Kupffer cells, expressing toll-like receptor 4 (TLR4), play a central role in hepatic ischemia/reperfusion (I/R) injury. Hyaluronic acid (HA) fragments, degradative products of high-molecular-weight HA (HMW-HA), acqui...Kupffer cells, expressing toll-like receptor 4 (TLR4), play a central role in hepatic ischemia/reperfusion (I/R) injury. Hyaluronic acid (HA) fragments, degradative products of high-molecular-weight HA (HMW-HA), acquire the ability to activate immune cells under inflammatory conditions. Here we inves- tigated whether HA fragments could activate Kupffer cells and analyzed the underlying mechanism. Kupffer cells were isolated from wild-type mice (WT, C3H/HeN) and TLR4 mutant mice (C3H/HeJ) and HA fragments were produced by the methods of enzyme digestion and chromatography. Then Kupffer cells were stimulated by HA fragments or other control stimuli. The activation of Kupffer cells was estimated as the release of pro-inflammatory cytokines. The activation of p38 MAPK pathway of Kupffer cells was checked and blocking experiments were done as well. The results indicated that HA fragments acquired the ability to activate Kupffer cells in vitro, which was TLR4 dependent and not due to contamination of lipopolysaccharide. Experiments of p38 MAPK kinase inhibition by SB-203580 verified p38 MAPK was required in HA fragments induced Kupffer cells activation. This suggests that HA fragments, degradative products of one of the major glycosaminoglycans of the extracellular matrix, play critical roles in Kupffer cell activation mediated by TLR4 signaling pathway, which is, at least partially, de- pendent on p38 MAPK activation.展开更多
AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expressi...AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently de展开更多
AIM:To labed Anti-hepatoma monoclonal antibody(mAb) fragment HAb18 F(ab')_2 was labeled with 188 Re for the pharmacokinetic model of ^(188)Re-HAb18 F(ab')_2 and to evaluate its pharmacokinetic parameters in he...AIM:To labed Anti-hepatoma monoclonal antibody(mAb) fragment HAb18 F(ab')_2 was labeled with 188 Re for the pharmacokinetic model of ^(188)Re-HAb18 F(ab')_2 and to evaluate its pharmacokinetic parameters in hepatoma- bearing nude mice. METHODS:HAb18 F(ab')_2 was directly labeled with ^(188)Re using 2-mercaptoethanol(2-ME)as reducing agents. Labeling efficiency and immunoreactivity of ^(188)Re-HAb18 F (ab')_2 were evaluated by Whatman 3MM paper chromatography and live cell assay,respectively. Biodistribution analysis was also conducted in nude mice bearing human hepatoma in which animals were sacrificed at different time points(1,4,18,24 and 24h)after ^(188)Re-HAb18 F(ab')_2 was injected through tail-vein into hepatoma-bearing nude mice.The blood and radioactivity of organs and mass were measured.The concentrations of ^(188)Re-HAb18 F(ab')_2 were evaluated with a pharrnacokinetic 3P97 software. RESULTS:The optimum labeling efficiency and immunoreactive fraction were 91.7% and 0.78%, respectively.The parameters of ^(188)Re-HAb18 F(ab')_2 were: T_(1/2),2.29h;Vd,1.49×10^(-9)L·Bq^(-1);AUC,20.49×10~9Bq·h· L^(-1);CL,0.45×10^(-3)L·h^(-1).^(188)Re-HAb18 F(ab')_2 could locate specially in hepatoma with high selective reactivity of HAb18 F(ab')_2.^(188)Re-HAbl8 F(ab')_2 was mainly eliminated by kidney.The maximal tumor to blood ratio was at 48h,and maximal tumor to liver ratio was at 18h. CONCLUTION:The pharmacokinetics of ^(188)Re-HAb18 F(ab')_2 fit a I-compartment model.^(188)Re-HAb18 F(ab')_2 can be uptaken selectively at the hepatoma site.展开更多
AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimag...AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging（RⅡ）.METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F（ab’）<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis（SDS-PAGE）and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate（ EDTA ）and L-cysteine（L-cys）solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma（HHCC）xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01（%ID/g）,respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F（ab’）<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.展开更多
Parameters identification of rockfill materials is a crucial issue for high rockfill dams. Because of the scale effect, random sampling and sample disturbance, it is difficult to obtain the actual mechanical propertie...Parameters identification of rockfill materials is a crucial issue for high rockfill dams. Because of the scale effect, random sampling and sample disturbance, it is difficult to obtain the actual mechanical properties of rockfill from laboratory tests. Parameters inversion based on in situ monitoring data has been proven to be an efficient method for identifying the exact parameters of the rockfill. In this paper, we propose a modified genetic algorithm to solve the high-dimension multimodal and nonlinear optimal parameters inversion problem. A novel crossover operator based on the sum of differences in gene fragments(So DX) is proposed, inspired by the cloning of superior genes in genetic engineering. The crossover points are selected according to the difference in the gene fragments, defining the adaptive length. The crossover operator increases the speed and accuracy of algorithm convergence by reducing the inbreeding and enhancing the global search capability of the genetic algorithm. This algorithm is compared with two existing crossover operators. The modified genetic algorithm is then used in combination with radial basis function neural networks(RBFNN) to perform the parameters back analysis of a high central earth core rockfill dam. The settlements simulated using the identified parameters show good agreement with the monitoring data, illustrating that the back analysis is reasonable and accurate. The proposed genetic algorithm has considerable superiority for nonlinear multimodal parameter identification problems.展开更多
AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the D...AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.展开更多
基金Supported by the National Natural Science Foundation of China (Grant No. 30500487 and 30700792)
文摘Kupffer cells, expressing toll-like receptor 4 (TLR4), play a central role in hepatic ischemia/reperfusion (I/R) injury. Hyaluronic acid (HA) fragments, degradative products of high-molecular-weight HA (HMW-HA), acquire the ability to activate immune cells under inflammatory conditions. Here we inves- tigated whether HA fragments could activate Kupffer cells and analyzed the underlying mechanism. Kupffer cells were isolated from wild-type mice (WT, C3H/HeN) and TLR4 mutant mice (C3H/HeJ) and HA fragments were produced by the methods of enzyme digestion and chromatography. Then Kupffer cells were stimulated by HA fragments or other control stimuli. The activation of Kupffer cells was estimated as the release of pro-inflammatory cytokines. The activation of p38 MAPK pathway of Kupffer cells was checked and blocking experiments were done as well. The results indicated that HA fragments acquired the ability to activate Kupffer cells in vitro, which was TLR4 dependent and not due to contamination of lipopolysaccharide. Experiments of p38 MAPK kinase inhibition by SB-203580 verified p38 MAPK was required in HA fragments induced Kupffer cells activation. This suggests that HA fragments, degradative products of one of the major glycosaminoglycans of the extracellular matrix, play critical roles in Kupffer cell activation mediated by TLR4 signaling pathway, which is, at least partially, de- pendent on p38 MAPK activation.
基金the grants No.KY951-Al-301 and No.KY95T-06-03 from the 9th Five Years Plan Key Research Programs of the Chinese Academy of Sciences.
文摘AIM: To establish a convenient immunoassay method based on recombinant antigen preS1(21-119aa) to detect anti-preS1 antibodies and evaluate the clinical significance of antibodies in hepatitis B. METHODS: The expression plasmid pET-28a-preS1 was constructed, and a large quantity of preS1(21-119aa) fragment of the large HBsAg protein was obtained. The preS1 fragment purified by Ni(2+)-IDA affinity chromatography was used as coated antigen to establish the indirect ELISA based on streptavidin-biotin system for detection of the anti-preS1 antibodies in sera from HBV-infected patients. For follow-up study, serial sera were collected during the clinical course of 21 HBV-infected patients and anti-preS1 antibodies, preS1 antigen, HBV-DNA and other serological HBV markers were analyzed. RESULTS: preS1(21-119aa) fragment was highly expressed from the plasmid pET-28a-preS1 in a soluble form in E.Coli (30mg.L(-1)), and easily purified to high purity over 90% by one step of Ni(2+)-IDA-sepharose 6B affinity chromatography. The purity and antigenicity of the purified preS1(21-119aa) protein was determined by 150g.L(-1) SDS-PAGE, Western blot and a direct ELISA. Recombinant preS1(21-119aa) protein was successfully applied in the immunoassay which could sensitively detect the anti-preS1 antibodies in serum specimens of acute or chronic hepatitis B patients. Results showed that more than half of 19 acute hepatitis B patients produced anti-preS1 antibodies during recovery of the disease, however, the response was only found in a few of chronic patients. In the clinical follow-up study of 11 patients with anti-preS1 positive serological profile, HBsAg and HBV-DNA clearance occurred in 6 of 10 acute hepatitis B patients in 5-6 months, and seroconversion of HBeAg and disappearance of HBV-DNA occurred in 1 chronic patients treated with lavumidine, a antiviral agent. CONCLUSION: The high-purity preS1(21-119aa) coated antigen was successfully prepared by gene expression and affinity chromatography. Using this antigen, a conveniently de
基金the National Natural Science Foundation of China,No.39700175
文摘AIM:To labed Anti-hepatoma monoclonal antibody(mAb) fragment HAb18 F(ab')_2 was labeled with 188 Re for the pharmacokinetic model of ^(188)Re-HAb18 F(ab')_2 and to evaluate its pharmacokinetic parameters in hepatoma- bearing nude mice. METHODS:HAb18 F(ab')_2 was directly labeled with ^(188)Re using 2-mercaptoethanol(2-ME)as reducing agents. Labeling efficiency and immunoreactivity of ^(188)Re-HAb18 F (ab')_2 were evaluated by Whatman 3MM paper chromatography and live cell assay,respectively. Biodistribution analysis was also conducted in nude mice bearing human hepatoma in which animals were sacrificed at different time points(1,4,18,24 and 24h)after ^(188)Re-HAb18 F(ab')_2 was injected through tail-vein into hepatoma-bearing nude mice.The blood and radioactivity of organs and mass were measured.The concentrations of ^(188)Re-HAb18 F(ab')_2 were evaluated with a pharrnacokinetic 3P97 software. RESULTS:The optimum labeling efficiency and immunoreactive fraction were 91.7% and 0.78%, respectively.The parameters of ^(188)Re-HAb18 F(ab')_2 were: T_(1/2),2.29h;Vd,1.49×10^(-9)L·Bq^(-1);AUC,20.49×10~9Bq·h· L^(-1);CL,0.45×10^(-3)L·h^(-1).^(188)Re-HAb18 F(ab')_2 could locate specially in hepatoma with high selective reactivity of HAb18 F(ab')_2.^(188)Re-HAbl8 F(ab')_2 was mainly eliminated by kidney.The maximal tumor to blood ratio was at 48h,and maximal tumor to liver ratio was at 18h. CONCLUTION:The pharmacokinetics of ^(188)Re-HAb18 F(ab')_2 fit a I-compartment model.^(188)Re-HAb18 F(ab')_2 can be uptaken selectively at the hepatoma site.
基金National Natural Science Foundation of China,No.39700175
文摘AIM To directly radiolabel an anti-hepatomamAb fragment HAb18 F（ab’）<sub>2</sub> with <sup>99m</sup>Tc bystannous-reduced method,and assess thestability,biodistribution and radioimmun-oimaging（RⅡ）.METHODS Immunoreactive fraction wasdetermined according to Lindmo’s method.Ellman’s reagent was used to determine thenumber of thiols in the reduced F（ab’）<sub>2</sub>.Labelingefficiency and homogeneity were measured bypaper chromatography,sodium dodecylsulphatepolyacrylamide gel electrophoresis（SDS-PAGE）and autoradiography.Challenge assay involvedthe incubation of aliquots of labeled antibody inethylenediaminetetraacetate（ EDTA ）and L-cysteine（L-cys）solutions with different molarratio at 37℃ for 1h,respectively.Investigationsin vivo utilized nude mice bearing humanhepatocellular carcinoma（HHCC）xenograftswith gamma camera imaging and tissuebiodistribution studies at regular intervals.RESULTS The labeling procedure was finishedwithin 1.5 h compared with the'pretinning'method which would take at least 21h.In vitrostudies demonstrated that the radiolabeled mAbfragment was homogeneous and retained itsimmunoreactivity.Challenge studies indicatedthat <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub> in EDTA is morestable than in L-cys.Imaging and biodistribution showed a significant tumor uptake at 24 h post-injection of <sup>99m</sup>Tc-labeled HAb18 F（ab’）<sub>2</sub>.Theblood,kidney,liver and tumor uptakes at 24hwere 0.56±0.09,56.45±11.36,1.43±0.27 and6.57±3.01（%ID/g）,respectively.CONCLUSION <sup>99m</sup>Tc-HAb18 F（ab’）<sub>2</sub> conjugateprepared by this direct method appears to be aneffective way to detect hepatoma in nude micemodel.
基金supported by the National Natural Science Foundation of China(Grant Nos.51379161&51509190)China Postdoctoral Science Foundation(Grant No.2015M572195)the Fundamental Research Funds for the Central Universities
文摘Parameters identification of rockfill materials is a crucial issue for high rockfill dams. Because of the scale effect, random sampling and sample disturbance, it is difficult to obtain the actual mechanical properties of rockfill from laboratory tests. Parameters inversion based on in situ monitoring data has been proven to be an efficient method for identifying the exact parameters of the rockfill. In this paper, we propose a modified genetic algorithm to solve the high-dimension multimodal and nonlinear optimal parameters inversion problem. A novel crossover operator based on the sum of differences in gene fragments(So DX) is proposed, inspired by the cloning of superior genes in genetic engineering. The crossover points are selected according to the difference in the gene fragments, defining the adaptive length. The crossover operator increases the speed and accuracy of algorithm convergence by reducing the inbreeding and enhancing the global search capability of the genetic algorithm. This algorithm is compared with two existing crossover operators. The modified genetic algorithm is then used in combination with radial basis function neural networks(RBFNN) to perform the parameters back analysis of a high central earth core rockfill dam. The settlements simulated using the identified parameters show good agreement with the monitoring data, illustrating that the back analysis is reasonable and accurate. The proposed genetic algorithm has considerable superiority for nonlinear multimodal parameter identification problems.
文摘AIM: To generate soluble single chain variable fragments (ScFv) of monoclonal antibody MC3 recognizing colorectal and gastric carcinomas. METHODS: mRNA was isolated from the hybridoma cell line producing MC3 and the DNAs encoding variable domains of heavy and light chains (VH and VL) of the antibody were amplified separately by RT-PCR and assembled into ScFv DNA with a linker DNA. The ScFv DNA was ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into E.coli TG1.The transformed cells were infected with M13KO7 helper phage to yield recombinant phages. After two rounds of panning with gastric carcinoma cell line AGS highly expressing MC3-binding antigen, the phage clones displaying ScFv fragments of the antibody were selected by ELISA. 4 phage clones showing strong signal in ELISA were used to infect E.coli HB2151 to express soluble ScFvs. The soluble ScFvs were identified by Dot blot and Western blot, and their antigen-binding activity was assayed by ELISA. The VH and VL DNAs of the ScFv DNA derived from phage clone 19 were sequenced. RESULTS: The VH,VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. After two rounds of panning to the recombinant phages, 18 antigen-positive phage clones were selected from 30 preselected phage clones by ELISA. All the soluble ScFvs derived from the 4 out of the 18 antigen-positive phage clones were about M(r)32000 and concentrated in periplasmatic space under the given culture condition. The soluble ScFvs could bind the antigen, and they shared the same binding site with MC3. The sequences of the VH and VL DNAs of the MC3 ScFv showed that the variable antibody genes belonged to the IgG1 subgroup,kappa-type. CONCLUSION: The soluble ScFv of MC3 is successfully produced, which not only provides a possible novel targeting vehicle for in vivo and in vitro study on associated cancers, but also offers the antibody a stable genetic source.
