Aim To prepare the monoclonal antibodies (mAbs) against human CD28 and to study its biological feature. Methods The hybridoma cell lines were obtained by fusing spleen cells of Blab/c mice that had been immunized with murine lymphoma cells transfected with full-length huaman CD28 cDNA to myeloma cells Sp2/0. Ascites were induced to produce the mAbs. The specificity and affinity of the mAb 18G8 was verified by CD28 competitive inhibitory test and FACS. Reactivities of mAb 18G8 to PBTC, U266, 8226, Jurkat and Daudi cell were studied by indirect immunofluorescence staining. mAb 18G8-inducing proliferation of peripheral blood T cells (PBTCs) was determined by [3H]thymidine incorporation test. Results Five hybridoma cell lines were obtained. mAb 18G8 secreted by one of the them, belong to mouse IgG2a. It recognized a epitope different from which recognized by the standard mAb(clone CD28.2). The Reactivitrates of the mAb 18G8 to PBTC, U266, 8266, Jurkat and Daudi cells were 70.2％ , 99.3％ , 98.6％ , 76.4％ and 1.9％ , respectively, similar with CD28.2. It was indicated that different antigen epitopes expressed on all above cells. mAb 18G8 could promote the PBTC proliferation in vitro(SI=7). It was indicated that The substitution of mAb 18G8 for B7-1 molecule could also mediate the costimulatory signals. Conclusion 18G8 is a specific and functional anti-CD28 mAb it may be of significant value in basic studies and clinical application.

Guanylate binding protein-1(GBP-1) is an interferon-induced protein. To observe its antiviral effect against Hepatitis B virus (HBV) and Coxsackie virus B3 (CVB3), we constructed an eukaryotic expression vector of human GBP-1(hGBP-1). Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector, then subcloned into a pCDNA3.1(-) vector. Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells, and inhibition effect of hGBP-1 against HBV replication was observed. Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3, and viral yield in cultures were detected. The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells. hGBP-1 inhibit CVB3 but not HBV replication in vitro. These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.

To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation,we constructed an expression plasmid which contained 1.3 copies of the HBV genome,and measured the level of viral replication after transient transfection in Huh7 cells.We then observed the effect of antiviral drug administration.1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion.The recombinant plasmid was trans ient transfected into Huh7 cells,HBsAg,HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively.The antiviral effect of adefovir,a novel anti-HBV nucleotide analogue,was evaluated in this cellular model system.The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells,adefovir could inhibit HBV replication in this cellular model,and the inhibition was dosage-dependent.The conclusion is HBV replicon,which can initiate viral replication efficiently in hepatoma cells,may be a useful tool in the study of HBV replication and antiviral drug.

BACKGROUND: During recent years, mononucleotide polymorphism of some genes is possibly related to affectability of osteoarthritis (OA). However, previous researches mainly compare the gene expression of synoviocytes between OA and rheumatoid arthritis (RhA); therefore, the correlation of gene expression between synovioblast and fibroblast in other tissues should be further studied as compared with OA.OBJ ECTIVE: To observe the differences of gene expression between OA synovioblast and skin fibroblast.DESIGN: Observational contrast analysis.SETTING: People's Hospital of Shenzhen City.PARTICIPANTS: Synovium tissue was derived from OA patients who received replacement of knee joint in the Department of Orthopaedics, People's Hospital of Shenzhen City. All OA patients met the diagnostic criteria of osteoarthritis established by American College of Rheumatology in 1995. Three patients including 1 male and 2 females aged more than 65 years old and they did not have cardiac and pulmonary disease and diabetes mellitus. Three male normal volunteers who aged 25 to 35 years did not have rheumatic disease, osteoarthritis and dermatosis. All subjects provided a confirmed consent. The main reagents were detailed as follows: RPMI1640 culture medium, fetal bovine serum and TRIZOL agent (Invitrogen Life Technologies Company, USA); pGEM-T pUC (Progema Company, USA);Display PROFILE-BASIC and Display PROFILE Probe kits (Qbiogen Company, USA).METHODS: The experiment was carried out in People's Hospital of Shenzhen City from January to June 2005. Synovium of OA patients were treated with primary culture to obtain synovioblast; meanwhile, skin fibroblast treated with primary culture from normal subjects was regarded as the control group. Restricted enzyme section differential display was used to separate the different-expressed genes of synovioblast and skin fibroblast in OA patients. In addition, blast technique was used to compare the resulted ranks with Genbank ranks.MAIN OUTCOME MEASURES: Differences of gene expression between synovioblast and skin fibroblast in OA patients.RESULTS: Gene expressions of superoxide dismutase (SOD), TFPI2, CXCL2, CXCL6 and transforming growth factor (TGF) were high in synovioblast of OA patients as compared with those in skin fibroblast of normal subjects.CONCLUSION: Gene expressions of SOD, TFPI2, CXCL2, CXCL6 and TGF are high in synovioblast of OA patients as compared with those in skin fibroblast of normal subjects. This suggests that gene may play a certain role in onset of OA.

Objective To explore the clinical characteristics,management and pregnancy outcome in patients with ovarian tumor and ovarian tumor like condition complicated with pregnancy.Methods The clinical data of 248 patients with ovarian tumor and ovarian tumor like condition eomplicated with pregnancy who treated,operated and pathology conducted from January 2003 to December 2009 was analyzed retrospectively.Results Among of 248 patients,8 patients were found by pelrioscopy,184 patients were found by sonography.The rate of ovarian tumor and ovarian tumor like condition during pregnancy was 0.49％ (248/50 652),about 52.82％(131/248)were diagnosed as benign tumor,most of them were ovarian mature teratoma(22.18％,55/248).About 45.56％(113/248)were diagnosed as ovarian tumor like condition,most of them were ovarian chocolatecyst(23.79％,59/248).Four patients(1.61％,4/248)were ovarian malignant tumor.Two hundred and sixteen received operation,term birth was in 164 patients,premature birth was in 19 patients,miscarriage was in 33 patients.Conclusions Pelvioscopy and sonogaraphy are principally important in the diagnosis and detection of ovarian tumor during pregnancy.Pregnancy complicated with ovarian tumor or ovarian tumor like condition should be treated by tumor resection.Suitable surgery intervention during second trimester is safe.

BACKGROUND: Despite the advent of molecular biology and immunogenetics, the biologic behaviors and disease entities of primary cutaneous B-cell lymphomas(pCBCL) have been undetermined. Moreover, rarity of pCBCL cases and the conflicting datas of current issues have contributed to the dilemmas in understanding of the biology of pCBCL. Until now, a study of the overall features of pCBCL in Korea has been rarely presented. OBJECTIVE: We performed this study in order to identify the histopathologic and immunogenetic characteristics of pCBCL in Korea. METHODS: The histopathologic, immunophenotypic and molecular analysis of preserved specimens of 15 cases with pCBCL were conducted. RESULTS: 1. Of the 15 patients with pCBCL, most common types are follicle center cell lymphomas(73.3%). In REAL classification, diffuse large B-cell lymphoma is most common(66.6%). 2. In bcl-2 immunohistochemical staining, 3 cases(20%) were positive. 3. Only one of 15 cases of pCBCL denoted bcl-2 gene rearrangement by t(14;18) in minor cluster region. 4. Immunohistochemical staining demonstrated overexpression of p53 protein in 3(20%) of 15 cases. 5. 2 cases(13.3%) with point mutations(one for exon 5; the other for exon 8) in p53 DNA sequencing analysis. CONCLUSION: t(14;18) translocation may be rare in pCBCL in Korea. This finding indicates that bcl-2 expression by tumor cells in pCBCL without t(14;18) may occur by different genetic dysregulation. It seems to be that overexpression of p53 protein might not correspond with p53 mutations in pCBCL.
B-Lymphocytes* ; Biology ; Classification ; Exons ; Genes, bcl-2 ; Humans ; Immunogenetics* ; Korea* ; Lymphoma, B-Cell* ; Molecular Biology ; Sequence Analysis, DNA

OBJECTIVETo study coronary artery stenosis, myocardial ischemia, and left ventricular dysfunction in dual source computed tomography (DSCT) coronary artery angiography.

METHODSTotally 32 patients underwent both DSCT and X-ray coronary angiography within one week to detect coronary artery stenosis separately. Meanwhile, the values of left ventricular myocardial mass (LVMM) , left ventricular ejection fraction (LVEF) , and left ventricular stroke volume (LVSV) were calculated using cardiac function software in DSCT. Electrocardiography was carried out to diagnose myocardial ischemia. The coronary artery stenosis, values of LVMM, LVEF, and LVSV, and myocardial ischemia were compared.

RESULTSThe results of DSCT and X-ray coronary angiography were not significantly different. LVMM, LVEF, LVSV, and myocardial ischemia were significantly different between two- or three-branch groups or between middle or severe groups (both P<0.05) . However, no such significant difference was observed between single and two branch groups and between mild and middle groups. There were no statistically different findings for LVEF and LVSV, but there was statistical deference between LVMM and myocardial ischemia (P<0.05) . For single branch and middle to severe stenosis in left anterior descending (LAD) coronary artery, right coronary artery, left circumflex coronary artery, only the values of LVMM,LVEF,and LVSV in LAD group showed significant difference (P<0.05) .

CONCLUSIONSMore stenotic branches and severer stenosis in coronary artery often are associated with higher incidence of myocardial ischemia and severer left ventricular dysfunction. The stenosis of LAD coronary artery has especially severe impact on cardiac functions. LVMM is a sensitive indicator for myocardial ischemia in coronary artery stenosis.

Aged ; Coronary Angiography ; methods ; Coronary Stenosis ; complications ; diagnostic imaging ; physiopathology ; Female ; Humans ; Male ; Middle Aged ; Myocardial Ischemia ; etiology ; Tomography, X-Ray Computed ; methods ; Ventricular Function, Left ; physiology

Objective To investigate the significance of monitoring P(c-a)CO2 (the gradients between transcutaneous PCO2 and arterial PCO2) in patients with septic shock.Method 31 patients with early septic shock were enrolled as the study group and 20 patients with stable hemodynamics as the control group from Fab.2013 to Sept.2014 in our Intensive Care Unit (ICU).The patients with septic shock were treated guided by early goal directed therapy (EGDT) within 6 hours since hospitalization.The differences of baseline P(c-a) CO2 levels and other index as arterial lactate (LAC) concentration between two groups and the variations of these indexes after EGDT in the study group were compared respectively.Results The baseline levels of P(c-a)CO2 and LAC in patients with septic shock were significantly higher than in patients of control group: (21.2 ± 10.1) mmHg vs.(7.5 ±4.6), P =0.000, and (4.0±2.4) mmol/ Lvs.(1.6 ± 0.5), P =0.000.The areas under receiver operator characteristic (ROC) curve (AUC) for baselineP(c-a)CO2 and LAC were 0.918 (95％ CI: 0.843-0.992) and 0.840 (95％ CI: 0.719-0.962) respectively.A threshold of 14.0 mmHg for P(c-a)CO2 and 2.1 mmol/L for LAC discriminated patients with septic shock from without shock with the same sensibility of 83.9％ and the same specificity of 90.0％, respectively.With regard to prognosis (Day 28), AUC for baseline P(c-a)CO2 and LAC were 0.739 (95％ CI: 0.562-0.917) and0.702 (95％ CI: 0.514-0.889) respectively.A threshold of 21.5 mmHg for P(c-a) CO2 and 3.9 mmol/L for LAC discriminated survivors from nonsurvivors with the same sensibility of 71.4％ and the same specificity of 70.6％ respectively.31 patients in the study group completed EGDT within 6 hours after the admission, 16 (51.6％) passed EGDT and 13 (81.3％) survived, 15 (48.4％) failed EGDT and 4 (26.7％) survived, and survival rates were significantly different, F =9.314, P =0.004.After EGDT, P(c-a) CO2 (18.8 ± 9.4) mmHg and LAC (3.3 ± 2.4) mmol/Lreduced significantly compared with the baselines, all P =0.000.AUC then for P(c-a) CO2 and LAC were 0.742 (95％ CI: 0.562-0.921) and 0.769 (95％ CI: 0.593-0.945), respectively.A threshold of 18.3 mmHg for P(c-a)CO2 and 3.1 mmol/L for LAC discriminated survivors from nonsurvivors with the same sensibility of 71.4％ and the specificity of 71.4％ and of 76.5％ respectively.P(c-a) CO2 and LAC of patients passed EGDT reduced significantly compared with those failed EGDT: (14.8 ± 7.5) mmHgvs.(23.6±9.6) mmHg (P=0.012)、 (2.5±1.5) mmol/L vs.(4.3±2.9) mmol/L (P=0.038), and so did with their baseline : (14.8±7.5) mmHgvs.(18.0±8.1) mmHg, (P=0.042)、 (2.5±1.5) mmol/Lvs.(3.2±1.8) mmol/L, P=0.043.In patients failed EGDT, P(c-a)CO2 and LAC changed little after EGDT, from (24.6 ± 9.2) to (23.6 ± 9.6) mmHg (P =0.238) and from (4.8 ± 2.5) mmol/L to (4.3 ± 2.9) mmol/L (P =0.629).When baseline levels were compared between patients passed EGDT with those failed EGDT, P(c-a) CO2 was (18.0 ±8.1) mmHg vs.(24.6 ± 9.2) mmHg (P =0.042), LAC was (3.2 ± 1.8) mmol/L vs.(4.8 ± 2.5) mmol/L (P =0.050).Conclusions P(c-a) CO2 ＞ 14.0 mmHg could play a role in recognizing early septic shock.EGDT was an effective therapy for the disease and P(c-a)CO2 level could reflect the efficacy of EGDT.P(c-a)CO2 ＞ 21.5mmHg before EGDT and P(c-a) CO2 ＞ 19.3 mmHg after EGDT both could predict the prognosis of patients with septic shock.All above correlated well with LAC and represented a new efficient technique to assess tissue microperfusion.

Objective To observe the effect of hippocampal DNA methyltransferases (DNMTs)on neonatal cognitive impairments induced by sevoflurance exposure.Methods Sixty-four 7-day old male Sprague-Dawley rats were randomly divided into the following four groups (n =1 6):control group (group C),sevoflurane group (group S),sevoflurane+NaCl group (group SN),and sevoflurane+5-AZA group (group SA).Sevoflurane animals received 3% sevoflurane plus 30% oxy-gen for 2 hours daily for 3 consecutive days,and rats in group C were placed into the same container, which contained 30% oxygen only.Animals in group SA were intracerebroventricularly injected with 5-AZA (1 mg/kg),while group SN same volume of NaCl one hour before sevoflurane exposure. Open field and Morris water maze were given the four weeks after anesthesia (n =8).Rats without any behavior tests from each group (n =8)were euthanized 4 weeks after the treatment and the hip-pocampus was harvested.Quantitative real-time PCR and Western blot were used to detect the mRNA and protein levels of DNMT1,DNMT3a and DNMT3b.Results In the open field test,no significant difference was observed in the distance travelled and the time spent in the center of the arena.Com-pared with the group C,group S showed an increase in the latency,decreased time spent in the target quadrant,and the mRNA and protein levels of DNMT3a and DNMT3b in the hippocampus were sig-nificantly increased (P < 0.05).Compared with the group SN,group SA showed a decrease in the la-tency,more time spent in the target quadrant,and the mRNA and protein levels of DNMT3a and DNMT3b in the hippocampus were decreased (P < 0.05).There was no significant difference in the expression of DNMT1 among the four groups.Conclusion Sevoflurane exposure induces neonatal cog-nitive impairments later in life,which was accompanied by the increased mRNA and protein levels of DNMT3a and DNMT3b in the hippocampus.By contrast,pretreatment of 5-AZA decreased hipp-ocampal DNMT3a and DNMT3b,and ameliorated cognitive impairments.These results suggest that DNMTs are involved in sevoflurane induced neonatal cognitive impairments.

ObjectiveTo evaluate the clinical efficacy of cutaneous branches of reverse second and third dorsal metacarpal artery fasciocutaneous flaps for repair of distal- and middle-segment finger soft tissue defects. MethodsA total of 14 patients with distal- and middle-segment finger soft tissue defects complicated by exposure of the phalanx or tendon were repaired by using cutaneous branches of second and third dorsal metacarpal artery fasciocutaneous flaps ranging between 2.0 cm × 4.5 cm and 3.0 cm × 7.0 cm.ResuitsAll of the skin flaps survived after surgery.Follow-up data during a 6-40 month period showed that the flaps exhibited a satisfactory appearance.They were not fat or clumsy,with a 2-point discrimination of 59 mm,and there was good recovery of finger function.The donor site was able to be directly sutured without dermoplasty.Pigmented linear surgical streaks appeared in the donor site.Conclusion The cutaneous branches of Second and third dorsal metacarpal artery fasciocutaneous flaps provide a good approach for the repair of distal- and middle-segment finger soft tissue defects and functional reconstruction because of convenient dissection,little trauma,sufficient use of the dorsal metacarpal artery.