Search PubMed⌕ Search

Biomedical subjects

Y Tani

Publications and source records attributed to Y Tani.

At least 19 recordsLinked to original sources
PubMed2007

Cotransduction of CCL27 gene can improve the efficacy and safety of IL-12 gene therapy for cancer.

Interleukin-12 (IL-12) is a potent antitumoral cytokine, but high doses are toxic. Herein, we demonstrate that combinational transduction of IL-12 and CC-chemokine ligand-27 (CCL27) genes into pre-existing murine OV-HM ovarian carcinoma and Meth-A fibrosarcoma, by using RGD fiber-mutant adenoviral vectors, could induce tumor regression and relieve systemic side effects more effectively than either treatment alone. The antitumor activity of the IL-12 and CCL27 combination treatment was T-cell-dependent, and development of long-term specific immunity was confirmed in rechallenge experiments. Immunohistochemical analysis of tumors transduced with CCL27 gene alone or cotransduced with IL-12 and CCL27 genes showed significant increases in numbers of infiltrating CD3(+) T cells, which included both CD4(+) and CD8(+) cells. Additionally, cotransduction with IL-12 and CCL27 genes could more efficiently activate tumor-infiltrating immune cells than transduction with CCL27 alone, as determined by the frequency of perforin-positive cells and expression levels of IFN-gamma. Furthermore, mice treated with the IL-12 and CCL27 combination compared with those treated with IL-12 alone showed milder pathological changes, for example, lymphocyte infiltration and extramedullary hematopoiesis, in lung, liver and spleen. Our data provide evidence that combinational in vivo transduction with IL-12 and CCL27 genes is a promising approach for the development of cancer immunogene therapy that can simultaneously recruit and activate tumor-infiltrating immune cells.

Adenocarcinoma↗
PubMed2006

Tumor suppressive efficacy through augmentation of tumor-infiltrating immune cells by intratumoral injection of chemokine-expressing adenoviral vector.

Our goal in the present study was to evaluate antitumor effects and frequency of tumor-infiltrating immune cells upon intratumoral injection of RGD fiber-mutant adenoviral vector (AdRGD) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1. Among eight kinds of chemokine-expressing AdRGDs, AdRGD-CCL19 injection most efficiently induced infiltration of T cells into established B16BL6 tumor parenchyma, whereas most of these T cells were perforin-negative in immunohistochemical analysis. Additionally, the growth of AdRGD-CCL19-injected tumors decreased only slightly as well as that of other tumors treated with each chemokine-expressing AdRGD, which indicated that accumulation of naive T cells in tumor tissue does not effectively damage the tumor cells. Tumor-bearing mice, in which B16BL6-specific T cells were elicited by dendritic cell-based immunization, demonstrated that intratumoral injection of AdRGD-CCL17, -CCL22, or -CCL27 could considerably suppress tumor growth and attract activated T cells. On the other hand, AdRGD-CCL19-injection in the immunized mice showed slight increase of tumor-infiltrating T cells compared to treatment using control vector. Collectively, although AdRGD-mediated chemokine gene transduction into established tumors would be very useful for augmentation of tumor-infiltrating immune cells, a combinational treatment that can systemically induce tumor-specific effector T cells is necessary for satisfactory antitumor efficacy.

Adenoviridae↗
PubMed2006

Brain site-specific gene expression analysis in Alzheimer's disease patients.

BACKGROUND: Alzheimer's disease (AD) is an age-related neurodegenerative disorder that is characterized by a progressive loss of higher cognitive functions. The brain of an individual with AD exhibits extracellular senile plaques (SPs) of aggregated amyloid-beta peptide (Abeta) and intracellular neurofibrillary tangles (NFTs). Given the critical role of neuronal transport of both proteins and organelles, it is not surprising that perturbation of microtubule-based transport may play a major role in the pathogenesis of AD. MATERIALS AND METHODS: We used the cDNA subtraction methodology and in vitro neural cell culture analyses to study the meaning of the brain site-specific gene expression pattern in cerebral tissue obtained from AD patients and also from control subjects at autopsy. RESULTS: We observed that cytoskeleton-associated proteins were down-regulated in AD subjects. We also noted an altered expression of the microtubule-associated protein 1B (MAP1B), the heat-shock protein (HSP)-90 (a key chaperone molecule), the tripartite motif-containing proteins (TRIM)-32/37 (an anti apoptotic enzyme with ubiquitin-protein ligase activity) and the Reticulon-3 (a modulator of the amyloid-precursor-protein (APP) cleavage) in AD brains. Additional molecular- and cell-biological studies revealed that small interfering RNA (siRNA)-mediated down-regulation of MAP1B expression leads to neuronal cell death in vitro. CONCLUSION: Altered expression of MAP1B, HSP90, TRIM32/37 and Reticulon-3 provides new clues by which the ubiquitin-proteasome-, the protein-chaperon- and the APP-processing systems are disturbed in AD, thus, leading to neuritic amyloid plaques and neurofibrillary tangles.

Aged↗
PubMed2006

Simultaneous five cell-lineage flow cytometric analysis system for detection of leucocyte antibodies.

Although flow cytometric (FCM) analysis is one of the most widely used approaches to screen the presence of leucocyte antibodies, it has several drawbacks. First, neutrophils and, especially, monocytes exhibit high background reactivity. Second, to determine antibody specificity, it is often necessary to examine not only neutrophils and monocytes but also other lineage cells including T cells, B cells and platelets. Therefore, we attempted to establish an FCM analysis system in which four lineages of leucocytes and platelets are simultaneously tested with low background. FCM analysis was performed using ethylene diamine tetraacetic acid-anticoagulated whole blood as cell sample without any cell preparation. Discrimination of five cell lineages was carried out based on the differences in forward vs. side scatter distribution and in the expression of CD4, CD20 and CD14. When anti-HNA (human neutrophil antigen) 1b antiserum was applied to HNA 1b-positive blood samples, only neutrophils were unambiguously positive. When anti-Naka (anti-CD36) antiserum was applied, only platelets and monocytes were positive. The background reactivity of neutrophils and monocytes was low enough. When anti-human leucocyte antigen (HLA) class II antiserum was tested, only B-lymphocytes and monocytes were positive. When anti-HLA class I antiserum was tested, all the five-lineage cells were positive.

Antibodies↗
PubMed2006

A single base insertion of the 4-alpha-galactosyltransferase gene led to the deficiency of Gb3 biosynthesis.

cDNAs for alpha 1,4 galactosyltransferase (A4GALT) have been isolated. To explore the molecular basis of the p phenotype in Japanese donors, we analyzed the A4GALT gene sequences of normal and p phenotype samples. The coding region in the A4GALT gene for DNA sequencing was amplified by PCR amplification. A4GALT expression vectors for individual were constructed by PCR amplification of the coding region using primers and subsequent subcloning into an expression vector. The expression of Gb3/CD77 antigen on the cell surface was evaluated by flow cytometry and by immunochemical techniques. All individuals with the p phenotype were found to have a single base insertion (A4GALT/insC) at the same nucleotide position. Neither the transfectant cells with a mutant gene (A4GALT/insC) of donor origin or those with a synthesized mutant gene (A4GALT/insC-Mu) expressed Gb3 antigen indicating that the presence of A4GALT/insC diminished the A4GALT enzyme activity. In addition, an allele-specific PCR (ASP) system was developed in which of the p phenotype with A4GALT/insC can be unambiguously discriminated from normal donors. Based on the finding that a single base insertion (A4GALT/insC) diminishes A4GALT activity, an ASP assay was developed to detect individuals with this particular p phenotype.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2005

Double heterozygosity for a novel missense mutation of Ile304 to Asn in addition to the missense mutation His280 to Pro in the integrin beta3 gene as a cause of the absence of platelet alphaIIbbeta3 in Glanzmann's thrombasthenia.

BACKGROUND: Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder characterized by a defect in the expression or the function of alphaIIbbeta3. OBJECTIVES: The purpose of the present study was to identify genetic defects in a GT patient. METHODS: The expression of alphaIIbbeta3 was determined by flow cytometric analysis and Western blotting. We analyzed the cDNA sequences of both alphaIIb and beta3, and performed transfection experiments using COS7 cells to confirm that a specific mutation was responsible for the GT case. RESULTS: Flow cytometric analysis and Western blotting showed remarkably reduced expression of alphaIIbbeta3. Sequence analysis of the patient's cDNA indicated a new missense mutation that led to the amino acid substitution of Ile304 (ATC) with Asn (AAC) in exon 6 of the beta3 gene. This was in addition to the missense mutation of His280 (CAT) to Pro (CCT) in exon 5, which had been previously reported. The missense mutation of Ile304 (ATC) to Asn (AAC) in beta3 was found to be responsible for this GT case. This was because transfection experiments using COS7 cells indicated that alphaIIbbeta3 possessing Asn304 in beta3 was not expressed on the surface of the transfected cells. In addition, immunoprecipitation analysis demonstrated that alphaIIbbeta3 was absent inside the transfected COS7 cells possessing Asn304 in beta(3). CONCLUSION: In this study, we describe a new missense mutation (ATC to AAC) at position 1009 in exon 6 that leads to an amino acid substitution (Ile304 to Asn) in beta3, which is responsible for this GT case.

Adult↗
PubMed2005

Insertion of a C in the exon 28 of integrin alphaIIb gene leading to a frameshift mutation is responsible for Glanzmann thrombasthenia in a Japanese case.

BACKGROUND: Glanzmann thrombasthenia (GT) is a hereditary bleeding disorder caused by a qualitative or quantitative defect in the integrin alphaIIbbeta3. OBJECTIVE: Our objective is to identify the gene mutation that resulted in GT. PATIENTS AND METHODS: The patient was a 66-year-old male with a history of frequent bleeding. The expression levels of the integrin proteins in the platelets were determined by flow cytometry and Western blot analysis. The sequences of genomic DNA and mRNA encoding for alphaIIb and beta3 were analyzed by the dye-terminator cycle sequencing method. For transfection experiments, expression vectors encoding for wild-type alphaIIb, mutated alphaIIb, beta3, green fluorescent protein (GFP) fusion wild-type alphaIIb, GFP fusion mutated alphaIIb and DsRed fusion beta3 were constructed. These vectors were transfected to COS-7 cells, and the expression levels were determined. RESULTS: The alphaIIb protein was remarkably reduced in the patient's platelets, and gene analysis showed that the patient possessed compound heterozygous mutations in the alphaIIb gene. One was a C --> G substitution at the splice acceptor site (- 3) of exon 26 (CAG -->GAG) and the other was the insertion of an additional C at the region including six C bases between 2911 and 2916 in exon 28 (InsC). Transfection experiments using COS-7 cells showed that alphaIIb containing InsC had expressed and formed a complex with beta3, but had not been transported to the Golgi apparatus. CONCLUSIONS: In the present study the novel mutation InsC, leading to a frameshift that affects the transmembrane domain and the cytoplasmic tail, was found to be responsible for GT.

Aged↗
PubMed2005

Epithelial-stromal tumor of the seminal vesicles in the transgenic adenocarcinoma mouse prostate model.

The transgenic adenocarcinoma mouse prostate (TRAMP) model, designed for researching human prostatic cancer, was genetically engineered to harbor a transgene composed of the simian virus 40 Large-T/small-t antigen promoted by the rat probasin gene. In addition to prostatic neoplasms, the TRAMP mouse develops tumors in the seminal vesicles. This study was conducted to evaluate the pathology and histogenesis of TRAMP seminal vesicle neoplasms. Tissues of accessory sex organs harvested from 72 TRAMP mice of various ages (11-40 weeks of age) were fixed in neutral buffered formalin and stained with hematoxylin and eosin, desmin, 5-bromo-2'-deoxyuridine (BrdU, treated animals only), and SV40 Large-T antigen (SV40-Tag). In the seminal vesicles, we found neoplastic stromal cells that emerged multicentrically just beneath the epithelium, densely packed between the epithelium and the smooth muscle layer. These stromal cells frequently exhibited mitotic figures and showed BrdU incorporation and SV40-Tag protein expression in the nuclei and immunopositivity for desmin. The proliferative mesenchymal cells were lined by cuboidal to columnar epithelium. Some of the larger papillary, polypoid lesions exhibited a phyllodes pattern resembling that seen in mixed epithelial-stromal tumors of the breast, prostate, and seminal vesicles of humans. Although the epithelium was negative for SV40-Tag and showed only occasional incorporation of BrdU, it clearly participated in the biphasic proliferation, forming papillary, cystic, and tubuloglandular structures. No conclusive evidence of malignancy (invasion or metastasis) was identified. Our recommended diagnosis of this lesion in the seminal vesicles is epithelial-stromal tumor.

Animals↗
PubMed2004

Antibacterial and antipeptide antibodies in Japanese and Finnish patients with rheumatoid arthritis.

It has been suggested that Proteus infection may be involved in the pathogenesis of rheumatoid arthritis (RA). Bacterial and peptide immune responses in patients with RA and other control subjects were investigated in two geographically different populations. Serum samples from Finnish patients with early ( n=72) and advanced ( n=27) RA and 30 Finnish healthy controls, as well as from Japanese RA patients from two different locations: Tokyo ( n=30) and Otsu ( n=30), 18 patients with systemic lupus erythematosus (SLE) and 23 Japanese healthy controls were all screened for the total, and class-specific (IgG, IgA and IgM) antibodies against Proteus mirabilis, Escherichia coli and Serratia marcescens by indirect immunofluorescence assay. These samples were also tested for the determination of levels of isotypic antibodies against the shared epitope involving 16-mer synthetic peptides containing the EQRRAA or ESSRAL sequences and compared to scrambled control peptide by using an enzyme-labeled immunosorbent assay method. Significantly elevated levels of IgG and IgM antibodies to P. mirabilis and antibodies against both EQRRAA and ESSRAL peptides were detected in sera of Finnish patients with early and advanced RA, and in Japanese patients from Otsu or Tokyo compared to their corresponding control groups. In contrast, no difference either in the total or in any of the isotypic antibodies were observed between these groups when serum samples were screened against each of E. coli and S. marcescens or against the control peptide. Furthermore, there was a significant correlation between the antibody levels against Proteus bacteria only and both EQRRAA and ESRRAL peptides. Our findings support the possibility for specific involvement of P. mirabilis in the etiopathogenesis of RA even in early cases.

Antibodies, Anti-Idiotypic↗
PubMed2003

Glanzmann's thrombasthenia due to a point mutation within intron 10 results in aberrant splicing of the beta3 gene.

Glanzmann's thrombasthenia (GT) is an hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. We attempted to identify genetic defects responsible for a case of GT in Korea. The patient was a 6-year-old boy who had suffered from hemorrhage and purpura. The cDNAs of alphaIIb and beta3 were amplified by reverse transcription (RT)-PCR and were sequenced. RT-PCR of COS7 cells transfected with Exontrap vectors containing the beta3 genomic DNA fragments were performed to verify an aberrant splicing. Transient expression of alphaIIbbeta3 on transfected-COS7 cells was determined by flow cytometry, and the presence of alphaIIbbeta3 was confirmed by immunoprecipitation. We discovered an abnormality with the insertion of 38 bp between exon 10 and exon 11, including a stop codon, in beta3 cDNA. Sequence analysis of genomic DNA showed a point mutation, G-->T, at the position 29 107 of intron 10. RT-PCR analyses of COS7 cells transfected with Exontrap vector containing the mutant beta3 gene revealed that the point mutation at the position 29 107 of intron 10, G-->T, was responsible for an aberrant splicing in the beta3 gene. The transfection experiments and immunoprecipitation revealed the absence of alphaIIbbeta3 in the COS7 cells transfected with mutant gene. The mutation at the position 29 107 of intron 10, G-->T, in the beta3 genomic DNA was found to induce an aberrant splicing and to be responsible for GT in this patient.

Alternative Splicing↗
PubMed2002

Section 1B: Rh flow cytometry. Coordinator's report. Rhesus index and antigen density: an analysis of the reproducibility of flow cytometric determination.

Fifty-seven IgG monoclonal anti-D antibodies were evaluated in the Rh flow cytometry section, in which 12 laboratories participated. Staining protocols and a fluorescein (FITC)-conjugated Fab fragment goat anti-human IgG (H + L) as a secondary antibody were recommended but not mandatory. A CcDEe red blood cell (RBC) sample that was shown to be homozygous for RHD by molecular methods was supplied and used as internal 'standard RBC' throughout all experiments. An RBC panel comprising two partial D and four weak D types was supplied as well. The use of standard RBC reduced the variability of the data among the laboratories and allowed the conversion of fluorescence data into epitope densities, which were compounded in an antigen density (antigen D per RBC). The highest antigen density was determined for DVI type III, followed by DVII and weak D type 3; the lowest antigen density were determined for weak D type 1 and type 2. Nine of the 12 participating laboratories discriminated three groups of aberrant RhD that had similar Rhesus indices (RI): D category VI with RI = 0; weak D type 2 and type 3 with an high RI; and D category VII and weak D type 1 with an intermediate RI. The antigen densities and the Rhesus indices obtained correlated well among the laboratories of this Workshop section despite different staining protocols, secondary antibodies and instrumentation.

Algorithms↗
PubMed2001

Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines.

System L is a major nutrient transport system responsible for the transport of large neutral amino acids including several essential amino acids. We previously identified a transporter (L-type amino acid transporter 1: LAT1) subserving system L in C6 rat glioma cells and demonstrated that LAT1 requires 4F2 heavy chain (4F2hc) for its functional expression. Since its oncofetal expression was suggested in the rat liver, it has been proposed that LAT1 plays a critical role in cell growth and proliferation. In the present study, we have examined the function of human LAT1 (hLAT1) and its expression in human tissues and tumor cell lines. When expressed in Xenopus oocytes with human 4F2hc (h4F2hc), hLAT1 transports large neutral amino acids with high affinity (K(m)= approximately 15- approximately 50 microM) and L-glutamine and L-asparagine with low affinity (K(m)= approximately 1.5- approximately 2 mM). hLAT1 also transports D-amino acids such as D-leucine and D-phenylalanine. In addition, we show that hLAT1 accepts an amino acid-related anti-cancer agent melphalan. When loaded intracellularly, L-leucine and L-glutamine but not L-alanine are effluxed by extracellular substrates, confirming that hLAT1 mediates an amino acid exchange. hLAT1 mRNA is highly expressed in the human fetal liver, bone marrow, placenta, testis and brain. We have found that, while all the tumor cell lines examined express hLAT1 messages, the expression of h4F2hc is varied particularly in leukemia cell lines. In Western blot analysis, hLAT1 and h4F2hc have been confirmed to be linked to each other via a disulfide bond in T24 human bladder carcinoma cells. Finally, in in vitro translation, we show that hLAT1 is not a glycosylated protein even though an N-glycosylation site has been predicted in its extracellular loop, consistent with the property of the classical 4F2 light chain. The properties of the hLAT1/h4F2hc complex would support the roles of this transporter in providing cells with essential amino acids for cell growth and cellular responses, and in distributing amino acid-related compounds.

Amino Acid Transport Systems↗
PubMed2001

Molecular events involved in up-regulating human Na+-independent neutral amino acid transporter LAT1 during T-cell activation.

We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, L-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells.

Amino Acid Sequence↗
PubMed2001

Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells.

We describe an application of gel microdroplet (GMD) and flow cytometry techniques to selective enrichment of non-growing Leuconostoc mesenteroides cells, which are well culturable on other media, from a mixture with Bacillus subtilis cells in nutrient broth. After encapsulating cells of the mixed population within GMDs and a brief incubation in nutrient broth, the inability of L. mesenteroides cells to form microcolonies within GMDs allowed their discrimination from B. subtilis cells. After staining the GMD mixture with 6-carboxyfluorescein diacetate, which showed no influence on cell viability, the GMDs containing single cells of L. mesenteroides were selectively collected using flow cytometry sorting based on differences in fluorescence intensity. The cells of L. mesenteroides retained viability during the process.

Bacillus subtilis↗
PubMed2001

Reactivity of autoantibodies of autoimmune hemolytic anemia with recombinant rhesus blood group antigens or anion transporter band3.

The specificity of autoantibodies in autoimmune hemolytic anemia (AIHA) has been studied using the serological procedure and immunoprecipitation technique with rare phenotype red cells. We attempted to analyze specificity using recombinant rhesus (Rh) blood group and band3 antigens expressed on erythroleukemic cell lines, KU812E. The autoantibody eluates were isolated by the acid elution procedure from the red cells of 20 AIHA patients. The recombinant Rh antigens, RhD, cE, ce, CE, and chimera antigens CE-D and D-CE, were obtained by retroviral cDNA transduction into KU812E cells, and the cell line expressing the antigens was cloned. Band3 cDNA was also obtained and introduced into KU812E and cloned KU812 expressing RhcE. The reactivities of AIHA eluates with recombinant Rh and band3 antigens were studied by flow cytometry. Fifteen eluates reacted with at least one of the RhcE, ce, or CE antigens, and four eluates reacted with RhD. Seven eluates with strong Rh specificity were studied further using chimera antigen. Five eluates showed reduced or lost reactivity, although two eluates reacted identically with the chimera antigens as wild type. These results indicated that conformational epitopes constituted by RhD or CE specific exofacial peptide loops are important for autoantibodies in most cases. Seven eluates reacted with band3, five exclusively. The coexpression study of RhcE and band3 did not enhance the expression of either antigen nor the reactivity with patient eluates, indicating that association of Rh and band3 was not involved in the appearance of autoantigen.

Anemia, Hemolytic, Autoimmune↗