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Biomedical subjects

N Kiyokawa

Publications and source records attributed to N Kiyokawa.

At least 19 recordsLinked to original sources
PubMed1999

Clonal dissemination of T-lymphocytes in scid mice from familial hemophagocytic lymphohistiocytosis.

BACKGROUND: Although familial hemophagocytic lymphohistiocytosis (FHL) has been considered a disorder of T-cell dysfunction, there is no evidence of the clonal origin of T-cells in this disease. PROCEDURE: We engrafted mononuclear cells (MNCs) from five FHL patients into scid mice and examined the infiltration of human cells in mouse organs. The characterization of human cells that infiltrated in the mouse organs was then performed. RESULTS: A diffuse infiltration of human lymphoid cells was detected in scid mice treated with 1 x 10(6) MNCs from one of the five patients. These cells were positive for HLA-DR and CD3, but negative for CD4, CD8, CD20, and CD68, suggesting the infiltration of double negative (DN) T-cells. The MNCs from the other four patients induced murine lymphoma-like disease; T-cell lymphoma in one and lymphoma of unknown origin in three. The characterization of these human DN T-cells was performed. The analysis of the Vbeta repertoire showed no preferential usage of the Vbeta family in MNCs, while the dominant expression of Vbeta13 was detected in T-cells infiltrating in the spleen and lung. A Jbeta analysis showed the restricted usage of Jbeta1.2 for Vbeta13 in these cells, and the clonality of Vbeta13-Jbeta1.2 fragment was confirmed by a single-strand confirmation polymorphism analysis. The analysis of the Valpha repertoire showed that Valpha24 was exclusively used in these DN T-cells, but no usage of JalphaQ for Valpha24 was observed. CONCLUSIONS: A clonal expansion of T-cells was induced in scid mice by the engraftment of MNCs from an FHL patient. The infiltration of DN alphabeta T-cells bearing invariant Valpha24 T-cell receptor in mouse organs may provide a useful clue to the pathogenesis of FHL. In the patients whose MNCs induced murine lymphoma-like disease, some cytokines or unknown factors that stimulate the growth and the tumorigenicity of murine lymphocytes might be produced by the MNCs engrafted in scid mice. Further study is needed to confirm the validity of our experimental approach and the findings observed in scid mice by using more FHL samples.

Animals↗
PubMed1999

The detection of Shiga toxins in the kidney of a patient with hemolytic uremic syndrome.

Infection of Shiga toxin (Stx)-producing Escherichia coli induces hemolytic uremic syndrome (HUS) in 10 to 15% of cases in infants and young children. Although the endothelial cell damage induced by Stx is widely believed to be a primary event of renal dysfunction in HUS, the precise mechanism remains to be elucidated. We were able to examine the kidney obtained at autopsy of a child who died after HUS associated with Stx-producing Escherichia coli O157:H7 infection, and immunohistochemistry indicated the deposition of Stxl and Stx2 in a portion of the distal tubular epithelia. To our knowledge, this is the first report to show the presence of Stx in human tissue of a patient with HUS, and the results obtained in this study provide evidence that Stx indeed migrates into the kidney and binds to renal tubules during Stx-producing Escherichia coli infection.

Bacterial Toxins↗
PubMed1998

Flow cytometric diagnosis of the cell lineage and developmental stage of acute lymphoblastic leukemia by novel monoclonal antibodies specific to human pre-B-cell receptor.

Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre-B-cell receptor (pre-BCR) composed of a mu heavy (muH) chain and a lambda5/VpreB surrogate light (SL) chain. HSL11 reacts with lambda5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro-B and pre-B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre-B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non-B- and non-T-ALLs (53 out of 56 cases) were found positive for either lambda5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic muH chain, indicative of pre-B-cell origin. Interestingly, 6 out of these 19 pre-B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, muH chain, pre-BCR, and light chain along the B-cell development.

Antibodies, Monoclonal↗
PubMed1998

Human microvascular endothelial cells are strongly sensitive to Shiga toxins.

We show here that the susceptibility of endothelial cells to Shiga toxin (Stx)s differs remarkably depending on their cellular origins. The concentration of Stx-1 required to reduce cell viability by 50% as measured by MTT assay was 30 and 300 fM for neonatal and adult human microvascular endothelial cells (HMVEC), respectively, and 30 pM for human coronary artery endothelial cells (HCAEC). Human umbilical venous endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC) showed no sensitivities to Stx-1. Surprisingly, Stx-2 was approximately 10-100 times more toxic to HMVEC than Stx-1. Moreover sodium butyrate sensitized HMVEC by 100-fold to the cytotoxic activity of Stxs. These results were found to reflect the amount of Gb3/CD77 on the cell surface on a per cell basis using flow cytometrical analysis. The high sensitivity of HMVEC to Stxs suggests their involvement in the pathogenesis of organ failure induced by Stx-producing Escherichia coli.

Adult↗
PubMed1998

Verotoxins induce apoptosis in human renal tubular epithelium derived cells.

Apoptosis mediated by verotoxins (VTs) has been identified in a renal carcinoma cell line, ACHN cells, which are an in vitro model of renal tubular epithelial cells. ACHN cells express the renal tubular marker CD24 as well as globotriaosyl ceramide/CD77, the receptor for VTs. VT binding to the ACHN cell surface was confirmed by positive staining with antibodies to the VTs. Treatment of ACHN cells with VTs induced prompt growth inhibition and cell death, and fragmentation of the genomic DNA in cells, typical of apoptosis, was observed. The expression of apoptotic antigen 7A6 detected by APO2.7 antibody in ACHN cells further supports the occurrence of apoptosis as a result of VT treatment. Cycloheximide enhanced VT-mediated apoptosis of ACHN cells, suggesting a strong correlation between the inhibition of protein synthesis and VT-mediated apoptosis. Moreover, tumor necrosis factor-alpha had a synergistic effect on VT-mediated apoptosis in ACHN cells. Considering the above evidence together with the clinical evidence showing the presence of apoptosis in the renal epithelium of a HUS patient, our results suggest a VT-induced apoptotic mechanism in normal renal tubular epithelium that may contribute to the pathogenesis of hemolytic uremic syndrome.

Antigens, Surface↗
PubMed1998

Induction of apoptosis in normal human renal tubular epithelial cells by Escherichia coli Shiga toxins 1 and 2.

The cytotoxicity of Shiga toxin (Stx) 1 and Stx2 produced by Escherichia coli to human renal cortical epithelial cells (HRCEC) in primary culture was investigated. HRCEC express CD24, the marker of renal distal tubules, as well as globotriaosyl ceramide/CD77, the receptor for Stxs. Binding of Stxs to HRCEC was confirmed by positive staining with specific antibodies to Stxs. Treatment of HRCEC with Stxs induced rapid cell death, which was reversed in the presence of neutralizing antibody specific for Stx. DNA fragmentation was found to be accompanied by Stx-mediated cell death in HRCEC, indicating that apoptosis was part of the process. These data and previous reports indicate that a variety of renal cell types, including tubular epithelial cells as well as glomerular capillary endothelial cells, may be targets for Stx-mediated apoptosis, which could contribute to the pathogenesis of hemolytic-uremic syndrome caused by Stx-producing E. coli infection.

Adult↗
PubMed1997

Involvement of cdc2-mediated phosphorylation in the cell cycle-dependent regulation of p185neu.

We previously reported cell cycle-dependent negative regulation of p185neu (decreased tyrosine phosphorylation and kinase activity, with electrophoretic mobility retarded by serine/threonine phosphorylation) in M phase and the escape of mutation-activated p185neu* from this regulation. Our present results showed that retardation of electrophoretic mobility occurs independently of the cells' transformed status. We found that normal p185neu lost its ability to dimerize in the M phase. We demonstrated a physical association between cdc2 (a serine/threonine kinase, active in M phase) and p185neu. We showed that the carboxy terminal portion of p185neu is phosphorylated in vitro by cdc2. Many phosphopeptides (at least three phosphoserine residues) unique to the M phase were identified, and the in vivo and in vitro phosphopeptide patterns were superimposable. In contrast, mutation-activated p185neu* dimerized in the M phase with no changes in electrophoretic mobility, failed to associate with cdc2 and no unique phosphoserine residues could be identified in the M phase (data not shown), consistent with the escape of p185neu* from cell cycle-dependent regulation. Our results suggest that this escape is an intrinsic property of the mutation-activated p185neu* independent of its ability to transform cells. Our results also suggest the involvement of serine/threonine kinases such as cdc2 in the cell cycle-dependent negative regulation of p185neu.

Animals↗
PubMed1997

Mitosis-specific negative regulation of epidermal growth factor receptor, triggered by a decrease in ligand binding and dimerization, can be overcome by overexpression of receptor.

The function of epidermal growth factor receptor (EGFR) was found to be negatively regulated in M phase in which it showed less phosphotyrosine content and reduced intrinsic kinase activity accompanied by retarded electrophoretic mobility owing to total hyperphosphorylation. Ligand-induced autophosphorylation and downstream signaling of EGFR were tightly suppressed in M phase due to a decrease in ligand binding affinity and the inability of epidermal growth factor (EGF) to induce receptor dimerization. There was no change in the number of surface-exposed EGF receptors between G0/G1 and M phases of the cell cycle. Hyperphosphorylation (due to serine and/or threonine phosphorylation) correlates with the unresponsiveness of cells to EGF-mediated stimulation of tyrosine phosphorylation in cells that express the normal or basal level of EGFR. This M phase-specific negative regulation was overcome by overexpression of EGFR, which was responsive to ligand throughout the cell cycle and revealed ligand-induced signaling in the M phase. These findings indicate that EGFR does not respond to ligand stimulation in M phase and suggest that a negative regulation of ligand-receptor interactions in M phase may control the normal function of receptor tyrosine kinase and that receptor overexpression will disrupt this cell cycle-dependent regulation of receptor tyrosine kinases.

3T3 Cells↗
PubMed1997

Strong correlation between c-erbB-2 overexpression and overall survival of patients with oral squamous cell carcinoma.

Overexpression of c-erbB-2 (also known as HER-2/neu) has been found in many human cancers, including head and neck squamous cell carcinoma (SCC). We therefore examined expression of the oncoprotein in oral SCC primary tumor samples and compared its relationship with clinical stages and survival rate. Out of 80 cases of oral SCC, high expression level (++ or +++) of c-erbB-2 was found in 41 cases. Of the 80 cases with follow-up information, 39 were further investigated for the correlation of expression level of c-erbB-2 and survival rate. Overexpression of the oncoprotein was significantly correlated with shorter overall survival, and the patients with low and no expression of c-erbB-2 had much higher survival rates. Overexpression of c-erbB-2 was also significantly correlated with nodal stage and metastasis. We found that high expression level of c-erbB-2 was frequently detected in oral cancer cell lines but not in the other head and neck SCC cell lines. Thus, we conclude that overexpression of c-erbB-2 is a frequent event in oral SCC and is correlated with poor survival and may be used as a poor prognostic factor.

Adolescent↗
PubMed1996

Lack of CD54 expression and mutation of p53 gene relate to the prognosis of childhood Burkitt's lymphoma.

Expression of the intercellular adhesion molecule-1 (CD54) as well as the mutations of p53 gene were studied in childhood Burkitt's lymphoma (BL). Expression of CD54 was identified in 6 of 15 fresh BL cases. Mutations of p53 gene, analyzed by polymerase chain reaction-single stranded chain polymorphism followed by sequencing, were found in 5 of 14 cases examined. Interestingly, all the cases with p53 mutation were CD54 negative. This high frequency of p53 mutation in the CD54 negative group prompted us to analyze the clinical features of these cases. Six of 15 cases died within 21 months after initiation of therapy and five of these were CD54 negative. In addition, four of these had p53 mutation. These results suggest that the lack of CD54 by BL cells may provide the background for the mutation of p53 gene to occur which could result in the transformation to a more aggressive phenotype.

Adolescent↗
PubMed1995

Cell cycle-dependent regulation of p185neu: a relationship between disruption of this regulation and transformation.

Structure and function of p185neu receptor tyrosine kinase were found to be regulated in a cell cycle-dependent manner. In M phase, p185neu is hyperphosphorylated at serine and/or threonine residues. The phosphotyrosine [Tyr(P)] content of p185neu is at its highest level in G0/G1 phase, decreases through S and G2 phases, and reaches its lowest level in M phase. Phospholipase C-gamma (PLC-gamma) and GTPase-activating protein (GAP), substrates of p185neu, also have a similar profile of Tyr(P) content during the cell cycle. These results, along with in vitro immune complex kinase assays, suggest that the tyrosine kinase activity of p185neu is least active in M phase. Interestingly, the mutation-activated neu oncogene (neu*)-encoded protein product, p185neu* escaped from cell cycle regulation. Taken together, we demonstrate in this report that the structure and function of p185neu are regulated in a cell cycle-dependent manner, yet p185neu* escapes from this regulation and remains active through the cell cycle. Disruption of this cell cycle regulation may define a mechanism for p185neu*-mediated cellular transformation.

3T3 Cells↗
PubMed1995

HER-2/neu oncogene characterization in head and neck squamous cell carcinoma.

OBJECTIVE: To characterize the HER-2/neu oncogene in head and neck squamous cell carcinoma (HNSCC) cell lines and tumor tissue specimens. DESIGN: Molecular analysis of HER-2/neu oncogene amplification and expression in HNSCC cell lines by Southern, Northern, and Western blot techniques, and HER-2/neu oncoprotein expression in HNSCC tumor tissue sections by immunohistochemical analysis. SPECIMENS: Eleven HNSCC cell lines, eight paired samples of frozen HNSCC tumor tissue specimens and adjacent nonmalignant mucosa, and 38 paraffin-embedded slides derived from HNSCC tumor specimens (including those from which the cell lines were derived) were analyzed. RESULTS: Southern blot analysis showed twofold HER-2/neu gene amplification in two (18%) of the 11 HNSCC cell lines, MDA-1386 and Tu-167. Northern blot analysis showed messenger RNA overexpression in the same two cell lines, and to a lesser degree in MDA-1483. Western blot analysis showed high levels of HER-2/neu oncoprotein expression in two (18%) of the 11 cell lines (MDA-1386 and Tu-167), a moderate level of protein expression in one cell line (9%) (MDA-1483), and low levels of protein expression in eight cell lines (73%). Some HER-2/neu protein expression was seen in all of the HNSCC cell lines. Immunohistochemical analysis of the tumor tissue sections from which the cell lines were derived corroborated the Western blot results. Western blot analysis of frozen primary tumor specimens showed HER-2/neu oncoprotein overexpression in two (25%) of eight specimens. Immunohistochemical analysis showed high levels of protein expression in six (16%) of the 38 tumor tissue slides, moderate levels in 12 (31%), and low levels in 20 (53%). CONCLUSIONS: The HER-2/neu oncogene is overexpressed in a subset of HNSCC tumors and cell lines. The results from Western blot and immunohistochemical analyses underscore a variable HER2/neu oncoprotein expression in HNSCC. Gene amplification was observed in a few of the cell lines, suggesting a potential mechanism of oncoprotein overexpression. Messenger RNA overexpression, however, can be seen in the absence of gene amplification, indicating that transcriptional or posttranscriptional control mechanisms must be involved. Further studies are indicated to determine the biologic role of HER-2/neu expression in the clinical progression of these lesions and to further define the molecular basis regulating its expression.

Aged↗
PubMed1991

[Successful treatment of metastatic and refractory osteosarcoma by ifosfamide, carboplatin and vindesine: case report].

A 13-year-old boy with unresectable pulmonary metastatic osteosarcoma, which was refractory to high dose methotrexate, adriamycin, cisplatin and combination of bleomycin, cyclophosphamide and actinomycin D, was treated by aggressive chemotherapy including the combination of ifosfamide (1 g/m2 x day 1-4), Carboplatin (100 mg/m2 x day 1-4) and Vindesine (4 mg/m2 x day 1). After 5 courses of the treatment, pulmonary metastasis regressed, respiratory symptoms resolved completely, and in this regimen no severe toxicity was observed. Thoracotomy for pulmonary metastatic osteosarcoma is an accepted treatment, but treatment for patients with unresectable disease has not been established. It is suggested that this regimen is relatively safe and very effective for refractory and unresectable osteosarcoma.

Adolescent↗
PubMed1990

Problems of mass screening for neuroblastoma: analysis of false-negative cases.

The Japanese mass screening (MS) system for neuroblastoma at 6 months of age has resulted in the earlier diagnosis of the tumor with excellent therapeutic results. However, some problems are involved in the present MS system. We present six false-negative cases, ages ranging from 1 year 11 months to 3 years 11 months. Neuroblastoma cell taken from four of these patients were studied biologically. These patients had advanced disease (one was stage III; five were stage IV). Three of the patients have died and one is terminally ill despite undergoing surgery combined with intensive chemotherapy. Cytogenetic analysis performed in three cases showed that all the cases had diploid chromosome mode associated with 1P-, double minutes (DMs), or marker chromosomes. N-myc oncogene analysis, performed in four cases, showed amplification in two; one patient had diploid chromosomes, but the other was not examined cytogenetically. These findings were strikingly different biologically from those of cases found by MS. The majority of neuroblastomas detected by MS were found to be triploid tumors without N-myc amplification. These findings suggest that the main reason for the false-negative results in the patients we examined is that they were tumor-free or the tumors were so small in size that they were unable to produce urinary vanillylmandelic acid and or homovanillic acid levels high enough to be detected at the time of MS. Therefore, we conclude that MS at 6 months of age is too early to detect neuroblastoma with a diploid chromosome mode and/or amplified N-myc oncogene. We propose that MS at the age of 1 year 6 months would be more effective to pick up these cases, because treatment strategies depend on the different biological characteristics of tumor cells.

Adrenal Gland Neoplasms↗
PubMed1990

Characterization of the common acute lymphoblastic leukaemia antigen (CD10) as an activation molecule on mature human B cells.

Distinct expression pattern of CD10 molecules during B cell activation was analysed using in vivo and in vitro systems. By two-colour flowcytometrical analysis, CD10 was found to be expressed at a specific stage of in vivo activating B cells. The expression of CD10 during B cell activation appeared to be unique from that of other activation-related B cell antigens including L29, MA6, OKT9 and OKT10. Although the expression of CD10 was associated with that of the activation-related B cell antigens, CD10+ B cells could be separated in the distinct fractions to those expressing other activation-related B cell antigens when fractionated by cell gravity. In particular, certain CD10+ B cells were detected positive for the resting B cell antigen, L30. In vitro studies revealed that CD10+ B cells arose from CD10- B cells at an early step of B cell activation, and disappeared lately when activated by Staphylococcus aureus Cowan I. Collectively, CD10 was an antigen transiently expressed at an early phase of B cell activation process. Expression of CD10 and other antigens on Burkitt's lymphomas (15 cases) was studied next. All cases were CD10+, and 87% (13 cases) were also L30+. In addition, six of CD10+ L30+ cases were L29+. This observation suggested that Burkitt's lymphomas were phenotypically similar to the B cells at an early phase of activation, those expressing CD10 and L30, simultaneously. The present study has dissected a precise expression pattern of CD10 on mature B cell activation in vitro and in vivo, and could be implicated for the histogenesis of one of the poorly characterized B cell lymphoma, namely Burkitt's lymphoma.

Antigens, CD↗
PubMed1988

Ki-1 lymphomas in childhood: immunohistochemical analysis and the significance of epithelial membrane antigen (EMA) as a new marker.

Two cases of Ki-1 lymphomas in childhood were analyzed immunohistochemically and immunoelectron microscopically. They expressed Hodgkin's disease associated antigen, Ki-1, interleukin-2 receptor (IL2R), OKT9, and HLA-DR. Histologically, the tumour cells were large in size with abundant cytoplasm and atypical nuclei. Lymph node involvement was characterized by parafollicular and marginal sinus infiltration. These features were identical to those reported in Ki-1 lymphomas. Electron microscopically tumour cells had abundant cytoplasmic organelles with pleomorphic nuclei but had no specific granules. Some tumour cells had marked interdigitation of cell membrane. Immunoelectron microscopically Ki-1 was positive on cell membrane. Tumour cells had no T-cell or B-cell antigens except for Leu-3 (T4). Unexpectedly they expressed epithelial membrane antigen (EMA) strongly. EMA was positive on cell membrane and in the cytoplasm. EMA was detected effectively in paraffin-embedded sections. Among the malignant lymphomas in childhood tested, two cases were EMA-positive. The pattern of EMA-reactivity and the histology were very similar to Ki-1 lymphomas. These results strongly suggest that Ki-1 lymphomas in childhood may arise from non-lymphoid haematopoietic cells and that EMA can be used as a new marker to distinguish certain type of Ki-1 lymphomas in childhood.

Antigens, Neoplasm↗
PubMed1988

Immunocytological and immunochemical analysis on the common acute lymphoblastic leukemia antigen (CALLA): evidence that CALLA on ALL cells and granulocytes are structurally related.

The common acute lymphoblastic leukemia antigen(CALLA) on acute lymphoblastic leukemia(ALL) cells and granulocytes were compared by newly developed and other anti-CALLA monoclonal antibodies(anti-CALLA). New anti-CALLA(IF-3 through IF-7) were effectively selected by immunostaining on kidney sections. By competitive binding three antigenic determinants were separated on ALL cells by IF and other anti-CALLA. All three determinants existed on granulocytes although the reactivity of each anti-CALLA was variable. Such a variability was partly due to the heterogeneous terminal sialic acid compositions. Although CALLA from granulocytes and ALL cells differed in molecular weight they showed identical peptide mapping patterns. These results strongly suggest that CALLA on ALL cells and granulocytes are structurally related although they are different in posttransulational modification.

Animals↗