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Background: The HAMA response is a major challenge when murine antibodies are repeatedly administered for antibody directed enzyme prodrug therapy in vivo. In this study we have achieved humanization of the anti-gamma-seminoprotein E(4)B(7) murine mAb by guided selection., Methods: Using optimal Ig Fab primers, human Fd and CL gene repertoires were amplified by RT-PCR from PBMCs of prostate cancer patients. The human Lc gene repertoire was first paired with the murine Fd gene of E(4)B(7) mAb to construct a pComb3X hybrid Fab display library. This hybrid library was screened with purified gamma-seminoprotein antigen. The human Fd gene repertoire was then paired with the selected human Lc to construct a fully human Fab library. After four more rounds of panning, completely human Fab antibodies specific for gamma-seminoprotein were selected and further identified., Results: First, using the E(4)B(7) Fd gene as a template, light chain shuffling was achieved by panning the hybrid library. Then, using the selected Lc as a template, a human Fab antibody against gamma-seminoprotein was produced through heavy chain Fd shuffling. Western blotting, ELISA, and flow cytometry results demonstrated that the resulting human Fab antibody resembled the parental E(4)B(7) mAb in that they both recognized the same epitope with similar affinities. Fluorescent cell staining and immunohistochemistry analysis further confirmed that this newly constructed human anti-gamma-seminoprotein Fab antibody indeed specifically bound prostate cancer cells and tissue., Conclusions: Through guided-selection, we successfully produced a human anti-gamma-seminoprotein Fab antibody. This work lays the foundation for optimal antibody-directed enzyme prodrug therapy of prostate cancer using a fully human Fab antibody.

Aim: To establish recombinant HEK293-16 cell strain expressing chimera receptor EpoR/LR-F3/HAb18GEF via site-specific integration expression system., Methods: The HAb18GEF gene fragment was amplified by PCR and digested with Sac I/Not I, and then cloned into the multiple clone site downstream of EpoR/LR-F3 gene of eukaryotic expression vector pCEL2f. The obtained pCEL2f/HAb18GEF vector, checked by partial nucleotide sequencing and restriction endonuclease digestion, was co-transfected with POG44 vector into HEK293-16 cells by mixing with Lipofect AMINE2000 reagent. After selection with hygromycin B for one month, transient and stable expression of EpoR/LR-F3/HAb18GEF were detected by indirect immunofluorescence staining, flow cytometry assay, and Zeocin test., Results: The recombinant pCEL2f/HAb18GEF vector, containing fused EpoR/LR-F3/HAb18GEF gene with correct open reading frame, was successfully constructed. It was confirmed that both EpoR and HAb18GEF were expressed efficiently on the membrane of co-transfeced HEK293-16 cells. 12 stably-expressed clones, sensitive to Zeocin, were finally obtained, which presented 99.93% EpoR-positive cells with strong immunofluorescence intensity of 1036.39, and simultaneously presented 99.51% HAb18GEF-positive cells with strong immunofluorescence intensity of 652.72., Conclusion: HEK293-16 cell strains stably expressing EpoR/LR-F3/HAb18GEF chimera receptor have been developed successfully, which lays foundation for MAPPIT (Mammalian protein-protein interaction trap) screening of binding molecule of hepatoma associated antigen HAb18G/CD147.

Unlabelled: Orthotopic liver transplantation (OLT) is the only curative therapy of HCC with underlying cirrhosis, but due to HCC metastasis and recurrence, its benefit is limited to a small population who meet the strict selection criteria. We previously reported that Licartin ([131I] mAb HAb18G/CD147) was safe and effective in treating HCC patients, and its antigen, HAb18G/CD147, was closely related to HCC invasion and metastasis. Here, we reported a randomized controlled trial to assess the post-OLT antirecurrence efficacy of Licartin in advanced HCC patients. We randomized 60 post-OLT patients with HCC, who were at tumor stage 3/4 and outside the Milan criteria before OLT, into 2 groups. Three weeks after OLT, the treatment group received 15.4 MBq/kg of Licartin, while the control group received placebo intravenously for 3 times with an interval of 28 days. At 1-year follow-up, the recurrence rate significantly decreased by 30.4% (P = 0.0174) and the survival rate increased by 20.6% (P = 0.0289) in the treatment group, compared with those in the control group. For the control group versus the treatment group, the hazard ratio for recurrence was 3.60 (95% confidence interval [CI], 1.50-8.60) and that for death was 3.87 (95% CI, 1.23-12.21). Licartin treatment also resulted in an earlier decreased AFP level and a longer time of normal AFP level than placebo (P = 0.0016). No Licartin-related toxic effects were observed., Conclusion: Licartin is a promising drug for preventing post-OLT tumor recurrence in advanced HCC patients excluded by the currently strict criteria for OLT. HAb18G/CD147 can be a good drug target.

Objective: To explore the Ilizarov mini-invasive distraction technique for the treatment of severer ankle and foot deformities secondary to ischemic contracture of the leg., Methods: Based on the tension-stress low of Ilizarov, a serial of adjustable three dimensions external distractive instrument was developed in our department. From April 2002 to March 2004, 8 patients with ankle and foot deformities secondary to ischemic contracture of the leg induced by trauma and fracture were treated with the distractive instrument. Of them, 4 patient were male and 4 female, aged from 13 to 31 years with an average of 23 years. Five affected legs were in the left and 3 in the right. Preoperative abnormal style included talipes equines in 6 feet and equinovarus in 2 feet, with extensive scar contracture in the legs. Five patients suffered from failure of soft tissue release before, two patients with severe bony deformity of the feet were underwent limited foot triple osteotomy in this department before the distractive correction. The distraction was begun from 7 d after operation and distractive time from 29 to 60 d with an average 46 d., Results: All of the 8 patients were followed up from 10 months to 29 months, with an average of 13 months. All of deformities in the feet were corrected satisfactorily, full feet contacted with the ground in stand or walking and achieved with good function. No complication, such as pin tract infection, skin necrosis and neurovascular injury was occurred in this group., Conclusions: Mini-invasive distraction technique for treatment of severe ankle and foot deformity secondary to ischemic contracture of the leg is safe and mini-injury, it is also an effective approach for the treatment of various kinds of rigid foot anomaly.

HAb18G/CD147, a membrane spanning molecule and highly expressed in hepatocellular carcinoma (HCC) cells, was shown to stimulate the production of matrix metalloproteinases (MMPs) in the interaction of tumor cells and fibroblasts. Studies on the EMMPRIN/CD147 showed that CD147 extracellular domain is involved in the induction of MMPs. To study the biological molecular function of HAb18G/CD147 extracellular domain (HAb18G/CD147-ED) on production of MMPs following mediated tumor cell invasion, we isolated four novel monoclonal anibodies (MAbs)-1B3, 3B3, HAb18Gedomab1, and HAb18Gedomab2-against HAb18G/CD147-ED by immunization of BALB/c mice with purified HAb18G/CD147-ED fragments, which were efficiently expressed in Escherichia coli. Gelatin zymography and Boyden chamber assays were used to identify the production of MMPs in the co-cultured human fibroblast and HCC cells, and to quantify the migrated cells in the presence of the generated MAbs. The results showed that two MAbs (1B3 and 3B3) inhibited [corrected] the secretion of MMP-2 and [corrected] the HCC cell invasion, whereas the other two MAbs (HAb18Gedomab1 and HAb18Gedomab2) had reverse function [corrected] FCM additive assay showed that four MAbs recognized different epitopes of HAb18G/CD147-ED. Taken together, the results suggest that various regions of HAb18G/CD147-ED participated in the regulation of MMP secretion.

Aim: To screen human anti-gamma-sm (gamma-seminoprotein) light chain (Lc) with guided selection of murine Fd fragment., Methods: The human Lc repertorie genes were amplified by RT-PCR from PBMC in patients with prostate cancer, and cloned into the phagemid vector pComb3X with murine Fd gene against gamma-seminoprotein to construct the human-mouse hybrid Fab antibody library. The size of the library, antibody gene recombinant percentage and diversity were identified by colony counting, plasmid digestion and colony sequence analysis, respectively. Purified gamma-sm was used as antigen to screen the displayed phage hybrid antibody library rescued by helper phage M13K07 for three rounds. The positive clones were selected by ELISA with pIII-fusion antibody and then the sequences of light gene in the positive clones were analyzed by IMGT-VQUEST., Results: A 1.2x10(7) CPU human-mouse Fab antibody library was constructed with 90% Lc gene recombinant and great diversity. After 3 rounds' panning with gamma-sm, 5 positive clones were selected by ELISA and 2 clones with higher affinity were selected. Sequence analysis suggested these two positive clones contained the same light gene with high V(L) homology to human germline gene IGKV4-1*01., Conclusion: Human anti-gamma-sm light chain was successfully screened by constructing mouse-human hybrid Fab phage antibody library with murine Fd-guided selection.

Objective: To shorten the time of external skeletal fixation on legs, and enhance quality of limb lengthening, avoid complications of shortening, bending, twisting and etc., Methods: Insert pin transcortical to attack external skeletal fixation simultaneously, put un-reaming locked intramedullary nail (do not insert distal locked screw) into endosteum of lengthening bone. After the legs achieved predetermined length, insert distal locked screw and then remove external skeletal fixation, locked intramedullary nail, then maintain consolidation of rehabilitation., Results: The group lengthened legs for 412 cases. The range of lengthening was 3 to 18 cm. Mean length was 7.6 cm. The mean time for needed external skeletal fixation was 20 d/cm. The mean time of osteogenesis was 56 d/cm. For complications, there were 3 tibias ununion cases and 1 varus ankle. All cases were treated undergoing twice., Conclusions: The method reduces the time for needed external skeletal fixation visibly, enhances the quality of limb lengthening remarkably, prevents complications of shortening new bone, deformity, bending and re-fracture which do not effect the healing time. This is a new choice of limb lengthening.

Aim: To compare characteristics of polyclonal anti-sera against extracellular domain of hepatoma-associated antigen HAb18G/CD147(HAb18GEF) generated by different immunization schemes., Methods: BALB/c mice were immunized with GST-HAb18GEF fusion protein expressed in E.coli (routine immunization method), recombinant eukaryotic expression plasmid pcDNA3/HAb18G (intramuscular injection) and pcDNA3/HAb18G plasmid followed by human hepatoma cells booster (DNA-cell booster), respectively. The titers and Ig subclasses of polyclonal anti-sera against denatured and natural HAb18GEF were detected by indirect ELISA and cell ELISA, respectively. The specific binding of polyclonal anti-sera prepared by different immunization schemes to denatured HAb18GEF was analyzed by Western blot. The specific binding of polyclonal anti-sera produced by DNA-cell booster immunization to natural HAb18G antigen on hepatoma cells was detected by immunofluorescence staining., Results: GST-HAb18GEF immunization could induce polycolonal antibody IgG1 with higher titer mainly against denatured or linear epitopes on HAb18GEF. Antibody induced by pcDNA3/HAb18G intramuscular immunization was IgG2a with lower titer against natural epitopes on HAb18GEF. DNA-cell booster immunization could induce generation of polycolonal antibody IgG2a and IgG1 of moderate titers against the native epitopes on HAb18G., Conclusion: The polycolonal sera with different titers against different epitopes on HAb18GEF can be induced by different immunization schemes.

Objective: To study the role of the synchronized springy lengthening apparatus for the tibia and calcaneal tendon designed by the author in preventing the clubfoot of secondary to the Ilizarov tibia lengthening., Methods: Based on the Ilizarov tibia lengthening apparatus, a special synchronized springy lengthening apparatus for the tibia and calcaneal tendon was designed. The tibial was made of distal and proximal 2 rings respectively and 4 threaded rods, and the calcaneal was made of a half ring, 2 hinges and a threaded rod with spring. The half ring was fixed to the calcaneus by 2 crossed wires. The fracture tibia and fibula, ankle joint, talocalcaneal joint were attached to the apparatus. At the same time of tibia lengthening, the soft tissue was simultaneously stretched, the ankle joint could move, and the leg could bear weight. If the clubfoot angle was larger, the percutaneous fasciotomy of calcaneal tendon was performed; if the angle was less than 20 degrees, the pes deformities were corrected only by the stretch of calcaneal tendon., Results: Seventy-seven patients' tibia were lengthened averagely 4.6 cm, with an average speed of 0.7 mm/d. The healing made tibia lengthened, and the index was 1.35 months/cm. There were not the secondary varus and valgus deformities and clubfoot in all the patients. The clubfoot with 100-400 angle of the 16 patients were corrected after tibia lengthening., Conclusions: The new apparatus coincides with the biomechanical principle and can effectively prevent the secondary deformities of foot such as clubfoot, talipes varus and valgus after tibia lengthening procedure.

Objective: To study the methods and effects of Ilizarov distraction technique in treating the flexion deformity of the knee of arthrogryposis multiplex congenita., Methods: Between August 1998 and February 2003, 6 patients (10 knees) with the arthrogryposis multiplex congenita were treated, 4 patients in double knees, 5 males, 1 female, mean age 8 years and 2 months, ranged from 3 years and 7 months to 13 years. The preoperative flexion degree was averagely 51 degrees. The patients accompanied 13 other parts malformation of limbs. The modified Ilizarov distraction apparatus of the knee was used. While installing the apparatus in the operation, the knees should be kept in the of location of maximum extension, the center of joint hinges on the apparatus should be placed towards the rotatory center of the knee, two groups of 2 mm K-wires were passed through the femur and tibia around the knee, who were fixed on the proximal and distal rings. Distraction was started after the surgical procedure 5 days via rotating the threaded rods at the posterior of the knee, at an average of 2 to 3 mm per day, at the first week, after 2 weeks with the rate modified to 2 mm per day, up to the knee extended to 0 degrees. The accompanied deformities of the hip and/or the foot might be corrected at the same time or next time. The average duration of the distraction was 37 days (23-48 days). During the correction all limbs might undergo weight. After 2 weeks at the end of distraction the fixator was removed and the patients could walk by a long-leg brace., Results: Ten knees with the flexion contracture were sufficiently corrected without severe complications. Nine knees of all were followed up at an average time of 1 years and 3 mouths, no recurrence of the deformity was seen in all patients, their function of walk was significantly improved., Conclusions: Ilizarov technique is a simply, safe and effective method for managing the flexion deformity of the knee of the arthrogryposis multiplex congentia. The procedure is conformable to the biological theories and microsurgical principles.

To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a + vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a + vector was reduced with following methods: (1) optimization of secondary structure (2) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.

Aim: To construct a anti-dodecane-tertraacetic acid-yttrium(DOTA-Y) immune Fab phage antibody library., Methods: BALB/c were immunized with BSA-Y-DOTA which was prepared by DOTA-conjugated BSA and chelated with Y. After determination of anti-serum, total RNA was extracted from splenic lymphocytes of immuned mice. The heavy chain Fd and light chain Kappa genes repertoires of immunoglobulin were amplified respectively by RT-PCR, and then the amplified products were cloned into the reconstructive phage vector pComb3M to construct anti-DOTA-Y Fab antibody. And then, the recombination rate, diversity and display of Fab antibody library were identified by restriction endonuclease digestion, DNA sequencing and ELISA., Results: BSA-Y-DOTA was prepared successfully, and a higher titer of immune sera was achieved. The amplified gene fragments of Fd and Kappa chain by RT-PCR were correct and the length was with about 650 bp, and were inserted exactly. The sink size of Fab phage display library reached 8 x 10(7), the re-combination rate was about 90%, and it possesed great diversity. In addition, ELISA detection showed that there was Fab expression on the phage library., Conclusion: An immune Fab phage antibody library of DOTA-Y has been constructed successfully, which lays a solid foundation for screening specific anti-DOTA-Y antibody.

Objectives: To explore the effect of IL-18 on peripheral blood monocytes (PBMCs) from chronic hepatitis B (CHB) patients and HBV DNA released by HepG2.2.15 cells, which were transfected with the gene of HBV., Methods: PBMCs were isolated from 25 healthy persons and 25 CHB patients, which were co-cultured with HBcAg and IL-18 at different concentrations for 72 hours. The level of IFN-gamma in the culture supernatant of PBMCs was determined by ELISA. One patient' PBMCs were co-cultured for 96 hours with various concentrations of IL-18 and HepG2.2.15 cells which had been cultured for 24 hours, the supernatant was collected to detect HBV DNA level by PCR., Results: When PBMCs were stimulated by HBcAg and IL-18 at various concentrations, the levels of supernatant IFN-gamma in the CHB group were much higher than those in the normal control group (at 0.2ng/ml: t=11.7, P<0.01; at 1.0ng/ml: t=16.19, P<0.01; at 5.0ng/ml: t=20.12, P<0.01), especially when the PBMCs were stimulated by HBcAg, IL-18 and IL-12 (1313.20pg/ml+-187.76pg/ml vs. 390.75pg/ml+-43.23pg/ml, t=23.94, P<0.01). The IFN-gamma level in the patients who were stimulated by HBcAg alone was much lower than the levels in the patients who were stimulated by HBcAg and IL-18 at various concentrations, and which were lower than those in the patients stimulated by HBcAg, IL-12 and IL-18 at the same concentrations (light: t=2.2, P<0.05; moderate: t=2.97, P<0.05). The HBV DNA content in the supernatant of co-cultivation with HepG2.2.15 cells and PBMCs was much higher than that of the two kinds of cells stimulated by HBcAg and IL-18 at various concentrations or HBcAg, IL-18 and IL-12/IFN-a1b., Conclusion: IL-18 can improve the PBMCs from CHB patients to produce a great deal of IFN-gamma, so it has a good application prospect in two aspects: immunoregulatory effects and increasing the ability to kill the cells infected with virus.

Objective: To identify the roles of serum IL-18, IL-10, TNF-alpha and sIL-2R in the pathogenesis of chronic hepatitis C and the effects of interferon on the mentioned serum cytokines., Methods: The levels of IL-18, IL-10, TNF-alpha and sIL-2R were detected in 10 healthy controls, 24 asymptomatic HCV carriers, and 27 patients with chronic hepatitis C (before and after IFN treatment) by enzyme linked immunosorbent assay (ELISA)., Results: The levels of IL-18, IL-10, TNF-alpha and sIL-2R in the patients of chronic hepatitis C were higher than those in the healthy controls (P<0.05) and in asymptomatic HCV carriers (P<0.05). The values of the mentioned cytokines showed a significant positive correlation to GPT. The levels of the mentioned cytokines decreased obviously after IFN treatment (P<0.05), while the serum levels of IL-10 and sIL-2R reduced in sequence in no-response group, partial-response group and complete-response group., Conclusions: IL-18, IL-10, TNF-alpha and sIL-2R co-participate in the pathogenesis of chronic hepatitis C, and are used to evaluate the effect of IFN on the immune state of organisms, and IL-10 and sIL-2R are important for predicting the anti-viral efficacy of IFN.

Keratinocytes from mouse skin were cultured for a short period in vitro following single or multiple treatments at low dose levels in vivo with the known chromosome-damaging agent triethylenemelamine (TEM). The chemical was applied to the skin of HRA/Skh hairless mice at concentrations corresponding to those reported to initiate cancer in initiation-promotion assays. A significant dose-related depression in keratinocyte cell recovery occurred over the dose range 0.3-1 mg TEM/mouse (single or multiple treatments). Under the same conditions, a dose-related induction of micronuclei was observed using the cytokinesis-block method with cytochalasin B. A similar frequency of micronuclei was detected in binucleate cells from mice treated with single or multiple applications of TEM. Mice held for 12-48 h post-treatment, before removal of skin for in vitro culture, yielded highest micronuclei frequencies. These results indicate that the same target cell population, skin keratinocytes, can be used to investigate both genotoxicity and carcinogenesis, and that micronucleus induction in these cells may be a sensitive signal of skin cancer initiation.