Jin-Kyoo Kim

Mapping the site on human IgG for binding of the MHC class I-related receptor, FcRn

The analysis of the pharmacokinetics of wild‐type and mutated Fc fragments derived from human IgG1 indicates that Ile253, His310 and His435 play a central role in regulating serum half‐life in mice. Reduced serum half‐life of the recombinant, mutated fragments correlates with decreased binding to the MHC class I‐related neonatal Fc receptor, FcRn. In addition, the analysis of an Fc fragment in which His435 is mutated to Arg435 demonstrates that the sequence difference at this position between human IgG1 (His435) and IgG3 (Arg435) most likely accounts for the shorter serum half‐life of IgG3 relative to IgG1. In contrast to His310 and His435, the data indicate that His433 does not play a role in regulating the serum half‐life of human IgG1. Thus, the interaction site of mouse FcRn on human and mouse IgG1 involves the same conserved amino acids located at the CH2‐CH3 domain interface of the IgG molecule. The sequence similarities between mouse and human FcRn suggest that these studies have direct relevance to understanding the factors that govern the pharmacokinetics of therapeutic IgG.

Crystal Structure of the Vα Domain of a T Cell Antigen Receptor

The crystal structure of the Vα domain of a T cell antigen receptor (TCR) was determined at a resolution of 2.2 angstroms. This structure represents an immunoglobulin topology set different from those previously described. A switch in a polypeptide strand from one β sheet to the other enables a pair of Vα homodimers to pack together to form a tetramer, such that the homodimers are parallel to each other and all hypervariable loops face in one direction. On the basis of the observed mode of Vα association, a model of an (αβ)2 TCR tetramer can be positioned relative to the major histocompatibility complex class II (αβ)2 tetramer with the third hypervariable loop of Vα over the amino-terminal portion of the antigenic peptide and the corresponding loop of Vβ over its carboxyl-terminal residues. TCR dimerization that is mediated by the α chain may contribute to the coupling of antigen recognition to signal transduction during T cell activation.

The stoichiometry and affinity of the interaction of murine Fc fragments with the MHC class I-related receptor, FcRn

The binding of recombinant wild type and mutant Fc-hinge fragments to soluble, FcRn expressed in insect cells has been analysed. The mutant Fc-hinge fragments are derived from murine IgG1 with mutation of residues located at the CH2-CH3 domain interface (Ile253, His31O, Gln311, His433 and Asn434; EU numbering). These mutant Fc-hinge fragments have previously been shown to be deficient in neonatal transcytosis in suckling mice and also have abnormally short serum half lives. The mutated residues are highly conserved in human and rodent gammaglobulins (IgGs) and are also involved in binding to staphylococcal protein A. This study demonstrates that the Fc mutants have lower binding affinities for recombinant FcRn and mutations in the CH2 domain have a greater effect than those in the CH3 domain. There is an excellent correlation between affinity and transcytosis or the control of catabolism, and this provides further evidence in support of the close overlap of the sites of IgG/Fc involved in these processes. The stoichiometry of the FcRn:Fc interaction has also been investigated and has been found to be 1:1, indicating that binding of FcRn to one CH2-CH3 domain interface site precludes an FcRn:Fc interaction at the second site.

Cloning and functional characterization of chicken interleukin-17D

The chicken interleukin-17D was cloned from a testis cDNA library prepared from the Korean native chicken. The full-length chicken IL-17D (chIL-17D) cDNA consisted of a 348 nucleotide sequence encoding an open reading frame of 116 amino acids with a predicted molecular mass of 13.3kDa. Comparison of the deduced amino acid sequence of chIL-17D with homologous proteins from human, mouse and opossum revealed 64%, 53% and 76% identity, respectively, including six conserved cysteine residues present in the mammalian polypeptides. The chIL-17D gene transcript was expressed in a wide range of tissues, and highest levels were in pancreas, thymus and lung. Following Eimeria maxima infection, levels of the chIL-17D mRNA were up-regulated in the intestinal jejunum, bursa, lung, and spleen but decreased in the thymus. Infected chickens also expressed greater levels of chIL-17D mRNA in CD4(+), CD8(+) and TCR1(+) intestinal intraepithelial lymphocytes while decreased expression was seen in TCR2(+) cells. Treatment of CHCC-OU2 fibroblasts with chIL-17D recombinant protein induced the expression of IL-6 and IL-8. Collectively, these results suggest that chL-17D has structural and functional similarities to mammalian IL-17Ds and that it plays an important role in local gut innate immune responses during experimental coccidiosis.

Humanization of an agonistic anti-death receptor 4 single chain variable fragment antibody and avidity-mediated enhancement of its cell death-inducing activity.

Development of agonistic monoclonal antibodies (mAbs) against the pro-apoptotic molecule death receptor 4 (DR4) [or tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptor 1] is an attractive anti-cancer strategy because of their potential for inducing tumor-specific cell death. In this study, we humanized an agonistic anti-DR4 AY4 scFv raised in mice (mAY4) by grafting the complementarity-determining regions (CDRs) onto a fixed human framework, while preserving the so-called Vernier zone residues, a group of framework (FR) residues directly underneath the CDRs, with the murine residues in the humanized antibody, hAY4. The humanized hAY4 scFv maintained the antigen binding affinity and epitope specificity of mAY4. To investigate how the valence of hAY4 scFv affects DR4-mediated cell death, bivalent and trivalent forms of hAY4 scFv were generated by linking a hinge region to the coiled-coil domain of a dimerizing leucine zipper and trimerizing isoleucine zipper, respectively. Compared to the monovalent and bivalent forms, the trivalent hAY4 scFv induced more potent caspase-dependent apoptotic cell death as evidenced by increased activation of caspase-8 and downstream pro-apoptotic molecules. Our results suggest that like other TNF family receptors, avidity-mediated oligomerization of DR4 augments the receptor-mediated apoptotic cell death by promoting intracellular cell death signaling.

Generation and characterization of recombinant ScFv antibodies detecting Eimeria acervulina surface antigens.

In our previous attempt to generate monoclonal antibodies (MAbs) against coccidia parasites that more accurately reflect the natural avian humoral immune response, we produced two chicken B-cell hybridomas, 5D11 and 2-1. While both cell lines secreted antibodies reactive with sporozoites of Eimeria acervulina, they were produced in yields too low to conduct meaningful in vivo studies. To circumvent this problem, we produced four single chain variable fragment (scFv) antibodies from the V(H) and V(L) genes of hybridomas 5D11 and 2-1. The concentration of these recombinant antibodies expressed in E. coli and purified to homogeneity was 5-6 mg/L. Three of the antibodies exhibited antigen binding specificity to Eimeria surface antigens equivalent to that of the native MAbs. Nucleotide sequence analysis of the V(L) genes from hybridomas 5D11 and 2-1 and genomic DNA revealed vestiges of gene conversion with V(lambda) pseudogenes. These recombinant scFv antibodies will prove useful for further characterization of natural Eimeria surface antigens as potential vaccine candidates.

Formation of specialized aerial architectures by Rhodococcus during utilization of vaporized p-cresol.

When grown with vaporized alkylphenols such as p-cresol as the sole carbon and energy source, several isolated Rhodococcus strains formed growth structures like miniature mushrooms, termed here specialized aerial architectures (SAA), that reached sizes of up to 0.8 mm in height. Microscopic examination allowed us to view the distinct developmental stages during the formation of SAA from a selected strain, Rhodococcus sp. KL96. Initially, mounds consisting of long rod cells arose from a lawn of cells, and then highly branched structures were formed from the mounds. During the secondary stage of development, branching began after long rod cells grew outward and twisted longitudinally, serving as growth points, and the cells at the base of the mound became short rods that supported upward growth. Cells in the highly fluffy structures were eventually converted, via reductive division, into structures that resembled cocci, with a diameter of approximately 0.5 microm, that were arranged in chains. Most cells inside the SAA underwent a phase variation in order to form wrinkled colonies from cells that originally formed smooth colonies. Approximately 2 months was needed for complete development of the SAA, and viable cells were recovered from SAA that were incubated for more than a year. An extracellular polymeric matrix layer and lipid bodies appeared to play an important role in structural integrity and as a metabolic energy source, respectively. To our knowledge, similar formation of aerial structures for the purpose of substrate utilization has not been reported previously for Gram-positive bacteria.

Up-regulation of the expression of major histocompatibility complex class I antigens by plasmid DNA transfection in non-hematopoietic cells

The effect of DNA on the surface expression of major histocompatibility (MHC) class I antigens was examined in non‐hematopoietic tumor cell lines. Transfection with plasmid DNA via liposome or electroporation significantly increased the surface expression of MHC class I molecules in a transient manner. Northern blot analysis showed that levels of MHC class I mRNA were increased by DNA transfection, probably via transcriptional activation. In contrast, the expression of the MHC class II and β‐actin genes was not affected, suggesting that the up‐regulation of MHC class I expression by plasmid DNA works in a gene‐specific manner.

Characterization of recombinant scFv antibody reactive with an apical antigen of Eimeria acervulina

The chicken monoclonal antibody (mAb), 6D-12-G10, reacts with an apical complex protein at the anterior tip of E. acervulina sporozoites that inhibits parasite invasion in vitro. Because this mAb was produced at low amount from the original hybridoma cells, an scFv antibody was constructed by amplification of the corresponding VH and VL genes and expressed in E. coli. The scFv antibody was produced at a minimum of 7 mg l−1 and exhibited virtually identical antigen reactivity as the original mAb.

Microstructure and electrical properties of cosubstituted BiFeO3 thin films prepared by a chemical solution deposition

5 mol % La 3 + , Nd 3 + or Y 3 + -cosubstituted BiFe 0.97 Cr 0.03 O 3 thin films (denoted by BLaFCr, BNdFCr, and BYFCr, respectively) have been successfully deposited on Pt (200)/TiO 2 /SiO 2 /Si substrates by a chemical solution deposition method. Well-saturated ferroelectric hysteresis loops and low leakage current densities have been observed in BLaFCr, BNdFCr, and BYFCr thin films with single-phase structure. The values of remanent polarization (P r ) and coercive field (E c ) were 64 μ C/cm 2 and 275 kV/cm, 61 μC/cm 2 and 290 kV/cm, and 31 μ C/cm 2 and 315 kV/cm for BLaFCr, BNdFCr and BYFCr thin films at the maximum electric field of 712 kV/cm, respectively. In the present study, the differences in values of P r seem to be related to the doped ionic radii.