• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.81-89
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• 2004

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.

• Journal of Microbiology and Biotechnology
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• v.22 no.8
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• pp.1066-1076
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• 2012

Previous observations demonstrated that various immunosuppressive agents and their combination therapies can increase allograft survival rates. However, these treatments may have serious side effects and cannot substantially improve or prolong graft survival in acute graft-versus-host disease (GVHD). To improve the therapeutic potency of divalent immunoadhesins, we have constructed and produced several tetravalent forms of immunoadhesins comprising each of cytotoxic T-lymphocyte-associated antigen-4 (CTLA4), CD2, and lymphocyte activation gene-3 (LAG3). Flow cytometric and T cell proliferation analyses displayed that tetravalent immunoadhesins have a higher binding affinity and more potent efficacy than divalent immunoadhesins. Although all tetravalent immunoadhesins possess better efficacies, tetravalent forms of CTLA4-Ig and LAG3-Ig revealed higher inhibitory effects on T cell proliferation than tetravalent forms of TNFR2-Ig and CD2-Ig. In vitro mixed lymphocytes reaction (MLR) showed that combined treatment with tetravalent CTLA4-Ig and tetravalent LAG3-Ig was highly effective for inhibiting T cell proliferation in both human and murine allogeneic stimulation. In addition, both single tetravalent-form and combination treatments can prevent the lethality of murine acute GVHD. The results of this study demonstrated that co-blockade of the major histocompatibility complex class (MHC)II:T cell receptor (TCR) and CD28:B7 pathways by using tetravalent human LAG3-Ig and CTLA4-Ig synergistically prevented murine acute GVHD.

• Toxicological Research
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• v.17
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• pp.305-307
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• 2001

Toluene diisocyanate (TDI) is widely used in the manufacture of polyurethanes and is a recognized cause of occupational asthma. Although extensive investigations have been undertaken, the molecular mechanism(s) of the disease is still unclear. We hypothesized that inflammatory cytokines are required during both the sensitization and elicitation phases of the disease and have utilized TNF-R knock-out (KO) mice to address the hypothesis. Black C57 TNFR knock-out mice were exposed to TDI by sc injection and challenged by inhalation of 100 ppb TDI vapor. Control animals included: wild type C57 animals, sham-exposed animals that were challenged with TDI, and animals that were injected with anti-TNF antibodies prior to sensitization and again prior to challenge. Total IgE was increased in the knock-out animals compared with the wild type sensitized and challenged animals whereas TDI-specific IgG antibodies did not differ significantly in KO and wild type animals. There was less inflammation in the nares and trachea in KO animals compared with the wild type animals exposed to TD1 as well as less goblet cell hyperplasia and epithelial damage. Airway reactivity was assessed in animals treated with anti-TNF$\alpha$ antibody and found to be substantially reduced compared with that in sensitized and challenged animals. These results indicate that TNF$\alpha$ plays a role in the immunologic and physiologic responses and in airways inflammation in this animal model and suggests a role for TNF in occupational asthma due to TDI.