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Biomedical subjects

K Udaka

Publications and source records attributed to K Udaka.

At least 19 recordsLinked to original sources
PubMed2006

Development of WT1 peptide cancer vaccine against hematopoietic malignancies and solid cancers.

Wild-type Wilms' tumor gene WT1 is highly expressed not only in hematopoietic malignancies, including leukemia and myelodysplastic syndromes (MDS), but also in various kinds of solid tumors. Human cytotoxic T lymphocytes (CTLs) which could specifically lyse WT1-expressing tumor cells with HLA class I restriction were generated in vitro. We have also demonstrated that mice immunized with the WT1 peptide or WT1 cDNA rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to normal organs which physiologically expressed WT1 in prophylactic and therapeutic models. Furthermore, we and others detected IgM and IgG WT1 antibodies in the patients with hematopoietic malignancies, indicating that WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing WT1-specific cellular immune responses were elicited in the patients. CD8+ WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of the findings mentioned above, we performed a phase I clinical trial of WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that WT1 peptide cancer vaccine had efficacy in the clinical setting, because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of TAA-derived cancer vaccine may be enhanced by combination with stronger adjuvants, helper peptide, or conventional treatments such as molecular-target-based drugs.

Animals↗
PubMed2002

WT1 as a novel target antigen for cancer immunotherapy.

Wild-type Wilms' tumor gene WT1 is expressed at high levels not only in most of acute myelocytic, acute lymphocytic, and chronic myelocytic leukemia, but also in various types of solid tumors including lung cancer. We tested the ability of the gene product (WT1) to serve as a target antigen for tumor specific immunotherapy both in human in vitro system and mouse in vivo system. In the latter, we can evaluate the efficacy and the side effects of WT1 vaccination in vivo. In the human in vitro system, two WT1 peptides that contain HLA-A2.1 binding anchor motifs were determined to bind to HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from an HLA-A2.1-psitive donor were repeatedly stimulated in vitro with TAP-deficient T2 cells pulsed with each of these two peptides, and CD8-positive cytotoxic T lymphocytes (CTLs) that specifically lyse WT1-expressing, HLA-A2.1-positive tumor cells were induced. Other groups also have succeeded in generating CTLs which specifically lyse WT1-expressing leukemia cells, and which do not inhibit colony-formation of normal hematopoietic cells that express WT1 at physiological levels. In the mouse in vivo system, immunization of C57BL/6 mice with one WT1 peptide with relatively high binding affinity for H-2D(b) molecules, which contain H-2D(b) binding anchor motifs, induced CTLs, which specifically lysed WT1-expressing tumor cells in an H-2D(b)-restricted manner. Furthermore, mice immunized with the WT1 peptide (peptide vaccination) or WT1 cDNA (DNA vaccination) rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to WT1-expressing normal organs by the induced CTLs. The WT1 protein has been identified as a novel tumor antigen and recent investigations provide a rationale for developing WT1-based adoptive T cell therapy and vaccination against various kinds of malignant neoplasms.

Animals↗
PubMed2000

NMR study on the interaction between MHC class I protein and its antigen peptide.

A major histcompatibility complex (MHC) class I protein H-2K(b) was expressed in a large scale as a fusion protein with thioredoxin and hexahistidine at the N-terminus to analyze the interaction with the antigen peptide SIYRYYGL. NMR spectra of the peptide in the mixture solution with the protein showed very broad signals, indicating the obviously clear existence of the dynamic interaction between the class I protein and the antigen peptide. The interaction of the protein and peptide was discussed as well as the surrounding atmosphere of the peptide in the complex.

Amino Acid Sequence↗
PubMed2000

Cancer immunotherapy targeting Wilms' tumor gene WT1 product.

The Wilms' tumor gene WT1 is expressed at high levels not only in acute myelocytic and lymphocytic leukemia and in chronic myelocytic leukemia but also in various types of solid tumors including lung cancers. To determine whether the WT1 protein can serve as a target Ag for tumor-specific immunity, three 9-mer WT1 peptides (Db126, Db221, and Db235), which contain H-2Db-binding anchor motifs and have a comparatively higher binding affinity for H-2Db molecules, were tested in mice (C57BL/6, H-2Db) for in vivo induction of CTLs directed against these WT1 peptides. Only one peptide, Db126, with the highest binding affinity for H-2Db molecules induced vigorous CTL responses. The CTLs specifically lysed not only Db126-pulsed target cells dependently upon Db126 concentrations but also WT1-expressing tumor cells in an H-2Db-restricted manner. The sensitizing activity to the Db126-specific CTLs was recovered from the cell extract of WT1-expressing tumor cells targeted by the CTLs in the same retention time as that needed for the synthetic Db126 peptide in RP-HPLC, indicating that the Db126-specific CTLs recognize the Db126 peptide to kill WT1-expressing target cells. Furthermore, mice immunized with the Db126 peptide rejected challenges by WT1-expressing tumor cells and survived for a long time with no signs of autoaggression by the CTLs. Thus, the WT1 protein was identified as a novel tumor Ag. Immunotherapy targeting the WT1 protein should find clinical application for various types of human cancers.

Animals↗
PubMed2000

An automated prediction of MHC class I-binding peptides based on positional scanning with peptide libraries.

Specificities of three mouse major histocompatibility complex (MHC) class I molecules, Kb, Db, and Ld, were analyzed by positional scanning using combinatorial peptide libraries. The result of the analysis was used to create a scoring program to predict MHC-binding peptides in proteins. The capacity of the scoring was then challenged with a number of peptides by comparing the prediction with the experimental binding. The score and the experimental binding exhibited a linear correlation but with substantial deviations of data points. Statistically, for approximately 80% of randomly chosen peptides, MHC-binding capacity could be predicted within one log concentration of peptides for a half-maximal binding. Known cytotoxic T-lymphocyte epitope peptides could be predicted, with a few exceptions. In addition, frequent findings of MHC-binding peptides with incomplete or no anchor amino acid(s) suggested a substantial bias introduced by natural antigen processing in peptide selection by MHC class I molecules.

Animals↗
PubMed2000

Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms' tumor gene (WT1 ) product.

The product of the Wilms' tumor gene WT1 is a transcription factor overexpressed not only in leukemic blast cells of almost all patients with acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia, but also in various types of solid tumor cells. Thus, it is suggested that the WT1 gene plays an important role in both leukemogenesis and tumorigenesis. Here we tested the potential of WT1 to serve as a target for immunotherapy against leukemia and solid tumors. Four 9-mer WT1 peptides that contain HLA-A2.1-binding anchor motifs were synthesized. Two of them, Db126 and WH187, were determined to bind to HLA-A2.1 molecules in a binding assay using transporter associated with antigen processing-deficient T2 cells. Peripheral blood mononuclear cells from an HLA-A2.1-positive healthy donor were repeatedly sensitized in vitro with T2 cells pulsed with each of these two WT1 peptides, and CD8(+) cytotoxic T lymphocytes (CTLs) that specifically lyse WT1 peptide-pulsed T2 cells in an HLA-A2.1-restricted fashion were induced. The CTLs also exerted specific lysis against WT1-expressing, HLA-A2.1-positive leukemia cells, but not against WT1-expressing, HLA-A2.1-negative leukemia cells, or WT1-nonexpressing, HLA-A2. 1-positive B-lymphoblastoid cells. These data provide the first evidence of human CTL responses specific for the WT1 peptides, and provide a rationale for developing WT1 peptide-based adoptive T-cell therapy and vaccination against leukemia and solid tumors.

CD3 Complex↗
PubMed2000

Cytotoxic T-lymphocyte responses elicited to Wilms' tumor gene WT1 product by DNA vaccination.

We recently have reported that Wilms' tumor gene WT1 is highly expressed not only in leukemias but also in various types of solid tumors and that WT1 protein is a novel tumor antigen against which cytotoxic T lymphocytes (CTLs) can be elicited by immunization with 9-mer WT1 peptides capable of binding to major histocompatibility complex (MHC) class I molecules. In the present study, plasmid DNA encoding murine full-length WT1 protein was injected intramuscularly into C57BL/6 mice. The mice vaccinated with the WT1 plasmid DNA elicited CTLs against the WT1 protein, and the CTLs specifically killed WT1-expressing tumor cells in a MHC class I-restricted manner. Furthermore, the vaccinated mice rejected the challenges of WT1-expressing tumor cells and survived with no signs of autoimmunity caused by the CTLs. These results demonstrated that vaccination with the WT1 plasmid DNA can elicit CTL responses specific for the WT1 protein, resulting in the acquisition of rejection activity against challenges of WT1-expressing tumor cells. This WT1 DNA vaccination may find clinical application for various types of solid tumors as well as leukemias.

Animals↗
PubMed2000

Purification, molecular cloning and ethylene-inducible expression of a soluble-type epoxide hydrolase from soybean (Glycine max [L.] Merr.).

A soybean protein was purified from mature dry seeds. Amino-acid sequencing of the nine internal peptides derived from this N-terminally blocked protein showed that it has a significant similarity to the soluble epoxide hydrolases known to date. A degenerate series of 23-mer oligonucleotides with sequences corresponding to an internal region of eight amino-acid residues was synthesized as a probe mixture for detection of a putative epoxide hydrolase cDNA in a developing cotyledon cDNA library. The 1332-bp cDNA obtained was found to have an open-reading frame encoding the seed epoxide hydrolase-like precursor consisting of 341 amino-acid residues, suggesting that 25 amino-acid residues upstream from the second methionine correspond to a transit peptide. Employing an Escherichia coli expression system, the putative mature epoxide hydrolase-like protein was overexpressed and purified to homogeneity. This recombinant protein was confirmed to exhibit its epoxide-diol converting activity using styrene oxide as substrate. The Vmax and Km values for styrene oxide are 1.36 micromol x min-1 x mg-1 and 1500 microM, respectively. Sedimentation equilibrium experiments showed that the active form of this epoxide hydrolase is monomeric in solution. Using the above cDNA as a probe, a 12-kb genomic clone was selected and the sequence of a 1933-bp fragment from this clone was found to cover the entire coding region together with 5'- and 3'-flanking regions of the soybean epoxide hydrolase gene. The coding region of the gene, interrupted by two short introns, was identical to the corresponding regions of the cDNA. Northern blot analyses showed that this epoxide hydrolase gene was expressed strongly at a very early stage (13 days after flowering) and then the level of expression gradually decreased and almost ceased at a very late stage (58 days after flowering) of seed development, whereas its expression was markedly up-regulated by ethylene treatment. In stems (hypocotyl portion), the epoxide hydrolase transcript was detected at significant levels and was also up-regulated in response to ethylene. On the other hand, it is hardly expressed in leaves, even though they were treated with the phytohormone. Overall, the results obtained may indicate that soluble-type epoxide hydrolase mRNA is expressed at the maximum level in an early stage of seed development. Later, oil bodies are formed and subsequently epoxy fatty acids, naturally occurring metabolites, accumulate within those bodies. The temporal induction of this epoxide hydrolase transcript in some tissues in response to ethylene also indicates that this epoxide hydrolase may play a crucial role in self-defense systems of plant.

Amino Acid Sequence↗
PubMed2000

Efficient induction of peptide-specific cytotoxic T lymphocytes by LPS-activated spleen cells.

Lipopolysaccharides of gram-negative bacteria are potent activators of B cells, dendritic cells and monocytes/macrophages. We have investigated the use of LPS-activated spleen cells as antigen-presenting cells to induce CD8+ cytotoxic T lymphocytes in vivo that are reactive to MHC class I binding peptides. Compared with resting spleen cells, CTL induction was more efficient and less variable for different peptides with LPS-activated spleen cells. Cytotoxic responses were specific for the immunized peptides and contained high affinity CD8+ T cells. The removal of dendritic cells and monocytes/macrophages by Sephadex G10 column did not show profound effects on CTL induction, indicating that B-cell blasts were largely responsible. This easily accessible method should facilitate the screening of MHC class I binding peptides to determine whether or not the host's T-cell repertoire contains reactive T cells.

Animals↗
PubMed1999

Molecular cloning and ethylene-inducible expression of Chib1 chitinase from soybean (Glycine max (L.) Merr.).

A soybean seed-specific PR-8 chitinase, named Chib2, has a markedly extended C-terminal segment compared to other plant Chib1 homologues of the PR-8 chitinase family known to date. To further characterize the molecular structure and the expression pattern of this chitinase family, we cloned two typical Chib1-similar cDNAs (Chib1-1 and Chib1-2) from soybeans by PCR-cloning techniques. The deduced primary sequence of Chib1-1 chitinase is composed of a signal peptide segment (26 amino acid residues) and a mature 273 amino acid sequence (calculated molecular mass 28,794, calculated pI 3.7). This Chib1-1 enzyme is more than 90% identical to Chib1-2 chitinase but is below 50% identical to Chib2 enzyme. Thus, we confirmed the occurrence of two distinct classes, Chib1 and Chib2 in the plant PR-8 chitinase family. The Chib1 genes, interrupted by one intron, were found to be up-regulated in response to ethylene in stems and leaves, but scarcely expressed in developing soybean seeds. Chib1 chitinases may be responsible for protecting the plant body from various pathogenic attacks.

Amino Acid Sequence↗
PubMed1998

Dystrophin acts as a transplantation rejection antigen in dystrophin-deficient mice: implication for gene therapy.

Duchenne muscular dystrophy is a lethal and common X-linked recessive disease caused by a defect in dystrophin. Normal myoblast transplantation and dystrophin gene transfer have been expected to correct the deficiency in the muscles, but their clinical application has been hampered by the limited preservation of dystrophin-positive myofibers. In this study we investigated the mechanism for immunologic rejection of normal C57BL/10 (B10) myoblasts transplanted into dystrophin-deficient mdx mice, an animal model of Duchenne muscular dystrophy. We found that mdx mice develop CTL specific for dystrophin itself, which were CD8 dominant and restricted by H-2Kb. We identified several antigenic peptides derived from dystrophin that bind to H-2Kb and are recognized by the mdx anti-B10 CTL. Immunologic tolerance against dystrophin was successfully induced by i.v. injection of these peptides before B10 myoblast transplantation, which resulted in sustained preservation of dystrophin-expressing myofibers in mdx mice. These results demonstrate that dystrophin is antigenic in dystrophin-deficient mice and that immunologic regimen would be necessary to achieve the persistent expression of introduced dystrophin in the muscles of dystrophin-deficient individuals.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and expression patterns of Cu/Zn-superoxide dismutases in developing soybean seeds.

Assuming that the amount of superoxide radicals generated in vivo correlates with the production of ergastic substances such as storage proteins, the coordinated response of detoxication enzymes such as superoxide dismutases is largely exploited to understand the self-defense systems of plant. Here we examined expression of the genes for superoxide dismutases during seed development of soybean. The cDNAs encoding a cytosolic copper/zinc form and an iron form of the above enzyme have been cloned and then employed as probes, separately. Northern blotting results suggested that both superoxide dismutase mRNAs are expressed at the maximum level, preceding a developmental stage when mRNA encoding glycinin, soybean 11S-storage protein, at the maximum.

Amino Acid Sequence↗
PubMed1997

Impaired induction of cytotoxic T lymphocytes by antagonism of a weak agonist borne by a variant hepatitis C virus epitope.

An epitope that acted as a weak agonist in the cytotoxicity assay was identified as part of the capsid protein of a hepatitis C virus (HCV) variant. In a low concentration, the variant epitope also had a weak antagonistic effect. When a minute amount of this variant epitope was added to the culture for induction, it selectively attenuated the expansion of major cytotoxic T cell populations and drastically reduced the cytotoxic responses against the wild-type epitope. Thus, antagonism to induction suppressed immune responses against both the wild type and the variant, thereby helping the persistence of not only variant itself but also the wild-type HCV. Because this variant was a weak agonist, most cytotoxic T cells induced with the wild-type epitope were cross-reactive with the variant and susceptible to the antagonism to induction. Only the T cells which were not cross-reactive with the variant and not susceptible to the antagonism survived the antagonism in induction. This implied that the specificity of the remaining immune response, if any, was directed exclusively to the wild-type epitope after the emergence of the variant. For viruses like HCV, being heterogeneous itself may contribute significantly toward persistent infection through antagonism to induction.

Antigenic Variation↗
PubMed1996

Self-MHC-restricted peptides recognized by an alloreactive T lymphocyte clone.

Alloreactive T lymphocytes are readily detected in unprimed animals although they have never encountered the alloantigen before. This well-established phenomenon is usually explained with the assumption that a self-MHC molecule complexed with a defined peptide resembles the allo-MHC molecule with another peptide and induces the corresponding T cell specificities. Here, for the first time and in support of this hypothesis, self-MHC-restricted peptides are described for a T cell clone that was induced with allo-MHC. The allo-MHC-specific CTL clone 2C was derived from a H-2b mouse and recognizes H-2Ld complexed with the naturally occurring endogenous peptide LSPFPFDL. H-2Kb was shown to be involved in positive selection of its TCR, and peptides associated with this MHC molecule are implicated in the process. To identify such peptides, positional scanning with random peptide libraries combined with an iterative approach was employed. Several active peptides were found and the most efficient, SIYRYYGL, was chosen for further studies. Recognition by 2C of the two MHC-peptide adducts H-2Ld + LSPFPFDL and H-2Kb + SIYRYYGL is mediated by the same TCR and appears to be similarly efficient as concluded from inhibition experiments with an Id-specific Ab. CTLs from SIYRYYGL-primed H-2b mice respond to H-2Ld + LSPFPFDL. This reciprocal cross-reactivity suggests that structural features are shared by the two MHC-peptide complexes.

Amino Acid Sequence↗
PubMed1996

A rapid purification method for soybean Bowman-Birk protease inhibitor using hydrophobic-interaction chromatography.

A protein fraction was isolated from defatted soybean flour by extraction at acid pH, 40% ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration chromatography. SDS-PAGE, under reducing conditions, confirmed it as a homogeneous preparation. This conclusion was consistent with N-terminal amino acid sequence data (20 cycles) which showed a major sequence identical to those reported for soybean Bowman-Birk-type protease inhibitor (BBI), and also indicated a minimum 95% purity based on recoveries of PTH-amino acid residues. The purified fraction inhibited both trypsin and chymotrypsin with average specific activities of 350 and 672 units mg(-1), respectively. Compared with classical BBI purification, this procedure is very rapid requiring only 72-96 h to achieve a yield of 37 mg purified BBI per 200 g starting material.

Amino Acid Sequence↗
PubMed1996

Decrypting class I MHC-bound peptides with peptide libraries.

Multiple peptides have been used as molecular tools to probe the selective, yet degenerative, recognition of peptides by class I major histocompatibility complex (MHC) molecules and T-cell receptors. The additive contributions from individual amino acids that have been identified experimentally can often be used to predict the binding of any peptide to class I MHC molecules, although other contributing factors will often result in this being an oversimplification.

Amino Acid Sequence↗
PubMed1995

Tolerance to amino acid variations in peptides binding to the major histocompatibility complex class I protein H-2Kb.

Major histocompatibility complex (MHC) class I molecules are cell-surface glycoproteins that bind peptides and present them to T cells. The formation of a peptide-MHC complex is the initial step in specific, T cell-mediated immune responses. But, unlike other receptor-ligand systems, peptides are essential for a stable conformation of the MHC proteins. To investigate the contribution of every amino acid of octapeptides to the stability and antigenic integrity of MHC proteins, complex octapeptide libraries with one defined amino acid and mixtures of 19 amino acids in the remaining seven positions were synthesized and tested for their capacity to stabilize the conformation of the mouse MHC class I molecule H-2Kb. Peptide transporter-deficient RMA-S cells were employed in this study. Amino acid preferences found for the eight sequence positions reveal constitutional, volumetric, and steric constraints that govern peptide selection by MHC molecules. The pattern of amino acid preferences indicates that the peptides behave as integral parts of the MHC proteins and follow rules established for the interrelationship of primary sequence and the conformation and stability of proteins in general.

Amino Acid Sequence↗