real-time PCR was used to determine the IFIT1 expression at transcript level. Peripheral blood of another 10 SLE patients was extracted to obtain specimens of white blood cell lysate. Molecular cloning and a modified gluthion S-transferase (GST)-pull down assay were used to capture the protein interacting with IFIT1 in the specimens of white blood cell lysate. MALDI-TOF mass spectrometry (MS) was preformed to identify the captured protein that could interact with IFIT1. Twenty-nine sex and age-matched healthy persons were used as controls., Results: By real-time PCR showed that the IFIT1Delta Ct value (x +/- s) was 2.344 +/- 1.200 in the SLE patients and was 3.734 +/- 1.274 in the controls (P < 0.001), showing a significant up-regulation in SLE patients. IFIT1 was cloned and GST-IFIT1 fusion protein was expressed in Escherichia coli. GST-IFIT1 fusion protein was further purified using Glutathione Sepharose 4B column, and was treated as bait to capture prey from peripheral white blood cell lysate of SLE patients. MALDI-TOF MS detected protein interaction between Rho/Rac guanine nucleotide exchange factor and IFIT1., Conclusion: IFIT1 may interact with Rho/Rac guanine nucleotide exchange factor, and regulate the activation of Rho/Rac proteins, thus being involved in the pathogenesis of SLE.

Objective: To localize susceptibility loci in Chinese systemic lupus erythematosus (SLE) cohort by linkage disequilibrium mapping the genomic interval in human chromosome 16 so as to understand whether the pathogenesis of human SLE is related to chromosome 16., Methods: Five microsatellite markers in chromosome 16 spanning from 57.79 cM to 65.1 cM were used for fluorescence based genotyping in 157 SLE families. Evidence for linkage disequilibrium was assessed using the extended transmission disequilibrium test (ETDT) program and Genehunter software. Susceptibility gene was searched in the positive locus, and real-time PCR method was used to detect gene expression., Results: With the ETDT, evidence for linkage disequilibrium of the marker D16S409 (P=0.0278) and D16S517 (P<0.0001) with SLE were found. It was observed that 271 bp allele of D16S517 was much in evidence for preferential transmission, while 277 bp allele was found to be transmitted less often than expected. Genehunter analysis is concordant with ETDT. By real-time PCR, OAZ(OLF1/ EBF-associated zinc finger protein) gene which is located in the positive locus showed higher expression in SLE patients than in normal controls., Conclusion: Results reveal that 16q12 (58.46 cM) is associated with Chinese SLE. OAZ is a likely susceptibility gene in this locus for SLE.

Systemic lupus erythematosus (SLE) is the prototype systemic autoimmune disease and genetic component seems to play an important role in disease susceptibility. Studies from murine models have shown that about 30 loci are related to the disease. Meanwhile, 50 loci have been found in linkage to SLE in human genomic studies, especially 1q23-24, 1q41-42, 2q37, 4p16-15.2, 6p21-11 and 16q13. A lot of candidate genes contribute to the disease susceptibility and different combinations of genes at multiple loci in individual patient may result in the development of diverse clinical features.

Objective: To study the association between bcl-2 gene polymorphism and systemic lupus erythematosus (SLE) in Chinese and detect the synergism between IL-10 and bcl-2 genotypes in determining susceptibility to SLE., Methods: The peripheral white blood cells from 232 nuclear families of SLE and 106 ethnically matched normal controls were collected and the DNA extracted. The allelic distribution of one microsatellite located in Bcl-2 gene was determined by using fluorescent-labeled primer and genotyping method., Results: (1) The distribution frequency of Bcl-2 alleles and its major genotypes were significantly different between SLE group and normal controls (P = 0.001 and 0.0029 respectively). (2) Transmission disequilibrium existed in Bcl-2 alleles. Bcl-2-195 bp allele was preferentially transmitted to affected offspring (t: non-t = 122:82, P = 0.0051) by TDT approach. Individuals carrying specific genotypes with both Bcl-2-195 bp allele and IL-10G138 bp allele were at higher risk of developing SLE than those carrying only Bcl-2-195 bp allele or IL-10G138 bp allele (OR = 3.00, 1.77, 1.07 respectively)., Conclusion: Bcl-2 gene polymorphism is associated with SLE and may be directly involved in the process of SLE or in linkage disequilibrium with some susceptibility loci nearby. Synergistic effect may exist between IL-10 and bcl-2 genotypes in determining susceptibility to SLE.

Objective: To establish an indirect immunofluorescent test so as to improve the sensitivity and specificity of examination of antibodies to beta(2)-glycoprotein., Methods: Full-length beta(2)GP cDNA was obtained from human hepatocellular cancer cell line HepG2 by RT-PCR and cloned into the mammalian expression vector pEGFP-C1. The recombinant plasmid pEGFP-beta(2)GP was transfected into HEp-2 cells. RT-PCR, immunoblotting (IBT), confocal fluorescence microscopy, and indirect immunofluorescent test (IIF) were used to confirm the expression, localization, and antigenicity of fusion protein of green fluorescent protein (GFP). Serum specimens from 19 patients suspected as with secondary antiphospholipid syndrome (APS), 1 patient diagnosed as with primary APS, and 10 normal persons were detected with IIF-IgG-beta(2)GP1, ELISA-IgG-ACL, and ELISA-IgG-beta(2)GP I simultaneously., Results: (1) The HEp-beta(2)GP I cells thus obtained retained their ability of expression of beta(2)GP-I-GFP for more than ten generations. This beta(2)GP-I-GFP showed the antigenicity of beta(2)GP-I with a characteristic feature. (2) Seven of the 20 serum specimens from APS patients showed characteristic immunofluorescent pattern. No serum specimen from normal persons showed immunofluorescent staining. The comparison of results of the three methods showed that the concordance between IIF-IgG-beta(2)GP I and ELISA-IgG-beta(2)GP I was the most perfect (Kappa = 0.886). (3) HEp-beta(2)GP I retained the immunofluorescent property of HEp-2 cell., Conclusion: As a new kind of substrate of IIF, beta(2)GP I transfectant can be used to detect anti-beta(2)GP-I antibodies. Transfeted HEp-2 cells keep the immunofluorescent property of HEp-2 cells in IFANA test and can be used as substrate for routine IFANA detection.