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Therapeutic antibodies (Abs) improve the clinical outcome of cancer patients. However, on-target off-tumor toxicity limits Ab-based therapeutics. Cluster of differentiation 147 (CD147) is a tumor-associated membrane antigen overexpressed in cancer cells. Ab-based drugs targeting CD147 have achieved inadequate clinical benefits for liver cancer due to side effects. Here, by using glycoengineering and hypoxia-activation strategies, we developed a conditional Ab-dependent cellular cytotoxicity (ADCC)-enhanced humanized anti-CD147 Ab, HcHAb18-azo-PEG 5000 (HAP18). Afucosylated ADCC-enhanced HcHAb18 Ab was produced by a fed-batch cell culture system. Azobenzene (Azo)-linked PEG 5000 conjugation endowed HAP18 Ab with features of hypoxia-responsive delivery and selective targeting. HAP18 Ab potently inhibits the migration, invasion, and matrix metalloproteinase secretion, triggers the cytotoxicity and apoptosis of cancer cells, and induces ADCC, complement-dependent cytotoxicity, and Ab-dependent cellular phagocytosis under hypoxia. In xenograft mouse models, HAP18 Ab selectively targets hypoxic liver cancer tissues but not normal organs or tissues, and has potent tumor-inhibiting effects. HAP18 Ab caused negligible side effects and exhibited superior pharmacokinetics compared to those of parent HcHAb18 Ab. The hypoxia-activated ADCC-enhanced humanized HAP18 Ab safely confers therapeutic efficacy against liver cancer with improved selectivity. This study highlights that hypoxia activation is a promising strategy for improving the tumor targeting potential of anti-CD147 Ab drugs., Competing Interests: The authors declare they have no conflicts of interest., (© 2024 The Authors. MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.)

Background: Hypoxia induces hepatocellular carcinoma (HCC) malignancies; yet it also offers treatment opportunities, exemplified by developing hypoxia-activated prodrugs (HAPs). Although HAP TH-302 combined with therapeutic antibody (Ab) has synergistic effects, the clinical benefits are limited by the on-target off-tumor toxicity of Ab. Here, we sought to develop a hypoxia-activated anti-M2 splice isoform of pyruvate kinase (PKM2) Ab combined with TH-302 for potentiated targeting therapy. Methods: Codon-optimized and hypoxia-activation strategies were used to develop H103 Ab-azo-PEG5k (HAP103) Ab. Hypoxia-activated HAP103 Ab was characterized, and hypoxia-dependent antitumor and immune activities were evaluated. Selective imaging and targeting therapy with HAP103 Ab were assessed in HCC-xenografted mouse models. Targeting selectivity, systemic toxicity, and synergistic therapeutic efficacy of HAP103 Ab with TH-302 were evaluated. Results: Human full-length H103 Ab was produced in a large-scale bioreactor. Azobenzene (azo)-linked PEG5k conjugation endowed HAP103 Ab with hypoxia-activated targeting features. Conditional HAP103 Ab effectively inhibited HCC cell growth, enhanced apoptosis, and induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions. Analysis of HCC-xenografted mouse models showed that HAP103 Ab selectively targeted hypoxic HCC tissues and induced potent tumor-inhibitory activity either alone or in combination with TH-302. Besides the synergistic effects, HAP103 Ab had negligible side effects when compared to parent H103 Ab. Conclusion: The hypoxia-activated anti-PKM2 Ab safely confers a strong inhibitory effect on HCC with improved selectivity. This provides a promising strategy to overcome the on-target off-tumor toxicity of Ab therapeutics; and highlights an advanced approach to precisely kill HCC in combination with HAP TH-302., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Atractylodes chinensis is one of the most commonly used bulk herbs in East Asia; however, root rot can seriously affect its quality and yields. In contrast to chemical pesticides, biological control strategies are environmentally compatible and safe. For this study, 68 antagonistic bacterial strains were isolated from the rhizospheres of healthy Atractylodes chinensis. Strain SY42 exhibited the most potent fungicidal activities, with inhibition rates against F. oxysporum, F. solani, and F. redolens of 67.07 %, 63.40 % and 68.45 %, respectively. Through morphological observation and molecular characterization, strain SY42 was identified as Paenibacillus polymyxa. The volatile organic components (VOCs) produced by SY42 effectively inhibited the mycelial growth of pathogenic fungi through diffusion. SY42 significantly inhibited the germination of pathogenic fungal spores. Following co-culturing with SY42, the mycelium of the pathogenic fungus was deformed, folded, and even ruptured. SY42 could produce cellulases and proteases to degrade fungal cell walls. Pot experiments demonstrated the excellent biocontrol efficacy of SY42. This study revealed that P. polymyxa SY42 inhibited pathogenic fungi through multiple mechanisms, which verified its utility as a biocontrol agent for the control of A. chinensis root rot., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Neurogenic limb deformity disorder (NLDD) refers to limb deformity disorders caused by various neurogenic disorders. However, there are no studies to systematically summarize and analyze these diseases in China, and we first proposed the concept of NLDD. We describe the epidemiological characteristics of NLDD in China based on the largest case database of limb orthopedics in China., Methods: This study analyzed parameters from the Qin Sihe Orthopedic Surgery Case Data (QSHOSCD). The database is based on the Rehabilitation Hospital affiliated to National Research Center for Rehabilitation, which has collected nearly 37,000 patients to date and includes a wide variety of limb deformities. The types of diseases are summarized and classified for all patients studied. Statistical analysis was based on the type of etiology, age, regional distribution, and historical surgical volume. Partial outcomes were statistically analyzed separately by common diseases (polio and cerebral palsy) and rare diseases (37 other diseases)., Results: From 1979 to 2019, 30,194 patients with NLDD were treated surgically for 39 neurogenic disorders. The male to female ratio was 1.48:1, the mean age was 19.65 years, and most patients (82.38%) were aged between 6 and 30 years. Patients included from 32 provinces and cities across China, mainly concentrated in populous central provinces and Heilongjiang Province. The peak of annual surgical procedures was from 1988 to 1994, and the number of annual surgical procedures for common diseases gradually decreased from 1994 onwards, but the trending is opposite for rare diseases., Conclusions: This study is the first to demonstrate the disease types, population characteristics and incidence trends of NLDD in China. It suggests that the prevention and treatment of NLDD should focus on the adolescent population and enhance the treatment of neurogenic diseases that cause limb deformities. The growth and adaption of the Ilizarov technique and its practice in Chinese orthopedic benefits the treatment of neurogenic limb deformity disorders., (© 2023. The Author(s).)

Purpose: To summarize the evolution of Ilizarov technology in China, highlight important milestones, introduce the atmosphere of the era concerning the first uses and development of this technology, and share Chinese modification and experience in this field., Method: A thorough interview with senior ASAMI members of China and literature search and physical books in libraries was undertaken to summarize the history of Ilizarov technology in China., Results: The formal development of Ilizarov technology began when professor Ilizarov himself came to Beijing (1991) and gave a speech. In the following 31 years, his technology was rapidly developed through China, with many symposiums held and associations established including ASAMI China (2003) and ILLRS China (2015). Today, Ilizarov technology has become the main treatment of complex fractures, defects, nonunion, infections, deformities, and chronic ischemic ulcers of the limbs. In those years, Chinese scholars also developed some special treatment methods and made many modifications to Ilizarov external fixators., Conclusion: Ilizarov technology has developed in China for 31 years. It revolutionized the treatment of complex limb traumas, deformities, and diseases. In the treatment of millions of patients, Chinese scholars had many unique experiences and made modifications to this technology which is worthy to share with the world., (© 2021. The Author(s).)

Background: Few mortality-scoring models are available for solid tumor patients who are predisposed to develop Escherichia coli-caused bloodstream infection (ECBSI). We aimed to develop a mortality-scoring model by using information from blood culture time to positivity (TTP) and other clinical variables., Methods: A cohort of solid tumor patients who were admitted to hospital with ECBSI and received empirical antimicrobial therapy was enrolled. Survivors and non-survivors were compared to identify the risk factors of in-hospital mortality. Univariable and multivariable regression analyses were adopted to identify the mortality-associated predictors. Risk scores were assigned by weighting the regression coefficients with corresponding natural logarithm of the odds ratio for each predictor., Results: Solid tumor patients with ECBSI were distributed in the development and validation groups, respectively. Six mortality-associated predictors were identified and included in the scoring model: acute respiratory distress (ARDS), TTP ≤ 8 h, inappropriate antibiotic therapy, blood transfusion, fever ≥ 39 °C, and metastasis. Prognostic scores were categorized into three groups that predicted mortality: low risk (< 10% mortality, 0-1 points), medium risk (10-20% mortality, 2 points), and high risk (> 20% mortality, ≥ 3 points). The TTP-incorporated scoring model showed excellent discrimination and calibration for both groups, with AUC being 0.833 vs 0.844, respectively, and no significant difference in the Hosmer-Lemeshow test (6.709, P = 0.48) and the chi-square test (6.993, P = 0.46). Youden index showed the best cutoff value of ≥ 3 with 76.11% sensitivity and 79.29% specificity. TTP-incorporated scoring model had higher AUC than no TTP-incorporated model (0.837 vs 0.817, P < 0.01)., Conclusions: Our TTP-incorporated scoring model was associated with improving capability in predicting ECBSI-related mortality. It can be a practical tool for clinicians to identify and manage bacteremic solid tumor patients with high risk of mortality., (© 2021. The Author(s).)

The first base-mediated intermolecular cyclization of arylaldehydes and terminal arylacetylenes for the synthesis of a wide range of pyrroles in a single step has been described. The developed methodology used commercially available starting materials and tolerated a broad range of functional groups affording 2,3,5-triaryl-substituted-1 H -pyrroles with good yields (up to 92% yield) under mild conditions. The possible mechanism was also discussed.

Objective: To analyze effect of accordion technique on bone mineralization of extended bone segment in treating tibial bone defect with bone transport., Methods: From May 2017 to October 2019, 22 patients with tibial bone defects were treated with Ilizarov bone-transport technique, and divided into two groups after bone-transport was completed, 11 patients in each group. In observation group, there were 9 males and 2 females aged from 20 to 60 years old with an average of (42.6± 13.3) years old;the length of bone defect ranged from 3 to 13 cm with an average of(6.4±2.6) cm;2 patients were suffered from upper tibial bone defects, 3 patients were middle and 6 patients were lower;patients were treated with accordion technique for 35 days. In control group, there were 10 males and 1 female aged from 41 to 60 years old with an average of (51.6±6.4) years old;the length of bone defect ranged from 3 to 10.7 cm with an average of (6.6±2.5) cm;1 patient was suffered from upper tibial bone defects, 3 patients were middle and 7 patients were lower;patients were treated with lock external fixator to waiting bone mineralization. The content of hydroxyapatite (HAP) extended bone segment was measured after bone-transport completed immediately, 35, 65 and 95 days after bone-transport was completed, respectively, then the mineralization time and healing time were compared between two groups, and the therapeutic effect of bone defect was evaluated by using Paley scoring criteria., Results: Twenty-two patients were followed up from 18 to 36 months with an average of (27.0±6.3) months. The wounds on the bone defects healed spontaneously during bone transport, and there were no wound complications such as skin infection or skin necrosis occurred. There were statisticaldifference in the content of HAP of the extended bone segments at 35, 65 and 95 days after bone-transport between two groups ( P <0.05). There were difference in mineralization time and healing time between two groups ( P <0.05). According to Paley standard evaluation, 10 patients got excellent results, and 1 good in observation group; while 9 patients got excellent results, 2 good in control group;and there was no differences between two groups ( Z =-0.607, P = 0.544)., Conclusion: Accordion technique and locking external fixator mineralization in prolonging bone segment healing after bone-transport have the equal clinical effect, while the accordion technique could significantly accelerate the growth rate of HAP and shorten the mineralization time and healing time.

Treating serious bone trauma with an osteo-inductive agent such as bone morphogenetic proteins (BMPs) has been considered as an optimized option when delivered via a collagen sponge (CS). Previous works have shown that the BMP concentration and release rate from approved CS carriers is difficult to control with precision. Here we presented the fabrication of a recombinant fusion protein from recombinant human-like collagen (HLC) and human BMP-2 (hBMP2). The fusion protein preserved the characteristic of HLC allowing the recombinant protein to be expressed in Yeast (such as Pichia pastoris GS115) and purified rapidly and easily with mass production after methanol induction. It also kept the stable properties of HLC and hBMP2 in the body fluid environment with good biocompatibility and no cytotoxicity. Moreover, the recombinant fusion protein fabricated a vertical through-hole structure with improved mechanical properties, and thus facilitated migration of bone marrow mesenchymal stem cells (MSCs) into the fusion materials. Furthermore, the fusion protein degraded and released hBMP-2 in vivo allowing osteoinductive activity and the enhancement of utilization rate and the precise control of the hBMP2 release. This fusion protein when applied to cranial defects in rats was osteoinductively active and improved bone repairing enhancing the repairing rate 3.5- fold and 4.2- fold when compared to the HLC alone and the control, respectively. There were no visible inflammatory reactions, infections or extrusions around the implantation sites observed. Our data strongly suggests that this novel recombinant fusion protein could be more beneficial in the treatment of bone defects than the simple superposition of the hBMP2/collagen sponge., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The basic fibroblast growth factor (bFGF) plays an important role in the wound repair process. However, lacking of better biomaterials to carry bFGF still is a challenge in skin repair and regeneration. In this study, the human-like collagen (HLC) cross-linked with transglutaminase (TG) to fabricate a HLC/TG hydrogel to load bFGF. The physical properties of hydrogel, such as interior structure, mechanical property, were characterized in vitro using scanning electron microscopy (SEM), rheometer. Then, the effects of the HLC/TG hydrogel on the bFGF and cell attachmentwere evaluated, and the results showed that the HLC/TG hydrogel has good biocompatibility towards bFGF and cells. Finally, skin wound healing test was performed for the evaluation of HLC/TG hydrogels with bFGF in a mouse model. All results of macroscopic and microscopic analysis indicated that not only our HLC/TG hydrogel provide a delivery of growth factors, but also the HLC/TG hydrogel with bFGF achieving better skin regeneration in the structure and function.

Objective: To explore the clinical effect of acupoint puncture combined with Ilizarov technique in the treatment of knee osteoarthritis in the elderly., Methods: From March 2015 to February 2016, 76 patients with primary knee osteoarthritis were treated with tibial osteotomy acupoint puncture grouop and Ilizarov technique anatomical puncture group, including 24 males and 52 females, aged 56 to 75 years old with an average of 61.4 years old, and a course of 3 to 17 years with an average of 5.2 years. Among them, 38 cases were treated with external fixation of acupoint puncture needle and 38 cases were treated with external fixation of anatomical puncture needle. Preoperative full-length X-ray of both lower limbs showed tibial varus deformity, narrowing of medial knee joint space and enlargement of lateral knee joint space. The force line of the affected knee and lower limb was moved inward by body surface measurement, and the KSS knee function score was decreased. Symptoms included medial knee pain, flexion and extension, and conservative treatment for more than 2 years., Results: The lower limb force lines of both groups were corrected and the osteotomy ends healed well. No nonunion of osteotomy, inadequate correction of lower limbs or recurrence of deformity were found. Seventy-five patients were followed up for 3, 6, 12 and 24 months after operation. There was no significant difference in knee joint mobility between the two groups before operation and on 6, 12, 24 months after operation( F =1.346, P >0.05). There were significant difference in KSS pain and total score between the two groups at 3 months after operation, acupoint puncture group was better than anatomical puncture group( P <0.05); there was no significant difference in KSS score at 12 months after operation( P >0.05)., Conclusions: The acupoint puncture group formed a potential acupuncture effect in the acupoint area by continuously tightening the steel needle on Ilizarov ring external fixator during the post-operative adjustment. Within three months after wearing external fixator, the knee pain symptoms of knee osteoarthritis were relieved rapidly, continuously and effectively, which was significantly better than that of the anatomical puncture group., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© 2019 by the China Journal of Orthopaedics and Traumatology Press.)

Background: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) is one of the main antimicrobial-resistant pathogens. Little data are available on how biofilm formation (BF) contributes to EC-caused bloodstream infection (BSI) in cancer patients. This study investigated the impact of BF on clinical outcomes of cancer patients with EC-caused BSI., Methods: Clinical outcome and microbiological characteristics including the presence of bla genes in ESBL-EC isolates were retrospectively collected from BSI cancer patients. Patients infected with ESBL-EC were compared with patients infected with third-generation cephalosporin-susceptible strains. Survival curves were generated by Kaplan-Meier analysis and the survival difference was assessed by the log-rank test. Risk factors for ESBL-EC infection, predictors of mortality, and outcome differences were determined by multivariate logistic regression and Cox regression analysis, respectively., Results: A high prevalence of ESBL-EC with dominant bla CTX-M-15 , bla CTX-M-15 plus bla TEM-52 genotype was found in BSI cancer patients. Independent risk factors for infection with ESBL-EC were cephalosporins, chemotherapy, and BF. Metastasis, ICU admission, BF-positive ESBL-EC, organ failure, and the presence of septic shock were revealed as predictors for mortality. The ESBL characteristic was associated with the BF phenotype, and the overall mortality was significantly higher in cancer patients with BF-positive ESBL-EC-caused BSI., Conclusion: bla CTX-M-15 type ESBL-EC is highly endemic among cancer patients with BSI. BF is associated with multi-drug resistance by ESBL-EC and is also an independent risk factor of mortality for cancer patients with BSI. Our findings suggest that the combination of BF-positive ESBL-EC isolates with other appropriate laboratory indicators might benefit infection control and improve clinical outcomes., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Objective: To study the taxonomy and distribution of Chinese medicinal centipedes., Methods: The species of Chinese medicinal centipedes were investigated in the light of their morphology. According to the feature of life, the distribution of centipedes were explored., Results: There were 12 centipede species in China, and seven of them were used for medical, the species could be effectively distinguished by the identification key. It was suggested that their characteristics were related to the climatic factors such as temperature, humidity, altitude and air pressure., Conclusion: The distribution of medicinal centipede is characteristics of "three river system distribution belts" and "three geographical distribution areas". The results provide the basis for the development and application of medicinal centipedes in China.

Background: The dosage of herb ultrafine particle (UFP) depended on the increased level of its dissolution, toxicity, and efficacy., Objective: The dissolution, antibacterial activity, and cytotoxicity of Coptidis rhizoma (CR) UFP were compared with those of traditional decoction (TD)., Materials and Methods: The dissolution of berberine (BBR) of CR TD and UFP was determined by high-performance liquid chromatography. The antibacterial activity of CR extract was assayed by plate-hole diffusion and broth dilution method; the inhibitory effect of rat serums against bacteria growth was evaluated after orally given CR UFP or TD extract. The cytotoxicity of CR extract was evaluated by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide assay., Results: The dissolution amount of BBR from CR UFP increased 6-8-folds in comparison to TD at 2 min, the accumulative amount of BBR in both UFP and TD group increased in a time-dependent manner. The minimal inhibitory concentrations and minimal bactericidal concentrations of CR UFP extract decreased to 1/2~1/4 of those of TD extract. The inhibitory effect of rat serums against bacteria growth decreased time-dependently, and no statistical difference was observed between two groups at each time point. The 50% cytotoxic concentrations of UFP extract increased 1.66~1.97 fold than those of TD., Conclusions: The antibacterial activity and cytotoxicity of CR UFP increased in a dissolution-effect manner in vitro, the increased level of cytotoxicity was lower than that of antibacterial activity, and the inhibitory effect of rat serums containing drugs of UFP group did not improve., Summary: Ultrafine grinding process caused a rapid increase of BBR dissolution from CR.The antibacterial activity and cytotoxicity of UFP extract in vitro increased in a dissolution-effect manner, but the cytotoxicity increased lower than the antibacterial activity.The antibacterial activity of rat serums of UFP group did not improve in comparison to that of TD group.

Numerous randomized controlled trials (RCTs) of acupuncture have been conducted in recent years. The results of several studies implied that acupuncture was only a powerful placebo; however, certain studies demonstrated that verum acupuncture had a greater effect than placebo and the mechanisms between a verum acupuncture group and a placebo/sham group were different. Researchers attempted to investigate the inherent factors that may potentially influence the results of trials. Certain problems observed in acupuncture RCTs also occurred in RCTs in other fields, including insufficient sample size, high dropout rates, inadequate follow-up and randomization. The study of acupuncture is so complex that specific methodological challenges are raised, which are frequently overlooked, including sham interventions, blinding, powerful placebo effects (even stronger than an inert pill) and variations in acupuncture administration. The aforementioned problems may contribute to bias, and researchers systematically attempt to solve these problems. The present review aimed to suggest techniques to design high-quality studies, minimize the placebo effect and optimize acupuncture administration in acupuncture studies. If these problems are addressed, then the results of acupuncture studies may be different.

Background: Botrytis cinerea, a haploid Euascomycete fungus that infects numerous crops, has been used as a model system for studying molecular phytopathology. Botrytis cinerea adopts various modes of infection, which are mediated by a number of pathogenicity and virulence-related genes. Many of these genes have not been reported previously., Objectives: This study aimed to investigate development and pathogenicity-related genes between a novel nonpathogenic mutant and the Wild Type (WT) in B. cinerea., Materials and Methods: Digital Gene Expression (DGE) tag profiling can reveal novel genes that may be involved in development and pathogenicity of plant pathogen. A large volume of B. cinerea tag-seq was generated to identify differential expressed genes by the Illumina DGE tag profiling technology., Results: A total of 4,182,944 and 4,182,021 clean tags were obtained from the WT and a nonpathogenic mutant stain (BCt89), respectively, and 10,410 differentially expressed genes were identified. In addition, 84 genes were expressed in the WT only while 34 genes were expressed in the mutant only. A total of 664 differentially expressed genes were involved in 91 Kyoto Encyclopedia of Genes and Genome pathways, including signaling and metabolic pathways., Conclusions: Expression levels of 1,426 genes were significantly up-regulated in the mutant compared to WT. Furthermore, 301 genes were down-regulated with False Discovery Rates (FDR) of < 0.001 and absolute value of log2 Ratio of ≥ 1.

Objective: To explore the surgical method and clinical curative effect of Ilizarov technique combined with limited surgical treatment for neurotrophic malnutrition equinovarus accompanied with weight-bearing area ulcer., Methods: From July 2004 to December 2011, 21 cases of neurological disorders equinovarus with skin ulcer in weight-bearing area were treated including 14 males and 7 females with an average age of 21.3 years old ranging from 8 to 32 years. Among them,19 cases with talipes equinovarus were on account of spina bifid and 2 cases on account of spinal cord injury of lumbar sacral segment. Nine cases were on the left foot and 12 cases were right foot. The ulcer area in out-below side of the fifth metatarsal bone was in 6 cases, in basement of the fifth metatarsal bone in 5 cases,in lateral of calcaneocuboid joint 2 cases, and in lateral of subtalar joint in 8 cases. Surgical procedure included plantar medial release technique, tendo-chilles lengthenning, and three-joint partial osteotomy. During operation,the skin and soft tissue of ulcer were resected and the incision was sutured when the ulcer was in the lateral of subtalar joint, and ankle Ilizarov external fixation for extension was installed after correcting talipes equinovarus partially. For the legacy skin ulcer in weight-bearing area,the Shenrg-ji cream (Chinese characters) was used after operation for one time per day until the wound healed. Adjusting Ilizarov external fixation for correcting residual deformity until it is satisfaction. During this process weight-bearing walking was keeping., Results: All cases were followed up from 6 to 52 months with an average of 28 months. All ulcers were healing and no recurrence. The ulcer healing time was 14 to 36 days postoperative (26 days in average). Eighteen deformity feet were corrected satisfactorily, and recovered fully plantar foot, 3 feet appeared mild deformity after 1 year. Ankle joint activity appeared limited in 15 cases and got completely rigidity in 6 cases. The AOFAS increased from preoperative 34.0±7.2 to 86.0±8.5 postoperatively; 8 feet got excellent, 10 good,3 fair., Conclusion: The clinical effect of Ilizarov technique combined with limited operation and the Sheng-ji cream in correction of the talipes equinovarus with skin ulcer in weigh-bearing area on account of neurotrophic malnutrition is satisfactory, the surgical method is simple and the treating course in security, and serious complications can be avoided.

At present, treatment for chronic radial head dislocation mainly focus on open reduction, and conservative treatment is invalid. While the surgical procedure generally applies to extend ulnar, short radial, reset radial head. Repair or reconstruction of annular ligament is the focus of study. For the dysfunction, radial head resection served as the final choice. Early diagnosis is the key to the treatment of chronic radial head dislocation. While ulnar or radial osteotomy and non-reconstruction of annular ligament is still the main trend of today.

Wolff law was an adaptable principle of bone, Tension-Stress Principle was equal to Distraction Osteogenesis or Distraction Tissue Regeneration, The Natural Reconstruction theory was a new orthopedic perspective proposed by Prof. QIN after deformity correction using Ilizarov technology. The thought about their relationship originated from a social phenomena, that the crowds and the confusion about export choice in Beijing's subway. Ilizarov technology and Wolff law were one concept related to Mechanics, and the former is completely in line with the latter. In other words, Ilizarov technology is an extension of Wolff law, is a repeated process of micro-trauma and continuous repair of bone trabecular initiated by moden engineering, just trabecular formed along the tension-stress direction. With adjustment of mechanical force,doctor can control the process of fracture healing and bone remolding to a certain extent. Natural Reconstruction theory enlarged the defined range of Wolff law obviously. Not only guided orthopedics clinical and basic research,but also related to the dialectical thinking of the doctor-patient relationship in sociology. There was an inevitable connection among Wolff law, Ilizarov technology and Natural Reconstruction theory. The history of discovery and understanding was a continuous process of thinking,practice and integration.

Objective: To study surgical strategies for ankle and foot deformities secondary to spina bifida and treatment methods for different types of deformities., Methods: From January 1990 and July 2009, 107 patients with ankle and foot deformities secondary to spina bifida were retrospectively analyzed. There were 44 males and 63 females, with an average age of 17.7 years (rangd from 1.3 to 52 years). Among the patients, 58 patients had double ankle deformities, 49 patients had unilateral deformities (22 cases on the left side, 27 cases on the right). Ninety-nine patients with equinus deformities were treated by achilles tendon lengthening and tendon transfering; 25 patients with talipes were treated by release of anterior tendon of ankle and tendon transfer; 17 patients with valgus and varus deformities were treated by tendon transfer and calcaneal osteotomy; 15 patients with flail deformities were managed treated by bone fusion between calcaneus and talus and shortening of achilles tendon; 9 patients with claw toe deformities were treated by bone fusion of interphalangeal joint or Ilizarovs distraction. AOFAS (American Orthopaedic Foot & Ankle Society) comprehensive scoring system was used to evaluate subjective pain and objective functional., Results: Seventy-nine (127 feet) of 107 patients were followed up, and the duration ranged from 48 to 180 months (averaged, 64 months). According to AOFAS scoring system, the results were rated as excellent in 89 feet, good in 26 feet, moderate in 9 feet and poor in 3 feet., Conclusion: Treatment strategies for ankle-foot deformities mainly included four principles, deformity correction, muscular balance, joint stability and reservation of foot elasticity. Different combined procedure was applied for different foot deformities and received good therapeutic effects.

Unlabelled: Tumor cells can move as individual cells in two interconvertible modes: mesenchymal mode and amoeboid mode. Cytoskeleton rearrangement plays an important role in the interconversion. Previously, we reported that HAb18G/CD147 and annexin II are interacting proteins involved in cytoskeleton rearrangement, yet the role of their interaction is unclear. In this study we found that the depletion of HAb18G/CD147 produced a rounded morphology, which is associated with amoeboid movement, whereas the depletion of annexin II resulted in an elongated morphology, which is associated with mesenchymal movement. The extracellular portion of HAb18G/CD147 can interact with a phosphorylation-inactive mutant of annexin II and inhibit its phosphorylation. HAb18G/CD147 inhibits Rho signaling pathways and amoeboid movement by inhibiting annexin II phosphorylation, promotes membrane localization of WAVE2 and Rac1 activation by way of the integrin-FAK-PI3K/PIP3 signaling pathway, and promotes the formation of lamellipodia and mesenchymal movement., Conclusion: These results suggest that the interaction of HAb18G/CD147 with annexin II is involved in the interconversion between mesenchymal and amoeboid movement of hepatocellular carcinoma cells., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Purpose: To evaluate the performances for detection of IgM and IgG antibodies to Orientia. tsutsugamushi (Ot) using a gold conjugate-based rapid diagnostic test (RDT)., Materials and Methods: The RDT employing mixture recombinant 56-kDa proteins of O. tsutsugamushi and the mIFA assay was performed on 33 patients from Fujian and Yunnan province respectively and 94 positive sera (36 from Hainan province and 58 from Jiangsu province) from convalescent stages of the patients with scrub typhus respectively and 82 negative sera from healthy farmers from Anhui province and Beijing City respectively in 2009. A comparison of the RDT and mIFA assay was performed by using the χ(2) test and the P level of ≤ 0.05 was considered to be significant., Results: Among these 94 positive sera from convalescent stages of the illness and 82 sera from control farmers, the specificity of RDT was 100% for both IgM and IgG tests. In 33 cases with scrub typhus, 5 cases were positively detected earlier by RDT than by mIFA for the IgM test, and 2 cases were positive for the IgG test. The sensitivities of RDT were 93.9% and 90.9% for IgM and IgG, respectively. Considering IgM and IgG together, the sensitivity was 100%. The geometric mean titre (GMT) of IFA and the RDT assay in diluted sera from confirmed cases were 1:37 versus 1:113 respectively (P<0.001) for IgM test and 1:99 versus 1:279 respectively (P<0.016) for IgG., Conclusions: The RDT was more sensitive than the traditional IFA for the early diagnosis of scrub typhus and was particularly suitable for use in rural areas.

Objective: To study therapeutic effects of limited internal fixation combined with external fixation in the treatment of deformity of distal end of femur, as well as to explore its effect on the function of knee joint., Methods: From June 2004 to June 2009, 38 patients with deformity of distal end of femur were treated with osteotomy at femoral supracondylar and internalfixation combined with hybrid external fixator. Among the patients, 19 patients were male and 19 patients were female, ranging in age from 14 to 58 years, with an average of 26 years. Six patients had genu valgum, 4 patients had varus of the distal end of femur, 2 patients had varus combined with rotation deformity,3 patients had genu recurvation and 23 patients had deformity of genuflex. The function of knee joint was evaluated according to Dimeglio clinical assessment after the treatment., Results: All the patients were followed up and the during ranged from 6 to 48 months, with a mean period of 23 months. All the deformity obtained satisfactory correction, without complications such as limitation of motion of the knee joint, under correction or over correction,displacement of the section of the osteotomy,internal fixation loosening and fracture. Nineteen patients got an excellent result, 12 good, 5 poor and 2 bad., Conclusion: Supracondylar femoral osteotomy and internal fixation combined with hybrid external fixator is a safe and effective surgery for the treatment of deformity of distal end of femur. This method combines the advantages of internal fixation and external fixation.

CD147 is a transmembrane glycoprotein overexpressed in human hepatocellular carcinoma (HCC) which could promote HCC progression and metastasis. Promoter methylation is one of the most important processes in gene regulation. In this study, we aim to investigate CD147 promoter methylation status and the correlation with clinicopathological features and prognosis in HCC. CD147 promoter methylation statuses and expression levels in normal and HCC cell lines and 54 paired HCC and adjacent non-tumour (ANT) tissues were, respectively, examined by bisulphite genomic sequencing, methylation-specific PCR, real-time RT-PCR, Western blot and immunohistochemistry. The correlations of promoter methylation statuses with CD147 expression level and the clinicopathological features were statistically analysed in HCC patients. Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines and tissues control. In vivo and in vitro analysis indicated that demethylation with 5-Aza-2'-deoxycytidine led to increased CD147 expression through enhancing Sp1 binding affinity, and methylation with methyltransferase reduced CD147 transcriptional activity through interfering Sp1 binding. CD147 promoter methylation level in HCC tissues (22.22%) was lower than that in ANT tissues (46.30%; P < 0.05). Within HCC tissues, a significant inverse correlation was observed between CD147 expression and methylation level (r=-0.615). Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1%versus 58.3%; P < 0.05) and death rate (83.3%versus 50.0%; P < 0.05) than patients with methylated CD147 promoter. In conclusions, promoter hypomethylation up-regulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Cell-penetrating peptides (CPPs) are widely used to deliver macromolecular cargoes to intracellular sites of action. Many CPPs have been demonstrated to rely on cell surface heparan sulfate proteoglycans (HSPGs) for efficient cellular entry and delivery. In this chapter, we describe methods for the study of PG involvement in CPP uptake. We provide descriptions of how to determine whether uptake of a CPP of interest is dependent on PGs. We also provide detailed protocols for the purification of PGs by anion-exchange chromatography as well as the characterization of the HSPG core protein composition of a cell line of interest. Finally, we present methods for modulating the expression level of specific HSPG core proteins as a means to determine the core protein specificity in the uptake of a particular CPP.

An increased understanding of cellular uptake mechanisms of macromolecules remains an important challenge in cell biology with implications for viral infection and macromolecular drug delivery. Here, we report a strategy based on antibody-conjugated magnetic nanoparticles for the isolation of endocytic vesicles induced by heparan sulfate proteoglycans (HSPGs), key cell-surface receptors of macromolecular delivery. We provide evidence for a role of the glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (hereafter termed "GRP75") in HSPG-mediated endocytosis of macromolecules. GRP75 was found to be a functional constituent of intracellular vesicles of a nonclathrin-, noncaveolin-dependent pathway that was sensitive to membrane cholesterol depletion and that showed colocalization with the membrane raft marker cholera toxin subunit B. We further demonstrate a functional role of the RhoA GTPase family member CDC42 in this transport pathway; however, the small GTPase dynamin appeared not to be involved. Interestingly, we provide evidence of a functional role of GRP75 using RNAi-mediated down-regulation of GRP75 and GRP75-blocking antibodies, both of which inhibited macromolecular endocytosis. We conclude that GRP75, a chaperone protein classically found in the endoplasmic reticulum and mitochondria, is a functional constituent of noncaveolar, membrane raft-associated endocytic vesicles. Our data provide proof of principle of a strategy that should be generally applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells.

Objective: To analyze the incidence, clinical features, deformity categories and orthopedic treatment of foot and ankle deformities caused by spinal bifida., Methods: The charts of the patients received surgical treatment between January 1990 and July 2009 were studied retrospectively, and the data were analyzed., Results: One hundred and seven cases of foot and ankle deformities caused by spinal bifida received surgical treatment and were included. There were 44 male and 63 female patients. The average age was 17.7 years (range, 1.3 - 52.0 years). And 50.5% (54/107) of cases were over 18 years old and had spinal bifida occulta, and the other 49.5% had spinal bifida manifesta. There was only one case of thoracic spinal bifida (T(3-8)), while the other 106 cases had lumbosacral vertebrae cleft (mainly L(3) to Sacrum). Among a total of 165 feet, unilateral involvement was found in 49 cases (22 cases on the left side, 27 cases on the right side), bilateral involvement in 58 cases. Combined ankle-foot deformities included 76 varus talipes, 23 talipes valgus, 15 flail feet, and 51 other foot deformities. Other site deformities, as a result of spinal bifida, included knee flexion or hyperextension deformity in 4 cases, hip deformity (hip adduction, flexion, or hip dislocation, pelvic tilt, lower extremity discrepancy, etc.) in 17 cases, and urinatory dysfunction and defecation in 30 cases. Twenty-nine of 54 cases with spinal bifida occulta failed to be diagnosed in other hospitals and the misdiagnosed rate reached 53.7% (29/54). Corrective surgery was performed in only 26 patients. And 50.5% (54/107) of patients (over 18 years old) had severe foot and ankle deformities due to a failure of prior surgical treatment., Conclusions: Spinal bifida is the most commonly found in the lumbosacral vertebrae. Although the main pathogenesis is developmental abnormalities of spinal cord and nerve, the secondary deformity is usually located on the foot and ankle. Some young orthopedic surgeons may not have enough awareness and treatment experience about this disease due to over-specialty of the orthopaedics, so the delay of early diagnosis and treatment is often found and many severe foot and ankle deformities occur.

HAb18G/CD147, a member of the immunoglobulin family enriched on the surface of tumor cells, is reported to be correlated with invasion, metastasis, growth, and survival of malignant cells. Here, we found that annexin II, a 36-kDa Ca(2+)- and phospholipid-binding protein and in vivo substrate for tyrosine kinase and PKC, is a new interaction protein of HAb18G/CD147 in human hepatocellular carcinoma (HCC) cells. In the present study, we explored the unclear role of annxin II in HCC invasion and migration and the interaction effects between HAb18G/CD147 and annexin II. Our data show that downregulation of annexin II in HCC cells significantly decreased the secretion of MMP, migration ability, and invasive potential, and affected the cytoskeleton rearrangement of tumor cells. The MMP-2 level and invasive potential of HCC cells were regulated by both annexin II and HAb18G/CD147. Also, interaction effects exist between the two molecules in tumor progression, including MMP-2 production, migration, and invasion. These results suggest that annexin II promotes the invasion and migration of HCC cells in vitro, and annexin II and HAb18G/CD147 interact with each other in the same signal transduction pathway working as a functional complex in tumor progression.

Cellular uptake of several viruses and polybasic macromolecules requires the expression of cell-surface heparan sulfate proteoglycan (HSPG) through as yet ill defined mechanisms. We unexpectedly found that among several cell-surface-binding single chain variable fragment (scFv) anti-HS antibody (alphaHS) clones, only one, AO4B08, efficiently translocated macromolecular cargo to intracellular vesicles through induction of HSPG endocytosis. Interestingly, AO4B08-induced PG internalization was strictly dependent on HS 2-O-sulfation and appeared independent of intact N-sulfation. AO4B08 and human immunodeficiency virus (HIV)-Tat, i.e. a well known cell-penetrating peptide, were shown to compete for the internalizing PG population. To obtain a more detailed characterization of this pathway, we have developed a procedure for the isolation of endocytic vesicles by conjugating AO4B08 with superparamagnetic nanoparticles. [(35)S]sulfate-labeled HSPG was found to accumulate in isolated, AO4B08-containing vesicles, providing the first biochemical evidence for intact HSPG co-internalization with its ligand. Further analysis revealed the existence of both syndecan, i.e. a transmembrane HSPG, and glycosyl-phosphatidyl-inositol-anchored glypican in purified vesicles. Importantly, internalized syndecan and glypican were found to co-localize in AO4B08-containing vesicles. Our data establish HSPGs as true internalizing receptors of macromolecular cargo and indicate that the sorting of cell-surface HSPG to endocytic vesicles is determined by a specific HS epitope that can be carried by both syndecan and glypican core protein.

A Setosphaeria turcica gene encoding the catalytic subunit of calcineurin was cloned using degenerated primers corresponding to conserved domains of Ser/Thr protein phosphatases and its complete cDNA (GenBank accession No. EF 407562) was obtained with RACE method. It's validated single copied model by southern hybridization. Furthermore, the CNA inhibitor Cyclosporin A (CsA) exhibited potent antifungal activity against conidial germination and appressorium formation of S. turcica. The inhibition ratio was positively correlated to CsA concentration. However, appressorium formation was more sensitive than conidium germination to the inhibitor at the same concentration. It was suggested that CNA might play an important role in the pathogenicity of S. turcica.

Considerable researches have been done about integrin alphanubeta6 and carcinomas, but little information has been shown about the relationship between integrin alphanubeta6 and apoptosis. In this study, we investigated the apoptosis and its related signal pathways to integrin alphavbeta6 in colon cancer cells. After we blocked the function of integrin alphavbeta6 in HT29 cells used the monoclonal antibody, the apoptotic cells increased markedly. Meanwhile, cytochrome C released from mitochondria into cytosol, Bcl-2 decreased while Bax increased significantly, and Fas and Fas-ligand had no change. The activities of caspase-3 and caspase-9 increased, while caspase-8 remained no change. Moreover, the expression of phosphorylated extracellular signal-related kinase (P-ERK) decreased. We confirmed that integrin alphavbeta6 acted as an important role in inhibiting apoptosis in colon cancer cells, and the signaling involved the mitochondrial pathway.

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.

Purpose: HAb18G/CD147 is a hepatocellular carcinoma (HCC)-associated antigen. We developed iodine (131I) metuximab injection (Licartin), a novel 131I-labeled HAb18G/CD147-specific monoclonal antibody Fab'2 fragment, and evaluated its safety, pharmacokinetics, and clinical efficacy on HCC in Phase I/II trials., Methods and Materials: In a Phase I trial, 28 patients were randomly assigned to receive the injection in 9.25-, 18.5-, 27.75-, or 37-MBq/kg doses by hepatic artery infusion. In a multicenter Phase II trial, 106 patients received the injection (27.75 MBq/kg) on Day 1 of a 28-day cycle. Response rate and survival rate were the endpoints., Results: No life-threatening toxic effects were found. The safe dosage was 27.75 MBq/kg. The blood clearance fitted a biphasic model, and its half-life was 90.56-63.93 h. In the Phase II trial, the injection was found to be targeted and concentrated to tumor tissues. Of the 73 patients completing two cycles, 6 (8.22%) had a partial response, 14 (19.18%) minor response, and 43 (58.90%) stable disease. The 21-month survival rate was 44.54%. The survival rate of progression-free patients was significantly higher than that of patients with progressive disease after either one or two cycles (p < 0.0001 or p = 0.0019)., Conclusion: Iodine (131I) metuximab injection is safe and active for HCC patients.

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.