title: A Human Anti-Toll Like Receptor 4 Fab Fragment Inhibits Lipopolysaccharide-Induced Pro-Inflammatory Cytokines Production in Macrophages.
abstract: The results of clinical and experimental studies suggest that endotoxin/toll-like receptor 4 (TLR4)-mediated proinflammatory and profibrotic signaling activation is critical in the development of hepatic fibrosis. However, studies examining the role of specific TLR4 inhibitor are still lacking. The present study was aimed to prepare a human anti-TLR4 Fab fragment, named hTLR4-Fab01, and to explore its immune activity. We screened the positive clone of anti-human TLR4 phagemid from a human phage-display antibody library using recombinant TLR4 protein, which was used as template cDNA for the amplification of variable regions of the heavy (VH) chain and light chain (VL), then coupled with highly conserved regions of the heavy chain domain 1 (CH1) and the light chain (CL), respectively. Thus, the prokaryotic expression vector pETDuet-1 of hTLR4-Fab01 was constructed and transformed into Escherichia coli (E. coli) BL21. The characteristic of hTLR4-Fab01 was examined by SDS-PAGE, Western blotting, ELISA, affinity and kinetics assay. Further, our data demonstrate that hTLR4-Fab01 could specifically bind to TLR4, and its treatment obviously attenuated the proinflammatory effect, characterized by less LPS-induced TNF-α, IL-1, IL-6 and IL-8 production in human macrophages. In conclusion, we have successfully prepared the hTLR4-Fab01 with efficient activity for blocking LPS-induced proinflammatory cytokines production, suggesting that the hTLR4-Fab01 may be a potential candidate for the treatment of hepatic fibrosis.
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing 210002, China.|Anhui Medical University Affiliated with Bayi Clinical College, Nanjing 210002, China.
authors.forename: Maorong
authors.initials: M
authors.lastname: Wang
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing 210002, China.
authors.forename: Wenkai
authors.initials: W
authors.lastname: Zheng
authors.affiliation: Huadong Medical Institute of Biotechniques, Nanjing 210002, China.|Department of Emergency Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
authors.forename: Xuhui
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing 210002, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.affiliation: Anhui Medical University Affiliated with Bayi Clinical College, Nanjing 210002, China.
authors.forename: Binggang
authors.initials: B
authors.lastname: Cai
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing 210002, China.
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Huadong Medical Institute of Biotechniques, Nanjing 210002, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Zheng
authors.affiliation: Department of Respiratory Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
authors.forename: Linfu
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing 210002, China.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Institute of Liver Disease, Nanjing Jingdu Hospital, Nanjing 210002, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Huadong Medical Institute of Biotechniques, Nanjing 210002, China.
authors.forename: Changjun
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Emergency Medicine, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China.
authors.forename: Shinan
authors.initials: S
authors.lastname: Nie
authors.affiliation: Huadong Medical Institute of Biotechniques, Nanjing 210002, China.|Department of Pathology, Key Laboratory of Antibody Technique of the Ministry of Health, NJMU, Nanjing 210029, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhu
mesh_terms: D045744:Cell Line, Tumor; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D008070:Lipopolysaccharides / Q000633:toxicity; D008264:Macrophages / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000276:immunology*
chemical_list: D016207:Cytokines; D007140:Immunoglobulin Fab Fragments; D008070:Lipopolysaccharides; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4