Objective To construct bispecific antibody BsAb1/17 against both IL-1β and IL-17A,express and purify the biologically active BsAbl/17 protein in prokaryotic system for further studies and applications. Methods VH1VL17-CL and VL1VH17-CH1 gene segments were constructed by overlap-PCR.Restriction enzyme sites Nco Ⅰ and BamH Ⅰ were designed at the 5'and 3' end primers respectively. The products of overlap-PCR were ligated to the Nco Ⅰ/BamH Ⅰ -prepared pET-27b vector. The recombinant plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 ( petB ) were transformed into E. coliRosetta separately. The expressing products were analyzed by SDS-PAGE and Western blot. Neutralization activity of the bispecific antibody for blocking the induction of IL-18 expression by IL-1β in human T cells was determined by real-time PCR. Neutralization activity of the bispecific antibody for blocking the induction of IL-6 expression by IL-17A in HeLa cells was determined by ELISA assay. Results The structure of the plasmids pET-27b-VH1 VL17-CL(petA) and pET-27b-VL1 VH17-CH1 (petB)was confirmed by DNA sequencing. After induction, the fusion proteins were expressed mainly as inclusion bodies. The purity of the both proteins exceeded 90%. SDS-PAGE analysis suggests the relative molecular mass of both products expressed by petA and petB were approximately 38× 103, which is in accordance with the theoretical value. The results of Western blot and ELISA test demonstrated that BsAb1/17 molecule had binding ability to both IL-1β and IL-17A. The BsAb1/17 could block IL-1β to stimulate human T cell to express IL-18 and block IL-17A to stimulate HeLa cell to express IL-6. Conclusion We successfully constructed a novel bispecific antibody BsAb1/17 against both IL-1 β and IL-17A, and expressed biologically active BsAb1/17 protein in prokaryotic system.

Objective To master the serum nickel change rule of acute nickel carbonyl poisoning in rats,and to provide laboratory support for clinical treatment of acute nickel carbonyl poisoning patients.Methods SPF rats were given nickel carbonyl (250mg/m3,500mg/m3) in static inhalation for 30min.Rats were anesthetized by ether for 15 seconds after exposure for 30min,2h,4h,8h,12h,24h,48h,72h and 7d respectively.Anatomized rats,kept blood collection 2-3mL,and separated serum 0.5-1mL.Serum nickel was detected by AA800 (American PE company).Results The average serum nickel levels of 250mg/m3 dose group at 30min,2h,4h,8h,12h,24h,48h,72h,7d were (33.69 ±2.59),(24.61 ±3.03),(27.83 ±5.69),(21.36 ±4.14),(20.39 ±4.14),(18.80 ±7.02),(14.51 ±8.21),(13.58 ± 5.78) and (12.83 ± 4.41)μg/L.30 minutes reached the peak,and was 5.30-fold of those in normal controls.There had significant differences compared with normal control(t =5.959,5.958,5.990,5.998,5.997,5.994,5.990,4.317,4.347,all P ＜ 0.01).The average serum nickel levels of 500mg/m3 dose group at 30min,2h,4h,8h,12h,24h,48h,72h,7d were (72.22 ± 1.62),(57.78 ± 12.99),(42.25 ± 7.25),(103.77 ± 11.11),(79.04 ±12.26),(26.35 ±6.56),(18.58 ±4.92),(17.22 ±9.73),(14.59 ±5.27) μg/L.8h reached the peak,and was 16.33-fold of normal controls.The differences were significant (t =5.960,5.947,5.978,5.927,5.948,5.959,3.143,2.447,2.440,all P ＜ 0.05).Meanwhile,there were significant differences between two groups of 30min,2h,4h,8h,12h(t =5.208,2.447,2.449,5.959,5.959,P =0.001,0.049,0.042,0.000,0.000),but there was no significant difference after 24h.Conclusion There was significant doses-effect relationship between serum nickel content of acute nickel carbonyl poisoning rats and the dosage.Nickel carbonyl or its metabolites were excreted mainly within 24h.

OBJECTIVETo establish a animal model of biliary leakage in pigs with laparoscopy.

METHODSEight healthy Bama minipigs were subject to laparoscopic surgery under general anesthesia.The cystic duct was resected at 0.5-1.0 cm from the root and the stump was left open with the gallbladder removed. Blood routine and hepatic functions of the pigs were tested before and 24 h after the surgery, and endoscopic retrograde cholangiography (ERC) was performed 24 h after the surgery. At 48 h after the surgery, the pigs were sacrificed for observation of the stump of the cystic duct.

RESULTSThe gallbladder, cystic duct, cystic artery, and the anatomical relations between the gallbladder and liver and between the cystic duct and common bile duct were fully exposed under laparoscopy. White blood cells, neutrophils and direct bilirubin increased significantly after the operation (P<0.05). The cystic ducts were resected at 0.5-1 cm away from the roots of the cystic ducts in 7 pigs and at 2 cm in 1 pig.

CONCLUSIONLaparoscopy is safe and feasible for establishing the porcine model of biliary leakage.

Animals ; Biliary Fistula ; Cystic Duct ; surgery ; Disease Models, Animal ; Female ; Laparoscopy ; Male ; Swine ; Swine, Miniature

Objective To establish a mouse model of systemic C. albicans infection by oral inoculation of the pathogen and observe the proliferation and distribution of C. albicans in vivo tissues. Methods Male ICR mice(n=46) were used as the experiment group(n=40) and blank group (n=6). Cotton swabs with C. albicans were used to infect the mice (7 × 106 CFU/mL), and the blank group with saline. The mice of the experiment group were randomly divided into two groups:model group A for clinical assessment (n=20) and model group B for tissue fungal burden detection (n=20). Clinical score, survival and autopsy were carried out among the model group A. Five mice were randomly killed from the model group B at 3 d, 5 d and7 d after infection, respectively ( blank group killed 2 mice each time) . Microbial load tablet method was used to detect the tissue fungal burdens in different tissues, meanwhile samples of tongue, esophagus, stomach, liver, kidney, lung of infected mice were taken for pathological examination. Results White spot appeared on the surface of tongue since 3 d postinfection and increased with time and finally caused death. The mortality reached over 50% at 5 d. C. albicans was not only detected from the tongue (87?5%), stomach (87?5%), liver (54?5%), kidney (50?5%), lung (20%) and heart (4%), but also was microscopically seen mycelia proliferation in the tongue, stomach, liver, and kidney , yet not seen in the control group, showing that C. albicans caused disseminated systemic infection through mucosal infection in mice. Conclusions C. albicans can induce opportunistic systemic infection by breakthrough the mucosal immune barrier, so as to increase the infection to death.

Adolescent ; Adult ; Endoscopy ; Ethmoid Sinus ; surgery ; Eyelids ; surgery ; Female ; Humans ; Male ; Middle Aged ; Orbital Fractures ; surgery ; Otorhinolaryngologic Surgical Procedures ; methods ; Young Adult

OBJECTIVETo evaluate the clinical value of multicolor melting curve analysis(MMCA) for detecting genetic mutations in G6PD deficiency.

METHODSA total of 402 peripheral blood samples(256 males and 146 females) were collected from suspected patients or their relatives at the Prenatal Diagnosis Center of Liuzhou Maternal and Child Health Hospital between March 2012 and May 2012. The samples were screened by G6PD/6PGD quantitative ratio testing. The reliability of the assay was evaluated by multiplex probe melting curve assay(which can detect 16 G6PD mutations) and DNA sequencing through a double blind study.

RESULTSOne hundred seventy cases with G6PD/6PGD ratio < 1.0 and 232 cases with G6PD/6PGD ratio ≥ 1.0 were detected by the enzymological method. DNA sequencing has identified 182 wild type samples, 151 hemizygous mutation samples, 5 female homozygous mutation samples, 54 female heterozygous mutation samples and 10 female double heterozygous mutation samples. Multicolor melting curve analysis has detected 185 wild type samples, 148 hemizygous mutation samples, 5 female homozygous mutation samples, 55 female heterozygous mutation samples and 9 female double heterozygous mutation samples. The specificity and sensitivity of G6PD gene mutation detection by multicolor melting curve analysis were 100%(182/182) and 98.6%(217/220), respectively. The positive predictive value and negative predictive value were 99.5%(216/217) and 98.4%(182/185), respectively, and the Youden's index was 0.986. The concordance rate of the sample detection between the melting curve assay and DNA sequencing was 99.0%(398/402). Twenty-one different genotypes were detected by the multicolor melting curve analysis and 24 different genotypes were detected by DNA sequencing. Four samples containing mutations(c.196T>A or c.406C>T) were not detected by multicolor melting curve analysis, which can be attributed to different technical settings of the two methods.

CONCLUSIONMulticolor melting curve analysis for G6PD gene mutation detection is a simple, rapid, sensitive and specific method, which can be used for clinical diagnosis of G6PD deficiency.

Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Glucosephosphate Dehydrogenase ; genetics ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

To investigate the effect of hSCGF-alpha on human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs), we obtained hSCGF-alpha using genetic engineering, hSCGF-alpha gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E. coli BL21 (DE3) was mainly existing in soluble form. The recombinant hSCGF-a was purified using NI-NTA affinity chromatography and the purity was up to 90%. The colony forming test revealed that combined use hSCGF-alpha and rmGM-CSF (recombinant murine GM-colony stimulating factor, rmGM-CSF) had granulocyte/macrophage (GM) promoting effects on murine bone marrow GM progenitor. In addition, the results indicated that hSCGF-alpha and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.
Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Drug Synergism ; Escherichia coli ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cell Factor ; biosynthesis ; genetics ; Umbilical Cord ; cytology

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.

OBJECTIVETo investigate the effects of different concentrations of paraquat (PQ) poisoning on the expression of voltage-dependent anion channel (VDAC) and caspase family in the mitochondria of rat lung tissue, and to explore possible mechanisms of acute lung injury induced by acute PQ poisoning.

METHODSTwo hundred healthy adult Wister rats with equal numbers of male and female ones were randomly and equally divided into control group and poisoned group. The control group received one-time gastric lavage with 1 ml of normal saline, and the poisoned group with PQ (50 mg/kg) diluted in 1 ml of normal saline. Twenty rats were collected at 1, 24, 72, 120, and 168 h after lavage with normal saline or PQ and dissected after anesthesia. Mitochondria were separated from rat lung tissue, and the content of VDAC and caspase-3, -8, and -9 were determined.

RESULTSThe expression of VDAC and caspase-3, -8, and -9 in the poisoned rats were significantly higher than that in the control group (P < 0.001). At 1, 24, 72, 120, and 168 h after exposure, acute diffuse damages were found in alveolar capillary endothelial cells, alveolar epithelial cells, and pulmonary interstitial cells. Inflammatory cell infiltration in the pulmonary interstitium, alveolar structural disorder, and substantially increased fibroblasts were also found in rat lung tissue.

CONCLUSIONPQ poisoning can up-regulate the expression of VDAC and caspase-3, -8, and -9 in mitochondria of rat lung tissue to induce acute lung injury.

Acute Lung Injury ; chemically induced ; pathology ; Animals ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Caspases ; metabolism ; Female ; Lung ; drug effects ; pathology ; Male ; Mitochondria ; drug effects ; metabolism ; Paraquat ; poisoning ; Rats ; Rats, Sprague-Dawley ; Voltage-Dependent Anion Channels ; metabolism