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Biomedical subjects

A Tsuboi

Publications and source records attributed to A Tsuboi.

At least 19 recordsLinked to original sources
PubMed2006

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway.

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(-)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(-)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.

Apoptosis↗
PubMed2006

Functional properties and regional differences of human masseter motor units related to three-dimensional bite force.

The aim of this study was to estimate numerically the properties of masseter motor units (MUs) in relation to bite force magnitude and direction three-dimensionally and to confirm the hypothesis that the properties differ between different parts of the muscle by means of simultaneous recording of MU activity along with the MU location and three-dimensional (3D) bite force. The MU activity of the right masseter of four healthy men was recorded using a monopolar needle electrode in combination with a surface reference electrode. The location of the needle electrode was estimated stereotactically with the aid of magnetic resonance images and a reference plate. The magnitude and direction of the bite force was recorded with a custom-made 3D bite force transducer. The recorded bite force was displayed on a signal processor, which enabled the participant to adjust the direction and magnitude of the force. The activities of 65 masseter MUs were recorded. Each MU had specific ranges of bite force magnitude and direction (firing range: FR) and an optimum direction for recruitment (minimum firing threshold: MFT). There was a significant negative correlation between MFT and FR width. There were functional differences in MU properties between the superficial and deep masseter and between the superficial layer and deep layer in the superficial masseter. These results indicate that the contribution of human masseter motor units to bite force production is heterogeneous within the muscle.

Action Potentials↗
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↗
PubMed2005

Th1-biased humoral immune responses against Wilms tumor gene WT1 product in the patients with hematopoietic malignancies.

The Wilms' tumor gene WT1 is highly expressed in leukemias and myelodysplastic syndrome (MDS), and WT1 expression levels increase along with the disease progression in chronic myeloid leukemia and MDS. We previously reported that IgM and IgG WT1 antibodies were detected with significantly higher detection rate and antibody titers in leukemias and MDS compared to those in healthy volunteers. In this study, whether IgG humoral immune responses against WT1 protein were Th1- or Th2-type were determined by measurement of four subclasses of IgG WT1 antibody, IgG1, IgG2, IgG3, and IgG4. In leukemias and MDS, Th1-type WT1 antibodies such as IgG1, IgG2, and IgG3 were significantly increased in both detection rate and antibody titers compared to those in healthy volunteers, whereas Th2-type WT1 antibody such as IgG4 did not increase. These results showed that Th1-biased humoral immune responses against WT1 protein were generated in leukemias and MDS. These results should allow us to consider that Th1-biased cellular immune responses against WT1 protein, which was essentially needed for cancer immunotherapy targeting WT1, should be elicited in patients with hematopoietic malignancies.

Antibody Formation↗
PubMed2004

Identification of a gene element essential for leukemia-specific expression of transgenes.

Leukemia-specific promoters and enhancers for gene therapy had never been reported. Since the Wilms' tumor gene WT1 is overexpressed in almost all types of leukemia, WT1 is an ideal target of leukemia-specific therapy. To explore the possibility of gene therapy for leukemia using WT1 promoter and enhancer, their activities in several kinds of cells were analyzed by using the enhanced green fluorescent protein (EGFP) gene as a reporter. First, we identified the best combination (654P/EGFP/int3- enh/3'-enh vector) of the 654-bp WT1 promoter and the two WT1 enhancers located in intron 3 and at the 3' end of the WT1 gene for inducing EGFP expression in K562 cells, which endogenously expressed WT1. When this was transfected into WT1-expressing leukemia cells (K562, HEL), WT1-nonexpressing hematopoietic cells (Daudi, U937), and WT1-expressing nonhematopoietic cells (TYK-nu-CPr, SW480, 293 T), 19.8, 22.9, 1.47, 1.43, 4.50, 4.16, and 1.09 times EGFP expression was induced, respectively, compared to that by the promoter-less EGFP vector. These results showed that the 654P/EGFP/int3-enh/3'-enh vector specifically induced high levels of EGFP expression in WT1-expressing leukemia cells. 654P/int3- enh/3'-enh vector containing transgenes such as suicide genes might become useful tools for leukemia-specific gene therapy.

Enhancer Elements, Genetic↗
PubMed2004

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.

ADP-ribosyl Cyclase↗
PubMed2004

Absence of mutations in the Wilms' tumor gene wt1 in de novo non-small cell lung cancers.

We recently demonstrated that the WT1 gene was overexpressed in the majority of de novo lung cancers regardless of cancer subtypes. Here, we examined WT1 genomic DNA in 38 cases of de novo non-small cell lung cancers (NSCLC) for mutations using direct sequencing. The sequencing analysis showed no mutations of WT1 genomic DNA in any of 38 de novo non-small cell lung cancers examined. These results indicated that the non-mutated, wild-type WT1 gene played an important role in de novo NSCLC.

Adult↗
PubMed2003

Successful treatment of bcr/abl-positive acute mixed lineage leukemia by unmanipulated bone marrow transplantation from an HLA-haploidentical (3-antigen-mismatched) cousin.

We describe a patient with bcr/abl-positive acute mixed lineage leukemia who successfully underwent transplantation in primary induction failure, using unmanipulated bone marrow from a human leukocyte antigen (HLA)-haploidentical cousin. The tumor burden was successfully reduced by the administration of imatinib mesylate (STI571) before transplantation. As graft-versus-host disease (GVHD) prophylaxis, a combination of tacrolimus and a short course of methotrexate, methylprednisolone, and mycophenolate mofetil was used. Hematopoietic reconstitution was rapid, and acute GVHD was limited to the skin (grade I). The patient is still in complete remission past day +400. This successful case suggests that HLA-haploidentical transplantation using unmanipulated marrow from a distantly related relative can be considered for patients in urgent situations who do not have HLA-identical donors.

Adult↗
PubMed2003

Neurons of the trigeminal main sensory nucleus participate in the generation of rhythmic motor patterns.

The trigeminal principal sensory nucleus (NVsnpr) contains both trigemino-thalamic neurons and interneurons projecting to the reticular formation and brainstem motor nuclei. Here we describe the inputs and patterns of firing of NVsnpr neurons during fictive mastication in anaesthetized and paralysed rabbits to determine the role that NVsnpr may play in patterning mastication. Of the 272 neurons recorded in NVsnpr, 107 changed their firing patterns during repetitive stimulation of the left or right sensorimotor cortex to induce fictive mastication. Thirty increased their firing tonically. Seventy-seven became rhythmically active, but only 31 fired in phase with mastication. The others discharged in bursts at more than twice the frequency of trigeminal motoneurons. Most rhythmic masticatory neurons were concentrated in the dorsal part, and those which fired during the jaw closing phase of the cycle were confined to the anterior pole of the nucleus. Most of these cells had inputs from muscle spindle afferents, whereas most of those firing during jaw opening had inputs from periodontal receptors. Non-masticatory rhythmical neurons had receptive fields on the lips and face. The majority of rhythmical masticatory units were modulated during fictive mastication evoked by both the left and right cortices and only four changed their phase of firing when switching from one cortex to the other. When coupled with the finding that NVsnpr neurons exhibit spontaneous bursting in vitro[Sandler et al. (1998) Neuroscience, 83, 891], the results described here suggest that neurons of dorsal NVsnpr may form the core of the central pattern generator for mastication.

Action Potentials↗
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↗
PubMed2001

Axonal projection of olfactory sensory neurons during the developmental and regeneration processes.

We have studied the projection of olfactory sensory neurons (OSNs), during the developmental and regeneration processes, using the transgenic mouse carrying the differently tagged odorant receptor genes, MOR28. We have found that the axon terminals of the two sets of MOR28-positive OSNs, one expressing the lacZ tag and the other expressing the green fluorescent protein gene, are dispersed and intermingled at early developmental or regeneration stages. Projection areas become more distinct and separated at later stages, however, two sets of axon fibers are not typically bundled or segregated during pathfinding. It appears that segregation of axons mainly occurs when they target at the olfactory bulb to form the glomerular structure.

Animals↗
PubMed2001

Monoallelic expression of the odourant receptor gene and axonal projection of olfactory sensory neurones.

BACKGROUND: We have previously generated transgenic mice carrying the murine odourant receptor gene, MOR28, tagged with lacZ. In this animal, the endogenous MOR28 is differently tagged with GFP. It was found that the transgenic and endogenous MOR28 genes are expressed in a mutually exclusive manner and that the two sets of olfactory sensory neurones (OSNs), each expressing either the transgenic or the endogenous MOR28, project their axons to separate glomeruli. RESULTS: Our fluorescent in situ hybridization (FISH) revealed that the two endogenous alleles of MOR28 are also mutually excluded for their transcriptional activation. Therefore, we studied whether there would be any segregation in the projection of the two subsets of OSNs: one set expressing the paternal and the other expressing the maternal allele. It was found that the OSNs for both alleles shared the same glomerulus for their projection, but the projection targets were segregated within the glomerular structure. CONCLUSION: Two subsets of neurones expressing either the transgenic or the endogenous MOR28 target their axons to two separate glomeruli based on the differences in the genetic backgrounds, nature of tagging, and chromosomal locations. In contrast, neurones expressing a maternal or paternal allele share the same glomeruli, but tend to target to segregated areas within the glomerular structure. The segregation was more prominent with increased differences in the genetic background between the two alleles.

Alleles↗
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

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

Mutually exclusive expression of odorant receptor transgenes.

To study the mutually exclusive expression of odorant receptor (OR) genes, we generated transgenic mice that carried the murine OR gene MOR28. Expression of the transgene and the endogenous MOR28 was distinguished by using two different markers, beta-galactosidase and green fluorescent protein (GFP), respectively. Double staining of the olfactory epithelium revealed that the two genes were rarely expressed simultaneously in individual olfactory neurons. A similar exclusion was also observed between differently tagged but identical transgenes integrated into the same locus of one particular chromosome. Although allelic inactivation has been reported for the choice between the maternal and paternal alleles, this is the first demonstration of mutually exclusive activation among non-allelic OR gene members with identical coding and regulatory sequences. Such an unusual mode of gene expression, monoallelic and mutually exclusive, has previously been shown only for the antigen-receptor genes of the immune system.

Animals↗
PubMed2000

The DNA-bending protein, HMG1, is required for correct cleavage of 23 bp recombination signal sequences by recombination activating gene proteins in vitro.

DNA-bending proteins are known to facilitate the in vitro V(D)J joining of antigen receptor genes. Here we report that the high-mobility group protein, HMG1, is necessary for the correct nicking of the 23 bp recombination signal sequence (23-RSS) by the recombination [corrected] activating gene (RAG) proteins, RAG1 and RAG2. Without HMG1, the mouse Jkappa1 23-RSS was recognized as if it were the 12-RSS and nicked at a site 12 + 7 nucleotides away from the 9mer signal, even though no 7mer-like sequence was evident at the cryptic nicking site. When increased amounts of HMG1 were added, the 23-RSS substrate was nicked correctly at a site 23 + 7 nucleotides from the 9mer, and nicking at the cryptic site disappeared. Unlike the 23-RSS, the 12-RSS did not require HMG1 for correct nicking, although HMG1 was found to increase the interaction between RSS and RAG proteins. Modification-interference assays demonstrated that HMG1 caused changes in the interaction between the 23-RSS and RAG proteins specifically at the 7mer and the cryptic nicking site.

Animals↗