Search

Xiaomeng Xu,1 Yan Zhao,2 Zhihong Zhao,1 Shuangshuang Zhu,1 Xinyu Liu,1 Chaomin Zhou,3 Xiaofei Shao,1 Yan Liang,1 Chongyang Duan,4 Harry Holthöfer,5 Hequn Zou1 1Department of Nephrology, The Third Affiliated Hospital of Southern Medical University, 2Blood Purification Center, The Third Hospital of Zhengzhou, 3Department of Nephrology, Guizhou Provincial People’s Hospital, 4Department of Biostatistics, Southern Medical University, Guangzhou, People’s Republic of China; 5Centre for BioAnalytical Sciences, Dublin City University, Dublin, Ireland Aim: To validate the association between visceral obesity and pathogenesis of chronic kidney disease (CKD) among individuals aged 40 years and above, and the potential of visceral adiposity index (VAI) to predict CKD.Methods: This study was based on a cross-sectional epidemiologic study in the People’s Republic of China. A total of 1,581 residents aged over 40 years were included and divided into four groups based on VAI quartile intervals, namely, Groups I, II, III, and IV (eg, Group I included patients with their VAIs in the lowest quartile). Logistic regression analysis was performed. Results: VAI is positively correlated with the albumin-to-creatinine ratio and the prevalence of CKD (P0.05], Group III 1.57 [P0.05), 1.59 (P0.05), 1.11 (P>0.05), and 1.68 (P>0.05), respectively. Conclusion: VAI is highly correlated with the prevalence of CKD in the population aged 40 years and above. It can be used to predict the pathogenesis of CKD, which is dependent on fasting blood glucose and blood pressure levels. Keywords: visceral adiposity index, visceral obesity, chronic kidney disease

To identify and prevent perioperative hypothermia, most surgical patients require a non-invasive, accurate, convenient, and continuous core temperature method, especially for patients undergoing major surgery. This study validated the precision and accuracy of a cutaneous zero-heat-flux thermometer and its performance in detecting intraoperative hypothermia. Adults undergoing major non-cardiac surgeries with general anaesthesia were enrolled in the study. Core temperatures were measured with a zero-heat-flux thermometer, infrared tympanic membrane thermometer, and oesophagal monitoring at 15-minute intervals. Taking the average value of temperature measured in the tympanic membrane and oesophagus as a reference, we assessed the agreement using the Bland-Altman analysis and linear regression methods. Sensitivity, specificity, and predictive values of detecting hypothermia were estimated. 103 patients and one thousand sixty-eight sets of paired temperatures were analyzed. The mean difference between zero-heat-flux and the referenced measurements was -0.03 ± 0.25 °C, with 95% limits of agreement (-0.52 °C, 0.47 °C) was narrow, with 94.5% of the differences within 0.5 °C. Lin's concordance correlation coefficient was 0.90 (95%CI 0.89-0.92). The zero-heat-flux thermometry detected hypothermia with a sensitivity of 82% and a specificity of 90%. The zero-heat-flux thermometer is in good agreement with the reference core temperature based on tympanic and oesophagal temperature monitoring in patients undergoing major surgeries, and appears high performance in detecting hypothermia., (© 2023. The Author(s).)

Banana fruit is highly vulnerable to chilling injury (CI) during cold storage, which results in quality deterioration and commodity reduction. The purpose of this study was to investigate the membrane lipid metabolism mechanism underlying low temperature-induced CI in banana fruit. Chilling temperature significantly induced CI symptoms in banana fruit, compared to control temperature (22 °C). Using physiological experiments and transcriptomic analyses, we found that chilling temperature (7 °C) increased CI index, malondialdehyde content, and cell membrane permeability. Additionally, chilling temperature upregulated the genes encoding membrane lipid-degrading enzymes, such as lipoxygenase (LOX), phospholipase D (PLD), phospholipase C (PLC), phospholipase A (PLA), and lipase, but downregulated the genes encoding fatty acid desaturase (FAD). Moreover, chilling temperature raised the activities of LOX, PLD, PLC, PLA, and lipase, inhibited FAD activity, lowered contents of unsaturated fatty acids (USFAs) (γ-linolenic acid and linoleic acid), phosphatidylcholine, and phosphatidylinositol, but retained higher contents of saturated fatty acids (SFAs) (stearic acid and palmitic acid), free fatty acids, phosphatidic acid, lysophosphatidic acid, diacylglycerol, a lower USFAs index, and a lower ratio of USFAs to SFAs. Together, these results revealed that chilling temperature-induced chilling injury of bananas were caused by membrane integrity damage and were associated with the enzymatic and genetic manipulation of membrane lipid metabolism. These activities promoted the degradation of membrane phospholipids and USFAs in fresh bananas during cold storage., 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 © 2023 Elsevier Ltd. All rights reserved.)

Background: To analysis the clinical outcomes of concurrent chemoradiotherapy (CCRT) alone based on 10-year results for loco-regionally advanced nasopharyngeal carcinoma (LANPC), so as to provide evidence for individualized treatment strategy and designing appropriate clinical trial for different risk LANPC patients., Methods: Consecutive patients with stage III-IVa (AJCC/UICC 8th) were enrolled in this study. All patients received radical intensity-modulated radiotherapy (IMRT) and concurrent cisplatin chemotherapy (CDDP). The hazard ratios (HRs) of death risk in patients with T3N0 was used as baseline, relative HRs were calculated by a Cox proportional hazard model to classify different death risk patients. Survival curves for the time-to-event endpoints were analyzed by the Kaplan-Meier method and compared using the log-rank test. All statistical tests were conducted at a two-sided level of significance of 0.05., Results: A total of 456 eligible patients were included. With 12-year median follow-up, 10-year overall survival (OS) was 76%. 10-year loco-regionally failure-free survival (LR-FFS), distant failure-free survival (D-FFS) and failure-free survival (FFS) were 72%, 73% and 70%, respectively. Based on the relative hazard ratios (HRs) of death risk, LANPC patients were classified into 3 subgroups, low-risk group (T1-2N2 and T3N0-1) contained 244 patients with HR < 2; medium-risk group (T3N2 and T4N0-1) contained 140 patients with HR of 2 - 5; high-risk group (T4N2 and T1-4N3) contained 72 patients with HR > 5. The 10-year OS for patients in low-, medium-, and high-risk group were 86%, 71% and 52%, respectively. Significantly differences of OS rates were found between each of the two groups (low-risk group vs. medium-risk group, P < 0.001; low-risk group vs. high-risk group, P < 0.001; and medium-risk group vs. high-risk group, P = 0.002, respectively). Grade 3-4 late toxicities included deafness/otitis (9%), xerostomia (4%), temporal lobe injury (5%), cranial neuropathy (4%), peripheral neuropathy (2%), soft tissue damage (2%) and trismus (1%)., Conclusions: Our classification criteria demonstrated that significant heterogeneity in death risk among TN substages for LANPC patients. IMRT plus CDDP alone maybe suitable for low-risk LANPC (T1-2N2 or T3N0-1), but not for medium- and high-risk patients. These prognostic groupings provide a practicable anatomic foundation to guide individualized treatment and select optimal targeting in the future clinical trials., (© 2023. The Author(s).)

Background: There are few studies on predictive biomarkers for hyperuricemia, and the predictive value of these biomarkers tends to be poor. Additionally, no reports have described the predictive value of retinol binding protein 4 (RBP4) for hyperuricemia., Purpose: This study was performed to evaluate the value of RBP4 for predicting the risk of hyperuricemia in a general population, determine whether RBP4 could be used alone or in combination with other factors to predict the risk of hyperuricemia in the general population, and establish an optimum predictive model., Methods: We conducted a population-based cross-sectional survey in 2018, involving a questionnaire, physical examination, and laboratory testing. We enrolled 2303 individuals by stratified random sampling, and 2075 were included in the data analysis after applying the eligibility criteria., Results: Serum RBP4 level had a highly significant association with hyperuricemia ( P <0.001). After adjusting for potential confounders, logistic regression indicated that the risk of hyperuricemia was highest in the highest RBP4 quartile (odds ratio: 7.9, 95% confidence interval [CI]: 4.18-14.84, compared to the lowest quartile). The area under the receiver operating characteristic (ROC) curve (AUC) for RBP4 was 0.749 (95% CI: 0.725-0.774, P <0.001), which was higher than that for all the other predictors assessed. The optimum model for predicting hyperuricemia in the general population consisted of RBP4, sex (male), body mass index, serum creatinine, high-sensitivity C-reactive protein, fasting blood glucose, insulin, and alcohol consumption. The AUC was 0.804 (95% CI: 0.782-0.826, P <0.001)., Conclusions: RBP4 is strongly associated with hyperuricemia, and its predictive value was higher than that of traditional predictors., Competing Interests: Author P-CG was employed by Nanjing CR Medicon Pharmaceutical Technology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hong, Shao, Li, Zhou, Ke, Gao, Li, Ning, Chen, Xiao, Xiong and Zou.)

Worldwide, nasopharyngeal carcinoma (NPC) is a rare head and neck cancer; however, it is a common malignancy in southern China. Radiotherapy is the most important treatment strategy for NPC. However, although radiotherapy is a strong tool to kill cancer cells, paradoxically it also promotes aggressive phenotypes. Therefore, we mimicked the treatment process in NPC cells in vitro. Upon exposure to radiation, a subpopulation of NPC cells gradually developed resistance to radiation and displayed cancer stem-cell characteristics. Radiation-induced stemness largely depends on the accumulation of the antiapoptotic myeloid cell leukemia 1 (MCL-1) protein. Upregulated MCL-1 levels were caused by increased stability and more importantly, enhanced protein synthesis. We showed that repeated ionizing radiation resulted in persistently enhanced reactive oxygen species (ROS) production at a higher basal level, further promoting protein kinase B (AKT) signaling activation. Intracellular ROS and AKT activation form a positive feedback loop in the process of MCL-1 protein synthesis, which in turn induces stemness and radioresistance. AKT/MCL-1 axis inhibition attenuated radiation-induced resistance, providing a potential target to reverse radiation therapy-induced radioresistance., (© 2022. The Author(s).)

Objective: To observe the effect of electroacupuncture (EA) preconditioning at heart meridian acupoints on the contents of dopamine (DA) and 5-hydroxytryptamine (5-HT) in lateral hypothalamus area (LHA) and cerebellar fastigial nucleus (FN) in the rats with acute myocardial ischemia-reperfusion injury (MIRI), and explore the role and mechanism of LHA and FN in the effect of EA at heart meridian acupoints against acute MIRI., Methods: Sixty SD rats were randomly divided into a sham-operation group, a model group, an EA heart meridian group and an EA lung meridian group, 12 rats in each group, as well as an LHA plus heart meridian group (damage of bilateral LHA) and an FN plus heart meridian group (damage of bilateral FN), 6 rats in each one. Three days after nucleus destruction, EA was applied to "Shenmen" (HT 7) and "Tongli" (HT 5) in the EA heart meridian group, the LHA plus heart meridian group and the FN plus heart meridian group and EA was applied to "Taiyuan" (LU 9) and "Lieque" (LU 7) in the EA lung meridian group, with 1 V in stimulating voltage and 2 Hz in frequency, lasting 20 minutes each time, once a day, for consecutively 7 days before model replication. Except in the sham-operation group, MIRI rat models were duplicated by ligation of the left anterior descending branch of the coronary artery in the rest groups. Using Power lab physiological recorder, ST segment displacement value was recorded before modeling, 30 min after ligation and 120 min after reperfusion separately. The high performance liquid chromatography-electrochemical detection and analysis system was adopted to determine the contents of DA and 5-HT in LHA and FN dialysate after rat modeling in each group., Results: In comparison of ST segment displacement value 30 min after ligation and 120 min after reperfusion among groups, the value in the model group was higher than that in the sham-operation group ( P <0.01), those in the EA heart meridian group, the LHA plus heart meridian group, the FN plus heart meridian group and the EA lung meridian group were lower than those in the model group successively ( P <0.01) and those in the EA heart meridian group were lower than those in the EA lung meridian group, the LHA plus heart meridian group and the FN plus heart meridian group successively ( P <0.01). The contents of DA and 5-HT in FN and LHA in the model group were lower than those in the sham-operation group ( P <0.01). Except in the groups with nucleus lesions, the contents of DA and 5-HT in FN and LHA of each intervention group were higher than those in the model group ( P <0.01), the contents of DA and 5-HT in FN in the EA heart meridian group were higher than those in the EA lung meridian group and the LHA plus heart meridian group ( P <0.01) and the content of 5-HT in LHA was higher than those in the EA lung meridian group and the FN plus heart meridian group ( P <0.01) separately, the content of DA in LHA was higher than that in the EA lung meridian group ( P <0.01)., Conclusion: EA preconditioning at heart meridian acupoints can effectively alleviate myocardial injury in acute MIRI rats, during which, DA and 5-HT in LHA and FN may be the important material basis.

BACKGROUND Klotho deficiency has been implicated in various kidney diseases and has been associated with renal fibrosis. However, the role of Klotho in renal allograft fibrosis still remains undetermined. MATERIAL AND METHODS A 24-week-old rat renal transplant model with chronic allograft dysfunction (CAD) was carried out by orthotopic kidney transplantation using F344 donor rats to Lewis recipient rats. Successful establishment of the model was verified by HE and Masson staining and renal allograft function assessment. HK-2 cells were cultured and treated with TGF-ß1 and/or siRNA-Klotho at various time points. Total proteins and RNA were extracted from the cultured cells and kidney tissues. Western blot assay and quantitative RT-PCR were used to analyze the expression of Klotho, fibronectin, and ß-catenin pathways. RESULTS We successfully established and identified a 24-week-old rat renal transplant model with CAD. Loss of Klotho was identified to be associated with epithelial-to-mesenchymal transition (EMT), renal allograft fibrosis, and CAD. In HK-2 cells, a significant decrease of Klotho protein was observed in the renal fibrosis induced by TGF-ß1 in a time-dependent manner. Moreover, intervention of siRNA-Klotho remarkably promoted the progression of renal fibrosis and activation of the Wnt/ß-catenin signaling pathway. CONCLUSIONS Our results show that Klotho has a significant protective role against EMT, renal allograft fibrosis, and CAD following kidney transplantation, which is mediated by inhibition of the Wnt/ß-catenin signaling pathway.

The true potential of the industry 4.0, which is a byproduct of the fourth industrial revolution, cannot be actually realized. This is, of course true, until the smart factories in the supply chains get connected to each other, with their systems and the machines linked to a common networking system. The last few years have experienced an increase in the adoption and acceptance of the industry 4.0's components. However, the next stage of smart factories, which will be the smart supply chains, is still in its period of infancy. Moreover, there is a simultaneous need to maintain a focus on the supply chain level implementation of the concept that industry 4.0 puts forth. This is important in order to gain the end to end benefits, while also avoiding the organization to organization compatibility issues that may follow later on. When considering this concept, limited research exists on the issues related to the implementation of industry 4.0, at the supply chain level. Hence, keeping in mind this lack of literature and research available, on a phenomenon that will define the future of business and industry, this study uses an exploratory approach to capture the implementation of industry 4.0 concepts across multiple tiers of the supply chain. Based on this research, the study proposes a multistage implementation framework that highlights the organizational enablers such as culture, cross-functional approach, and the continuous improvement activities. Furthermore, it also highlights the staged implementation of the advanced tools, starting from the focal organization with the subsequent integration with the partner organizations., (© 2020 Elsevier Inc. All rights reserved.)

Following the adaptation of the Paris Agreement at COP21, it was noted that the traditional measures of carbon emissions have several limitations; and a reliable and relevant carbon emissions measurement is important to formulate a response to the challenge of climate change. This study, therefore, explores the relationship between international trade and consumption-based carbon emissions, which is a trade adjusted indicator; and measures the outflow and the inflow of emissions through exports and imports separately. We also include technological innovation in the model to understand its impact on consumption-based carbon emissions. The results show that exports and consumption-based carbon emissions are negatively associated, and technological innovation helps reducing the adverse effect of CO 2 growth. In contrast, Imports and gross domestic product are positively linked with consumption-based carbon emissions. The findings also suggest the countries which embraced the Paris Climate Agreement must focus on consumption-based carbon emissions rather than the production-based carbon emissions., 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 © 2020. Published by Elsevier Ltd.)

Osteoarthritis (OA) is a common clinical degenerative disease characterized by the destruction of articular cartilage, which has an increasing impact on people's lives and social economy. The pathogenesis of OA is complex and unclear, and there is no effective way to block its progress. The study of the pathogenesis of OA is the prerequisite for the early diagnosis and effective treatment of OA. To define the pathogenesis of OA, this review considers the pathological mechanism of OA that involves microRNA, lncRNA, and exosomes. More and more evidence shows that microRNA, lncRNA, and exosomes are closely related to OA. MicroRNA inhibits the target gene by binding to the 3'- untranslated region of the targets. LncRNA usually competes with microRNA to regulate the expression level of downstream genes, while exosomes, as a carrier of intercellular information transfer, transmit the biological information of mother cells to target cells, and the effect of exosomes secreted by different cells on OA are different. In this review, we emphasized that different microRNA, lncRNA, and exosomes have different regulatory effects on chondrocyte proliferation and apoptosis, extracellular matrix degradation and inflammation. Besides, we classified and analyzed these molecules according to their effects on the progress of OA. Based on the analysis of the reported literature, this review reveals some pathogenesis of OA, and emphasizes that microRNA, lncRNA, and exosomes have great potential to assist early diagnosis and effective treatment of OA., (© 2020 The Authors. Orthopaedic Surgery published by Chinese Orthopaedic Association and John Wiley & Sons Australia, Ltd.)

Purpose: Metabolic syndrome (MetS), characterized by a constellation of insulin resistance, central obesity, hypertension, and hyperlipidemia, is a global health threat. High-sensitivity C-reactive protein (hs-CRP) has been shown to be associated with type 2 diabetes and cardiovascular disease; however, its association with incident MetS is less known. Therefore, the aim of this study was to examine the prospective association between hs-CRP and MetS among a Chinese population in a 5-year follow-up study., Patients and Methods: The levels of hs-CRP were measured using serum samples collected at baseline recruitment in 2012 from 886 participants without MetS. Follow-up interviews were conducted in 2018, and MetS was diagnosed by 2017 criteria from the Chinese Diabetes Society. Multivariate logistic regression models were used to assess the overall and sex-specific associations between hs-CRP and incident MetS. The odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were computed with adjustment for demographic, socioeconomic, clinical, and lifestyle factors., Results: After a mean follow-up duration of 5.40 ± 0.56 years, 116 (13.3%) participants developed MetS. In the total study population, increased hs-CRP levels were associated with a higher risk of MetS (OR comparing extreme quartiles of hs-CRP: 4.06 [95% CI: 1.91-8.65]) in the fully-adjusted model. When stratified by sex, the positive association was only observed in women (OR: 4.82 [1.89-12.3]) but not in men (OR: 3.15 [0.82-12.1]; P -interaction = 0.039)., Conclusion: In this study of a Chinese population, a positive association between hs-CRP and incident MetS was found only in women and not in men. Sex-specific prediction and intervention of MetS using hs-CRP as a target should be further evaluated., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Hong et al.)

Acute lung injury (ALI) is one of the severe complications in patients with traumatic brain injury (TBI), contributing to the high mortality. Ghrelin has protective effects against various inflammatory diseases, but the effects of Ghrelin on TBI-induced ALI and its mechanisms remain unknown. In this study, Ghrelin administration was performed on the mice with TBI, then histological change in cortex and lung tissues, lung vascular permeability and macrophage number in bronchoalveolar lavage fluid (BALF) were examined, respectively. Simultaneously, the alterations of proinflammatory factors and pyroptosis-related proteins in lung tissues were detected. As a result, TBI-induced ALI was ameliorated after Ghrelin treatment, which was demonstrated by improved histology, reduced lung vascular permeability, and peripheral macrophage number. Furthermore, Ghrelin decreased the mRNA levels of proinflammatory factors (IL-1β, IL-6, TNF-α and IL-18), the protein levels of pyroptosis-related proteins (NLRP3, Caspase1-P20, HMGB1 and Gasdermin D), and the phosphorylation levels of NF-κB in lung tissues. These results showed that Ghrelin attenuating TBI-induced ALI might be via ameliorating inflammasome-induced pyroptosis by blocking NF-κB signal, which are important for the prevention and treatment of TBI-induced ALI., Competing Interests: Declaration of Competing Interest No potential conflicts of interest were disclosed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The associations of vegetable and fruit intake with liver cancer risk have been inconsistent based on epidemiological studies. The present study aimed to quantitatively evaluate these associations with prospective cohort studies. A systematic literature search was performed with PubMed and Scopus databases up to June 2019. Multivariate-adjusted relative risks (RRs) with a corresponding 95% confidence interval (CI) for the highest versus lowest category were pooled by using a random-effects model. Pre-specified subgroup and univariate meta-regression analyses were performed to identify the sources of heterogeneity. Dose-response analysis was conducted by using the variance weighted least squares regression model. Nine independent prospective cohort studies with 1703 liver cancer events and 1 326 176 participants were included for data synthesis. The summary estimates showed that higher vegetable intake was associated with a 39% (95%CI: 0.50, 0.75) reduction in liver cancer risk, with no significant between-study heterogeneity (P = 0.057). Dose-response analysis indicated that the risk of liver cancer was reduced by 4% (95%CI: 0.97, 0.95; P for trend <0.001) with a 100 gram per day increment of vegetable intake. Subgroup analysis showed that higher intakes of vegetables were associated with a 50% (95%CI: 0.35, 0.72) reduction of liver cancer risk in males, but not in females. However, a non-significant association was found between fruit intake and liver cancer risk. The present study provides strong evidence that higher intakes of vegetables would have beneficial effects on the prevention of liver cancer, especially for males.

Objective: Casein kinase 2 interacting protein-1 (CKIP-1) has exhibited multiple functions in regulating cell proliferation, apoptosis, differentiation, and cytoskeleton. CKIP-1 also plays an important role as a critical regulator in tumorigenesis. The aim of this study is to further examine the function of CKIP-1 in glioma cells., Methods: The expression level of CKIP-1 protein was determined in gliomas tissues and cell lines by immunohistochemistry stain and western blotting while the association of CKIP-1 expression with prognosis was analyzed by Kaplan-Meier method and compared by log-rank test. CKIP-1 was overexpressed or silenced in gliomas cell lines. CCK-8, colony formation assay, and BrdU incorporation assay were used to determine cell proliferation and DNA synthesis. Cell cycle and apoptosis rate were determined with fluorescence-activated cell sorting (FACS) method. Then, expression of key members in AKT/GSK3 β / β -catenin pathway was detected by western blot analysis., Results: In the present study, we reported new evidence that CKIP-1 was reversely associated with the proliferation of glioma cells and survival in glioma patients. Additionally, the overexpressed CKIP-1 significantly inhibited glioma cell proliferation. Further experiments revealed that CKIP-1 functioned through its antiproliferative and proapoptotic activity in glioma cells. Importantly, mechanistic investigations suggested that CKIP-1 sharply suppressed the activity of AKT by inhibiting the phosphorylation, markedly downregulated the phosphorylated GSK3 β at Ser9, and promoted β -catenin degradation., Conclusions: Overall, our results provided new insights into the clinical significance and molecular mechanism of CKIP-1 in glioma, which indicated CKIP1 might function as a therapeutic target for clinical treatment of glioma.

Ras association domain family member 6 (RASSF6) has been shown to act as a tumor suppressor and predictor of poor prognosis in renal cell carcinoma (RCC). However, little is known about the effects of RASSF6 on sorafenib resistance or the underlying mechanism. Here, we show that RASSF6 expression positively correlates with sorafenib sensitivity in RCC cells and human samples. Stable ectopic overexpression of RASSF6 in RCC cell lines reduces resistance to sorafenib in vitro and in vivo. At a molecular level, RASSF6 activates the JNK signaling pathway, which further contributes to Mcl-1 inhibition. Suppression of the JNK pathway can partially restore Mcl-1 expression and sorafenib resistance. Together, these findings suggest that RASSF6 inhibits sorafenib resistance by repressing Mcl-1 through the JNK-dependent pathway. RASSF6 may serve as a novel regulator for sorafenib therapy in RCC., (Copyright © 2018. Published by Elsevier B.V.)

Bacillus cereus was isolated from ready-to-serve brine goose, identified by 16S rRNA gene sequencing analysis and treated with a commercial microwave sterilization condition (a power of 1,800 W at 85°C for 5 min). The influence of microwaves on the morphology, the permeability of membrane and the expression of total bacterial proteins was observed. Microwave induced the clean of bacterial nuclear chromatin, increased the permeability and disrupted the integrity of membrane. Twenty-three proteins including 18 expressed down-regulated proteins and 5 expressed up-regulated proteins were identified by HPLC-MS/MS in the samples treated with microwave. The frequencies of proteins changed after microwaves treatment were labeled as 39.13% (synthesis and metabolism of amino acid or proteins), 21.74% (carbohydrate metabolism), 8.70% (anti-oxidant and acetyl Co-A synthesis), and 4.35% (the catalyst of catabolism of bacterial acetoin, ethanol metabolism, glyoxylate pathway, butyrate synthesis and detoxification activity), respectively. This study indicates that microwaves result in the inactivation of Bacillus cereus by cleaning nuclear chromatin, disrupting cell membrane and disordering the expression of proteins.

Objective: To investigate the relationship between hypertriglyceridemic waist to height ratio phenotype (HWHtR) and chronic kidney disease (CKD) in a community population in South China., Methods: A cross sectional study was conducted among 2142 residents in Zhuhai (Guangdong Province, China) from June to October of 2012. The HWHtR phenotype was defined as a waist to height ratio(WHtR) ≥0.55 and triglyceride level ≥2.0 mmol/L, based on which the participants were divided into HWHtR group and nonHWHtR group. CKD was defined as an eGFR<60 mL/min per 1.73 m2 or an ACR ≥30 mg/g. A logistic regression model was established to investigate the relationship between chronic kidney disease and HWHtR phenotype., Results: Compared with the nonHWHtR group, the HWHtR group had a higher prevalence of chronic kidney disease (11.1% vs 33%, P<0.001). Analysis using the logistic regression model showed that HWHtR was significantly associated with CKD in the unadjusted analyses (OR=3.23, 95% CI: 2.32-4.48, P<0.001). After adjustment for age, sex, history of hypertension, history of diabetes, systolic blood pressure, diastolic blood pressure, drinking, physical exercise, education and current smoking, HWHtR was significantly associated with CKD (OR=2.36, 95% CI: 1.52-3.67, P<0.001); the association of HWHtR and CKD was still significant after further adjustment for BMI (OR=2.12, 95%CI: 1.34-3.35, P<0.001)., Conclusion: Our finding suggests that HWHtR is associated with CKD in this community population.

The aim of this study was to evaluate the inhibiting effect of apigenin on liver cancer in vivo based on the optical molecular imaging method. Subcutaneous liver tumor models were established using respective 1 × 10 6 firefly luciferase (fLuc) and green fluorescent protein (GFP) labeled human hepatocellular carcinoma cells (HepG2-fLuc and HepG2-GFP cells) in 20 BALB/c nude mice which were randomly divided into two groups, 10 in each group. After the tumor cells were implanted 15 days, apigenin was administered through intraperitoneal injection in group B, the other ten mice as control group A. Bioluminescence imaging (BLI) and fluorescence molecular imaging (FMI) were carried out for the follow-up of subcutaneous tumor model. As time goes on, intensity and distribution of bioluminescence and fluorescence of tumours increased gradually with the growth of tumours little by little. The whole process of observation was in accordance with known activities of HCC in the human liver. The tumor volume and tumor weight were significant lower in group B than in group A (p < 0.05), Subcutaneous tumours in the apigenin treatment group B based on BLI and FMI were significantly inhibited compared to the control group A (p < 0.05). Apigenin could be expected as a new drug to treat hepatocellular carcinoma. Optical molecular imaging technology enabled the non-invasive and reliable assessment of anti-tumor drug efficacy on liver cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To investigate the relationship between the clinical and pathological findings in IgA nephropathy with or without IgG deposition in the glomerular mesangial area., Methods: The data were collected from 122 patients with a diagnosis of IgA nephropathy by renal biopsy in the Third Affiliated Hospital of Southern Medical University between November, 2009 and February, 2016. All the samples were examined by light microscopy, immunofluorescence and electron microscopy. According to the results of immunofluorescence assay, the patients were divided into IgA group (n=63) and IgA-IgG group (n=59). The pathological classification of IgA nephropathy was analyzed according to Oxford classification and Lee's classification. The clinical and pathological findings were compared between the two groups., Results: Compared with the patients with IgA nephropathy but without IgG deposition, patients with IgA nephropathy with IgG deposition had higher serum creatinine, higher 24-h urine protein, higher blood uric acid, higher triglyceride levels (P<0.05) and lower eGFR (P<0.05); more of these patients were in Lee's grade IV-V, had renal tubular atrophy and/or interstitial fibrosis, and had MEST scores more than 3 (P<0.05)., Conclusion: Patients with IgA nephropathy with IgG deposition in the glomerular mesangial have severer clinical symptoms and more serious pathological changes. Measures should be taken to control IgG deposition in patients with IgA nephropathy to delay the progress of the disease.

Objective: To explore the relationship between waist-to-hip ratio (WHR) and chronic kidney disease (CKD) in non-diabetic subjects and compare the difference between male and female subjects., Methods: We performed a cross-sectional survey among 2142 community-based southern Chinese participants without diabetes from June to October 2012. We divided all the participants into 4 groups according to the gender-specific quartiles of WHR. Logistic regression models were used to explore the associations of WHR with CKD in these subjects., Results: In the unadjusted model, WHR was significantly associated with CKD in women (OR=7.29, 95% CI: 3.56-16.32, P<0.001), and the association was still significant (OR=6.13, 95% CI: 2.56-15.20, P=0.003 ) after adjustment for the potential confounders (including age, history of hypertension, coronary heart disease, current smoker, physical inactivity, education level, systolic blood pressure, diastolic blood pressure, serum triglyceride, serum high density lipoprotein, blood glucose, and BMI). The odds ratio (OR) for having CKD in the highest versus lowest quartile of WHR levels was 2.44 (95% CI: 0.98-4.97, P=0.103) in men in the unadjusted model., Conclusion: WHR levels are associated with CKD in non-diabetic women but not in non-diabetic male subjects.

The effect of different concentrations (0, 0.1 and 0.5%, v/v) of black pepper essential oil (BPEO) on thiobarbituric acid reactive substances (TBARS), meat color, the percentage of metmyoglobin (MetMb%), microbiological parameters and total volatile basic nitrogen (TVB-N) of pork loins stored at 4°C for 9days was evaluated. BPEO treatments showed lower TBARS, MetMb%, yellowness (b*) values, Pseudomonas spp. and Enterobacteriaceae count and TVB-N values and higher lightness (L*) and redness (a*) values than the control during storage; the effectiveness of BPEO was dose-dependent. The retardation of the formation of MetMb by adding BPEO ensured higher L* and a* values and lower b* values than the control at 6 and 9days; the MetMb content has a similar trend to the lipid oxidation. The lower TVB-N value of BPEO treatments than the control could be attributed to the inhibition of Pseudomonas spp. and Enterobacteriaceae. Gram-negative bacteria were more sensitive than Gram-positive bacteria to BPEO., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The prevalence of chronic kidney disease (CKD) and prediabetes has increased in China, and at different rates in different locations. Therefore a community-based screening research was conducted in order to determine the prevalence of CKD and prediabetes, and to analyze associated risk factors of CKD and prediabetes in a city of Southern China., Methods: A total of 7801 community residents aged 18 year and older from 6 communities were screened by a stratified random cluster sampling method. An estimated glomerular filtration rate (eGFR), albuminuria, fasting plasma glucose (FPG), and homeostatic model assessment of insulin resistance (HOMA-IR) were measured. Age-standardized prevalence was calculated by the direct method with the use of data on the population distribution in China in 2006. Multivariate logistic analysis was used to analyze the risk factors of CKD and prediabetes, and association of insulin resistance (IR) with CKD and prediabetes was analyzed., Results: The age-standardized prevalence of CKD was 12.5%, eGFR < 60 ml×min(-1)×1.73 m(-2) was 2.7% and ACR (albumin to creatinine ratio) > 30 mg/g was 10.3%. The age-standardized prevalence of prediabetes was 12.1%. Logistic regression suggests that IR was a common independent risk factor of CKD and prediabetes. Further analysis show that HOMA-IR was increased with the aggravation of kidney injury and FPG., Conclusion: CKD and prediabetes have become a major public health problem in Zhuhai, Southern China; insulin resistance may be an important risk factor.

ENHANCIN is an enhancing protein chiefly found in insect baculoviruses. One ENHANCIN homologue was identified, by blast method, in Agrotis Segetum granulovirus (AgseGV) genome, named enhancin-like. Sequence analysis indicated that this gene includes the conserved domains, conserved in other ENHANCIN, and it has no signal peptide or a-transmembrane helix. A proline-rich domain, which is similar to those of mammals, is present at its C-terminal. To analyze the synergistic function of AgseGV enhancin-like gene, prokaryotic expression vectors of its whole gene and the 5'-truncated fragment (1, 017bp) were constructed. Expression product of truncated fragment was purified by chromatography, and then it was used to prepare antibody. The expression product of whole gene was identified by Western blot with specific antibody and anti-His-Tag antibody. Bioassay proved that the expression product of whole gene can increase the mortality with 16.25% to 3th instar larvae of Helicoverpa armigera (HaNPV: 1.17 x 10(2) PIBS/mL), while the truncated fragment has no obvious synergistic function.

Objective: To investigate the role of Ara-C in regulating anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells., Methods: The diabody of anti-CD3/anti-Pgp was purified by E-tag affinity chromatography. K562 and K562/A02 cells were treated with Ara-C. The expressions of B7-1 and B7-2 on K562 and K562/AO2 cells were detected by FACS. The cytotoxicity of T-lymphocytes combined with anti-CD3/anU-Pgp plus Ara-C was analyzed by CytoTox 96 nonradioactive method., Results: The expressions of B7-1 and B7-2 on K562 and K562/A02 cells treated by Ara-C was significantly higher than those untreated. The effect/target ratio was from 0.39:1 to 25:1, and the killing rate of activated T cells to anti-drug-resistant leukemia cells was from (16.44 +/- 1.20)% to (60.49 +/- 2.90)%. The killing rates were increased gradually, with both the effect/target ratio and the antibody concentration increasing (P < 0.05)., Conclusion: Ara-C may be an important adjuvant for improving anti-CD3/anti-Pgp mediating T-lymphocytes activities against multi-drug resistant leukemia cells.

Objective: To study the synergistic mechanism between PH II -7 and doxorubicin against multi-drug resistant HL-60/ADR cells and its parent HL-60 cells., Methods: The anti-tumor activity of doxorubicin alone and combined with PH II -7 were measured by MTT assay. RNA was extracted from the cells treated with PH II -7 for different times or doses then the expression of MRP gene was measured by RT-PCR. Confocal laser scanning microscopy and FACS were used to detect the intracellular cumulation of doxorubicin in PH II -7 treated HL-60 and HL-60/ADR cells., Results: PH II -7 has anti-tumor effect with IC50 of (0.83 +/- 0.08) micromol/L and (1.74 +/- 0.56) micromol/L for HL-60 and HL-60/ADR, respectively. It could potentiate the anti-tumor effect of doxorubicin with CDI of 0.7 and 0.43 for HL-60 and HL-60/ADR, respectively. PH II -7 and doxorubicin act synergistically in inhibiting the proliferation of HL-60 and HL-60/ADR cells and down-regulating the expression of MRP gene in a dose and time dependent manner. PH II -7 restored the intracellular cumulation of doxorubicin in HL-60/ADR cells to 55% of that in HL-60 cells., Conclusion: PH II -7 can significantly hasten the cytotoxicity of doxorubicin to HL-60 and HL-60/ADR cells through down-regulating the expression of MRP. The synergistic effect was more obvious in HL-60/ADR cells.

Objective: To investigate the related factors of systemic lupus erythematosus (SLE) and the situation of diagnosis and treatment after onset., Methods: 203 SLE patients, 11 males and 192 females, with a male/female ratio of 1:17, average onset age of (32 +/- 14), and average course of 3 years, were followed up and the related factors such as risk factors, family history and the situation of diagnosis and treatment were studied., Results: 63 patients (31%) had the history of overworking before falling ill; 47 patients (23.2%) had suffered from infection before SLE onset. Other risk factors, such as solarization, emotional fluctuation, menstrual disorder, abortion, dyeing, and chemical drug contacting accounted for 14.3%, 14.3%, 9.4%, 6.9% , 4.9%, and 1.5% respectively. 16 patients (7.9%) had positive family history. 120 patients got the correct diagnosis at the first visit. 83 patients had been misdiagnosed for less than 1 year to 14 years. 47 of the 83 patients (23.2%) failed to get the correct diagnosis for more than one year. 140 of the 203 patients (69.0%) were given glucocorticoid and/or immunosuppressant as the first choice treatment measures after the diagnosis was confirmed. 39 of the 203 patients (19.2%) chose traditional Chinese medicine or folk prescription as the first choice. And 24 of the 203 patients (11.8%) used anti-inflammatory, and antipyretic drugs. 128 patients (63.1%) failed to continue to work., Conclusion: Overworking, infection, solarization, and emotional fluctuation may be related to the onset of SLE. A great part of SLE patients can not be diagnosed early and treated promptly. SLE brings bad influence on the life quality of patients.

Objective: To prepare monoclonal antibody (McAb) against anti-CD3 ScFv for purifying and detecting serum anti-CD3 antibody concentration., Methods: McAb against anti-CD3 ScFv was prepared by hybridoma technique and used to prepare affinity chromatography column, which was used to purify anti-CD3 ScFv and Diabody [CD3 x Pgp] without E-tag. The binding activities of anti-CD3 ScFv, Diabody [CD3 x Pgp] without E-tag, and Diabody [CD3 x Pgp] purified by anti-CD3 affinity chromatography column or anti-E-tag affinity chromatography column against K562/A02 cell and Jurket cells were detected by fluorescence activated cell sorting (FACS) method. ELISA was used to identify the specificity of the McAb., Results: McAb against anti-CD3 ScFv specifically detected serum anti-CD3 ScFv without reacting with sera. The anti-CD3 ScFv purified by anti-CD3 affinity chromatography column and purified by anti-E-tag affinity chromatography column had the same specific binding activity with Jurkat cells. The positive binding rates of Diabody [CD3 x Pgp] without E-tag to K562/A02 and Jurkat cells were 89.87% and 83.95%, respectively. In the competitive binding experiments with K562/A02 and Jurkat cells, the binding rates of Diabody [CD3 x Pgp] without E-tag decreased to 56.30% and 43.78%, respectively., Conclusion: The McAb against anti-CD3 ScFv prepared in our lab can be used to purify and detect serum anti-CD3 antibody concentration.

Aim: To investigate the role of the extracellular domain of human 4-1BBL (ex4-1BBL) in regulating the in vitro activities of peripheral blood lymphocytes (PBL)., Methods: Viable cells were quantified with Trypan-blue exclusion assay. CytoTox 96 Nonradioactive Cytotoxicity Assay Kit was used for measuring LDH levels of supernatant. ELISA kit was used for measuring IL-2 level. In vitro cytotoxity of PBL combined with anti-CD3/anti-Pgp bispecific diabody plus ex4-1BBL was analyzed with CytoTox 96 nonradioactive method., Results: Ex4-1BBL can increase the proliferation of PBL, reduce cell death, promote IL-2 secretion, and the experimental group with ex4-1BBL showed obviously enhanced cytotoxic effect toward K562/A02 cells., Conclusion: Ex4-1BBL may be an important adjuvant for improving activities of PBL.

Aim: To construct and express a diabody [CD3 x Pgp] without Etag and analyse its biological activity., Methods: In this study, the diabody [CD3 x Pgp] was obtained by PCR and restriction cleavage, and expressed in E.coli 16C9. The product was purified by anti-anti-CD3 scFv affinity chromatography and verified through SDS-PAGE electrophoresis. Flow cytometry(FCM) was used to analyse the bingding properties and competitive bingding capacity., Results: The sequence of diabody [CD3 x Pgp] without Etag was correct. It migrated as two bands with the expected molecular weight(25 kD and 26 kD) in SDS-PAGE. The binding rate to CD3 and Pgp antigen was 83.95% and 89.87% respectively. The competitive bingding rate to CD3 and Pgp was 43.78% and 50.25% respectively., Conclusion: The diabody [CD3 x Pgp] without Etag has been successfully constructed, expressed and purified. The product can bind to CD3 and Pgp antigen specifically, and its biological activity doesn't decrease.

In this work, nano-CdS was successfully prepared. The nano-CdS was also modified with L-cysteine. Absorption and fluorescence spectra of CdS nanoparticles for different pH values, reaction times and different Cys/Cd2+/S ratios were investigated. Meanwhile a fluorescence enhancing effect was observed between trace zinc ions and the functionalized L-Cys-CdS nanoparticles. The response is linearly proportional to the concentration of zinc ions from 1.0 to 15 micromol x L(-1). The functionalized nanoparticles are hopeful of use as fluorescence probes in detecting trace elements in biological samples.

In the present paper, a fluorescence method was used to study at different pH the fluorescence quenching of bovine serum albumin (BSA) by its interaction with balofloxacin (BLFX). The interaction association constants of BSA and BLFX were determined from a double reciprocal line Weaver-Burk plot. According to the Forster dipole-dipole energy transfer, the distance to be measured between the BLFX and tryptophane is 5.09 nm. From thermodynamical coordination it can be judged that the binding power between BLFX and BSA is electrostatic effect. The effect of BLFX on the conformation of BSA was also analyzed by using synchronous fluorescence spectroscopy.

Objective: To study the specific cytotoxicity mediated by anti-P-gp/anti-CD(3) diabodies in multidrug resistant solid tumor using P-gp as target., Methods: The anti-P-gp/anti-CD(3) diabodies were secreted from E. coli strain 16C9 containing the expression plasmid PAYZDCP, grown at 30 degrees C in a shaker flask; the diabodies were purified by affinity chromatography and identified by SDS-PAGE; the effect of the anti-P-gp/anti-CD(3) diabody mediated lysis of P-gp-expressing tumor cells was assayed by (51)Cr release assay in vitro, and by human KB nude mouse xenograft models in vivo., Results: The diabodies were generated by bacteria as a soluble functional form and purified by one-step affinity chromatography with a yield > 4 mg/L culture medium. In (51)Cr release assay, the diabodies targeted human activated T cells to lyse P-gp(+)-KB/MDR cells in a dose-dependent manner. It suggested that the diabody was able to induce an efficient lysis of the target cells by human T cells in vitro. When combined with activated human T cells, the diabody significantly inhibited the growth of KB/MDR, but had no effect on KB xenografts., Conclusion: The anti-P-gp/anti-CD(3) bispecific antibody is a potent agent for targeting human T lymphocytes to lyse solid tumor cells overexpressing P-gp in vitro and in vivo.

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.

Objective: To study the specific targeting cytotoxicity to drug-resistant leukemia cells mediated by anti-Pgp/anti-CD3 diabody., Methods: The diabody was purified by affinity chromatography and identified by SDS-PAGE and FACS. The effect of the anti-Pgp/anti-CD3 diabody mediated lysis of Pgp-expressing tumor cells was assayed by human leukemia nude mice xenograft model in vivo., Results: The diabody was produced in E.coli in a soluble functional form and could bind both Jurkat cells (CD3(+)) and K562/A02 cells (Pgp(+)). The binding rates were 86.25% and 86.26%, respectively. It could inhibit tumor growth by 98.57% and prolonged the survival of mice bearing xenografted K562/A02 cells., Conclusion: The diabody was proved to be a potent agent for mediating T lymphocyte cytotoxicity to lyse Pgp expressing tumor cells in vitro and in vivo.

Objective: To prepare a neutralizing monoclonal antibody (McAb) against vascular endothelial growth factor receptor KDR and study its biological activity., Methods: Extracellular immunoglobulin (Ig)-like domain III of KDR (KDR III) was expressed in E. coli and purified by affinity chromatograph. Monoclonal antibody against KDR III was prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [(3)H]-TdR incorporation assay were also used to detect the activity of anti-KDR McAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on VEGF-induced mitogenesis of human endothelial cells., Results: McAb Ycom1D3 against KDR III was prepared which bound specifically to both the soluble KDR III and the cell-surface expressed KDR. It effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated activation of KDR expression on human endothelial cells. Furthermore, Ycom1D3 efficiently neutralized VEGF-induced mitogenesis of human umbilical vascular endothelial cells., Conclusion: McAb Ycom1D3 against KDR III may suppress the action of VEGF by blocking native vascular endothelial growth factor receptor KDR. It has potential clinical applications in the treatment of cancers and other diseases where pathological angiogenesis is involved.

Aim: To construct and express a single chain antibody (scFv) against human vascular endothelial growth factor (VEGF) receptor KDR and characterize its biological activity., Methods: The restriction enzyme sites were added to the previously cloned V(H) and V(L) genes of mAb Ycom1D3 against KDR by PCR. The anti-KDR scFv gene was constructed by the splicing overlap extensive (SOE) PCR and then inserted into fusion expression vector pAYZH. The recombinant protein was expressed in E.coli 16C9 and purified with His-tag affinity chromatography. The specificity of the purified scFv was examined by ELISA and FACS., Results: DNA sequencing indicated that the cloned scFv gene consisted of 729 bp, encoding 243 amino acids. After induction in low-phosphate medium of AP5, a new protein band with relative molecular mass (M(r)) of 30 000 appeared on gel of SDS-PAGE and on nitrocellulose membrane of Western blot, which was consistent with the theoretically predicted value. Anti-KDR scFv was expressed in the form of inclusion body, which accounted for 20% of total bacterial protein. ELISA and competitive immunofluorescence binding test showed that the anti-KDR scFv had the same binding activity as mAb Ycom1D3 and that it could block VEGF/KDR interaction effectively., Conclusion: Recombinant anti-KDR scFv gene has been successfully constructed and expressed in E.coli 16C9, which lays the foundation for its diagnostic and therapeutic application.

Aim: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domain III of vascular endothelial growth factor receptor KDR and study its biological activity., Methods: Soluble KDR Ig domain III (KDR-III) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-III were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial cells., Results: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-III protein. Ycom1D3 bound specifically to both the soluble KDR-III and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells., Conclusion: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved., (Copyright 2004 Acta Pharmacologica Sinica)

The genes encoding for the light and heavy chain variable regions (V(H) and V(L)) has been cloned by RT-PCR from a murine hybridoma that produced monoclonal antibody (mAb) Ycom1D3, which was against domain III of human vascular endothelial growth factor receptor II (KDRIII) and were then connected to each other by a short peptide linker containing 15 amino acids (Gly4Ser)3 using splice-overlap extensive PCR. The recombinant Ycom1D3-ScFv gene was cloned into the expression vector pAYZ and induced to express in E. coli 16C9. SDS-PAGE and Western blot analysis showed that the recombinant Ycom1D3-ScFv gene was expressed in E. coli 16C9 and the relative molecular weight of the fusion protein is 30kD which was consistent with the theoretically predicted value. ScFv expression was in the form of an inclusion body and the purified fusion protein was obtained after a series of purification steps including cell breakage, inclusion body solubilization, TALON metal affinity chromatography and protein refolding. Flow cytometric analysis showed that the ScFv fragment can react with human umbilical vein endothelial cells (HUVECs) which express KDR on the cell surface. In Conclusion, Recombinant Ycom1D3-ScFv gene has been successfully constructed and expressed in E. coli 16C9, which could be useful in both diagnostic and therapeutic applications.

Background & Objective: The anti-CD20 antibodies and fragments have been applied for treatment of non-Hodgkin's lymphomas (NHL) in clinic. The new anti-CD20 antibodies and their fragments (unmodified or radiolabeled) yet have been exploited for those patients with incomplete response to rituximab. The chimeric antibody fragments Fab and F(ab) '(2) derived from HI(47)(a mouse anti-CD20 antibody) have been constructed. The present study was designed to determine the effect of HI(47) and chimeric anti-CD20 antibody fragments on growth inhibition and apoptosis of lymphoma cells., Methods: The binding of anti-CD20 antibodies to CD20 positive human B cell lymphoma Raji cells was examined using immunofluorescence assay. MTT method was used to evaluate the effect of chimeric antibody fragments on Raji cells growth. Annexin V staining and DNA ladder were used to examine the apoptosis of Raji cells induced by chimeric antibody fragments., Results: HI(47) and its chimeric antibody fragments were capable of binding to CD20 positive Raji cells with the binding rate of above 90%. HI(47) did not compete with rituximab in binding with Raji cells. The growth of Raji cells was inhibited by HI(47) and its fragments with the inhibition rates of (57+/-1.5)%, (65.2+/-2.5)%,and (77.2+/-3.2)%, respectively, at the concentration of 100 microg/ml. The monovalent antibody fragment Fab (20 microg/ml) induced the apoptosis of Raji cells with early apoptosis rate of 17%., Conclusion: The chimeric anti-CD20 antibody fragments derived from HI(47) have inhibitory effect on Raji cells and can induce apoptosis of Raji cells.

The anti-CD3 antibody can improve success rate of organs transplant. HIT3a, a mouse anti-CD3 antibody, was chimerized by using gene engineering methods to decrease its immunogenity. The anti-CD3 genes, heavy chain and light chain, were cloned using PCR from the vector pCANTAB 5E containing anti-CD3 scFv gene fragment, and two PCR fragments were recombined into the expression vector pKN100 with human antibody light constant domain and pG1D105 with human antibody heavy constant domain, respectively. The two vectors were co-transfected into CHO cells using liposome. The anti-CD3 antibody was detected by ELISA and Western blot assay in supernatant of transfected CHO cells culture. The primary results of competitive assays by FACS showed that anti-CD3 antibody could partially block the sites through which parent antibody (HIT3a) bind to CD3+ Jurkat cells. The result of 3H-TdR incorporation showed that the chimeric anti-CD3 antibody could stimulated proliferation of peripheral blood mononuclear cells (PBMC) as the parent antibody. In this thesis, the results of some experiments indicated that the chimeric anti-CD3 antibody expressed in CHO cells was an antibody with native biological activity, and it is possible to apply to in clinic in the future.

The use of tumor antigen specific antibodies for the delivery of therapeutic agents offers the possibility of targeting therapy with reduced toxicity to normal tissues compared to conventional treatments. However, several factors restrict the use of anti-PGP monoclonal antibodies(Mabs). First, Pgp is expressed in normal tissues, particularly in epithelial and endothelial cells of the gastrointestinal tract, liver, kidney, blood brain barrier, choroids plexus and other organs. It plays a significant role to transport drugs and toxins in these organs. Therefore, anti-PGP antibodies in combination with cytotoxic compounds or radiolabelled antibodies should neither inhibit the activity of PGP, nor harm the cells which expressed PGP normally. BiMab exploit the specificity of Mab and ensures activation of cellular cytotoxic mechanisms which kill tumor cells only, but not harm normal cells. It will provide a strategy for resistant cancer therapy using anti-PGP antibodies. Second, Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and appro ximately 90% following a second treatment. In an effort to reduce the toxicity and antigenicity, we focus to produce anti-PGP antibodies which have the binding activity only, but not inhibit the function of the "pump", and to construct a small and partially humanized recombinant molecule with dual specificity for both PGP and CD3 complex to activate the host immune response toward the tumour. PCR and overlap PCR were used to construct anti-CD3/ anti-Pgp Diabody. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by both the detection of western blot and size exclusion chromatography; its antigen-binding activity was examined by FACS, cellular RIA. Plasmid pAYZDCP which expressed the anti-CD3/anti-Pgp Diabody was constructed correctly. The diabody was recovered in high yield( up to 2mg/ L) after E-taq purification and predominantly(90%) as a dimer. The diabody can bind to Jurkat cells (CD3+) and K562/A02 cells(Pgp+). The affinities of the diabody were similar with the anti-CD3 ScFv or anti-Pgp ScFv, respectively. The anti-CD3/ anti-Pgp BsF(ab')2 was first recast into the diabody format and succeeded to obtain high level expression. The results of some biological activity experiments indicated that the diabody could bind to Jurkat cells and K562/A02 cells. Multidrug resistance can be reversed experimentally by a variety of drugs, among which the best known are verapamil and trifluoperazine, which unfortunately are of limited use in practice due to severe collateral cardiac toxicity. Anti-PGP x anti-CD3 diabody will provide another therapeutic strategy against multidrug resistance cancer.

Monoclonal antibodies (mAb) directed against CD20, either unmodified or in radiolabeled forms, have been successfully exploited in clinic as effective therapeutic agents in the management of non-Hodgkin's B-cell lymphoma. The antibody fragment is a potential agent in image and therapy of tumor. To further improve the soluble expression of anti-CD20 antibody Fab' fragment, PCR was used to mutate the anti-CD20 VL and VH genes and its biological activity was identified. The expression vector of chimeric antibody Fab' was constructed and expressed in E. coli. The data of mutant clone DNA sequence showed that the amino acid of light chain gene of the parent anti-CD20 antibody (H47) was successful mutated as Ser (GAG)-Asn (CAG). The soluble expression of mutated anti-CD20 Fab' (CD20-7) was 3.8 mg/g dry cell weight, while the parent (CD20-2) was 1.3 mg/g dry cell weight. The affinity constant Ka of CD20-7 was 2.2 x 10(9) L/mol. The primary results of competitive assays by FACS showed that CD20-7 could partially block the sites through which parent antibody (HI47) bind to Raji cells. There was difference in the Raji cells (CD20+)-binding activity between the mutant CD20-7 and parent CD20-2. The site mutation of anti-CD20 Fab' gene make it possible that the anti-CD20 antibody fragment was succeeded to obtain higher expression. In this thesis, we succeeded in completing mutation and expression of anti-CD20 Fab' genes, distinguishing its biological activity, and obtaining its highly expression. These period results will lay a foundation for development of other kind of anti-CD20 engineering antibody (for instance: Fab' Diabody and miniantibody), and make it possible for anti-CD20 antibody to be applied to tumor therapy in civil in the future.

Bone marrow specimens from 100 cases of acute leukemia (AL) diagnosed by MIC were detected with fluorescence microscopy for their mdr-1 expression using monoclonal antibody JSB-1 against P-glycoprotein (P-170). The results showed that almost all subtypes of AL had P-170 expression and M5 of ANLL had a significantly higher expression rate in the newly diagnosed group. The MDR expression highly correlated with the clinical drug resistance and prognosis. The Positive rate of P-170 (20.8% +/- 14.9%) and MDR expression (78.9%) of refractory group were significantly higher than newly diagnosed group (7.5% +/- 9.8% and 18.2% respectively). Cases with MDR expression had poor response to chemotherapy and bad prognosis.