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Biomedical subjects

H Shiku

Publications and source records attributed to H Shiku.

At least 199 records · Page 11Linked to original sources
PubMed1995

Outcome of disseminated intravascular coagulation in relation to the score when treatment was begun. Mie DIC Study Group.

We examined 395 patients with disseminated intravascular coagulation (DIC) divided into two groups: non-leukemic and leukemic. In 58% of the patients as a whole, treatment of DIC resulted in complete or partial remission, while exacerbation and death occurred in 31%. The efficacy of DIC treatment in the non-leukemic group was less than that in the leukemic group, indicating that the outcome of DIC depended, in part, on the underlying disease. We examined hemostatic indicators in relation to DIC score: prothrombin time (PT) ratio, FDP, platelet count, and fibrinogen levels were found to be important indicators for the diagnosis of DIC, but not for Pre-DIC. Plasma levels of fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin-plasmin inhibitor complex (PPIC) were significantly increased in pre-DIC. The efficacy of treatment in relation to the DIC score when the treatment was begun showed that greater efficacy was achieved in pre-DIC than in DIC patients. The outcome was poorer with increasing DIC score, suggesting that early diagnosis and early treatment are important. On examining the relationship between outcome and hemostatic indicators, we found that the PT ratio and the levels of antithrombin, plasminogen, PPIC, the PPIC/TAT ratio, and thrombomodulin were related to outcome, suggesting that very high consumption of blood coagulation factors, liver dysfunction, hypofibrinolysis, or organ failure caused a poor outcome. Although the outcome in DIC patients may not depend substantially on plasma levels of TAT and fibrin-D-dimer, we can use these indicators to treat DIC patients at an early stage.

Antifibrinolytic Agents↗
PubMed1994

Isolation of GD3 synthase gene by expression cloning of GM3 alpha-2,8-sialyltransferase cDNA using anti-GD2 monoclonal antibody.

For the isolation of ganglioside GD3 synthase (EC 2.4.99.8) cDNA, we developed an expression cloning approach that used an anti-GD2 monoclonal antibody for selection. A host recipient cell line that we have named KF3027-Hyg5 was also utilized. This cell line expresses high levels of GM2 as well as GM3 but no GD3 or GD2 and was constructed from mouse B16 melanoma cells transfected with the polyoma large tumor antigen gene (KF3027) and the previously cloned beta-1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) cDNA. Four rounds of transfection, monoclonal antibody 3F8 panning, and Hirt extraction resulted in the isolation of two cDNA clones, transfection of which directed the expression of GD3 in KF3027 and B16 melanoma cells and GD3 and GD2 in KF3027-Hyg5 cells. The cDNA contained a 1650-bp insert and a single open reading frame. The deduced amino acid predicted a type II membrane topology consisting of cytoplasmic (14 aa), transmembrane (18 aa), and catalytic (309 aa) domains. The sequence also predicted the presence of a sialyl motif similar to that found in the other sialyltransferases cloned so far. As expected, mRNA of this gene (2.6 kb) was strongly expressed in human melanoma lines.

Amino Acid Sequence↗
PubMed1994

Selective inhibition of human T-lymphotropic virus type I-transformed human T-cell growth by a tax-targeted conditionally cytotoxic recombinant retrovirus.

Adult T-cell leukemia (ATL), a disorder associated with high mortality rates, arises from human T-lymphotropic virus type I (HTLV-I)-infected CD4+ T cells. We designed a retroviral vector-based gene therapy approach to ATL. The long terminal repeat (LTR) of HTLV-I is transactivated by the viral tax protein. We constructed a hybrid gene consisting of herpes simplex virus thymidine kinase (HSV TK) under the control of the HTLV-I LTR and inserted it into a retroviral vector. When HTLV-I-transformed and tax-expressing human T-cell lines were infected with this recombinant retrovirus (LNLTK alpha virus), they expressed high levels of HSV TK and exhibited increased sensitivity to acyclovir, a nucleoside analog that is converted to the toxic anabolite after phosphorylation by the HSV TK. On the other hand, the retroviral infection had little effect on acyclovir-induced cytotoxicity in HTLV-I-negative human hematopoietic cell lines. Our data may provide the prospect of the gene therapy for ATL by tax-targeted selective elimination of leukemic cells.

Acyclovir↗
PubMed1994

Isolation of human nm23 genomes and analysis of loss of heterozygosity in primary colorectal carcinomas using a specific genomic probe.

Genomic clones of nm23-H1 and -H2 were isolated. The nm23-H1 and -H2 genes were located in a tandem array 4 kilobases apart. Each genome contained 5 exons and most of the splicing sites in the exon-intron junctions of two isotypes were essentially identical. A probe derived from intron 4 of nm23-H1 was used to examine the loss of heterozygosity (LOH) in primary colorectal carcinomas. Twenty-nine of 42 samples were informative and LOH was found in 3 of these 29. In all three samples with LOH, cancer tissues expressed lower levels of nm23-H1 mRNA when compared with those without LOH.

Amino Acid Sequence↗
PubMed1994

Expression of human nm23-H1 and nm23-H2 proteins in hepatocellular carcinoma.

BACKGROUND: The expression of nm23-H1 and nm23-H2 proteins in 25 hepatocellular carcinomas was studied immunohistochemically. METHODS: Tissue specimens were reacted with anti-human nm23-H1 and nm23-H2 monoclonal antibodies (MoAb) (H1-229 and H2-206, respectively) and then stained by the biotin-streptoavidin complex method. RESULTS: Adjacent nontumorous tissues were intensely stained with nm23-H1 and nm23-H2. Of the 25 hepatocellular carcinomas, 60% were positive for MoAb H1-229, and 68% were positive for MoAb H2-206. These immunoreactivities were most common in the cytoplasm of tumor cells. There was no significant correlation between the expression of nm23-H1 protein and tumor size, Edmondson's histopathologic classification, or invasion of the capsule. However, the authors observed an inverse relationship between nm23-H1 expression and intrahepatic metastases of hepatocellular carcinomas. There was no significant correlation between the expression of nm23-H2 protein and clinicopathologic findings. Only a short survival period was observed in patients with hepatocellular carcinoma with reduced nm23-H1 or nm23-H2 proteins. CONCLUSIONS: The results suggest that nm23-H1 protein plays a role in the suppression of intrahepatic metastasis of hepatocellular carcinoma and that the combined expression of nm23-H1 is associated with favorable prognosis.

Carcinoma, Hepatocellular↗
PubMed1994

Production and characterization of monoclonal antibodies against the polyamine, spermine: immunocytochemical localization in rat tissues.

Two monoclonal antibodies of types IgG2b and IgG2a, anti-spermine-(Spm)-1 (ASPM-1) and anti-Spm-2 (ASPM-2) respectively were found among five clones of murine monoclonal antibodies, which were raised against Spm conjugated with bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy) succinimide (GMBS). Antibody specificity was evaluated by a recently developed ELISA binding test, and led to the study of tissue sections by immunocytochemistry (ICC). ASPM-1 showed exclusive immunoreactivity with Spm, with the exception of a negligible cross-reactivity (2.0%) with spermidine (Spd). ASPM-2, on the other hand, reacted almost equally with acetylspermine (Ac-Spm) and N1-acetylspermidine (N1-Ac-Spd) but with none of the other polyamine-related compounds tested. Complete agreement was obtained with the results of immunoblot analysis. Furthermore, results for antibody specificity obtained with the ELISA inhibition test and ICC model experiments using Sepharose gel beads strongly suggested that ASPM-1 recognizes the Spm molecule possessing at least a free terminal primary amino group, while ASPM-2 recognizes the Spm molecule acylated at both the terminal primary amino groups. An ICC method using ASPM-2 produced strong staining for polyamines (PAs) in the cytoplasm (but very few in the nuclei) of two different tumor cell lines and protein- or peptide-secreting cell systems, including exocrine and endocrine cell types; ASPM-1 showed immunoreactivity only with the tumor cell lines. These results strongly suggest that ASPM-2 may be useful for studies on actively proliferating and neoplastic cells, supporting our previously proposed idea that in immunocytochemistry PAs were converted to a variety of PA derivatives during the fixation process.

Animals↗
PubMed1994

Long-term survival of orthotopic Lewis liver grafts in Wistar Furth rats. Elimination or inactivation of effector CTL and altered antigenicity as possible reasons for tolerance.

After orthotopic transplantation of rat livers from Lewis (LEW) donors into Wistar Furth (WF) recipients, a rejection response occurs 2 weeks after transplantation, followed by indefinite survival of the transplant. Spleen cells from WF rats bearing long-term LEW liver grafts responded in MLR and CML assays like spleen cells from normal WF rats after in vitro stimulation with mitomycin C-treated LEW spleen cells, but did not generate CTL after stimulation with paraformaldehyde-treated LEW spleen cells. Transient damage to the long-term grafted livers was induced by inoculation of recipients with homogenized normal LEW liver. CD8+ CTL were detected in the spleen at the time of this liver damage. These findings indicate that CTL precursors are present in WF rats bearing LEW liver transplants. These CTL precursors are capable of differentiating into effector CTL in vitro and in vivo in response to donor antigen. However, once generated, effector CTL in WF rats are eliminated or become unresponsive to the transplanted LEW liver. Homogenized LEW liver from grafted donors did not induce transient liver damage when inoculated into recipient WF rats bearing LEW liver transplants. The demonstration of large amounts of antibody bound to either parenchymal or nonparenchymal cells in the donor graft suggests that reduction (or loss) of antigenicity could also be responsible for the inability of LEW liver to elicit an immune response in WF-recipient rats. Thus, elimination or inactivation of effector CTL and altered antigenicity could both be responsible for maintaining tolerance after allogeneic liver transplantation.

Animals↗
PubMed1994

Expression of Kirsten-ras p21 in gastric cancer correlates with tumor progression and is prognostic.

The purpose of this study was to determine the correlation between expression of ras oncoproteins and the tumor stage or outcome of patients with gastric carcinoma. After the specificity of each anti-ras monoclonal antibody was confirmed by protein immunoblot analysis, immunohistochemical assays for a common-ras antigen present in N-, Harvey- and Kirsten (K)-ras oncoproteins, as well as for K-ras specific antigen, were performed on paraffin-embedded carcinoma tissue from 110 patients who underwent curative resection. By Western blot analysis, there was more p21 in fresh cancer specimens than in normal specimens. K-ras expression distinguished advanced from early gastric carcinoma and correlated with depth of cancer invasion. Among the 110 patients, survival rates of those with carcinomas positive for the common-ras or K-ras antigens were significantly lower than of those with antigen-negative carcinomas (p < 0.05). In a multivariate analysis, nodal involvement (p = 0.002), serosal invasion (p = 0.012) and K-ras p21 expression (p = 0.044) were independently predictive of the recurrence. These results suggest that K-ras p21 is a useful marker of tumor progression and poor prognosis after curative resection.

Aged↗
PubMed1994

GD2 ganglioside-specific monoclonal antibody reacts with murine cytotoxic T lymphocytes reactive with FBL-3N erythroleukaemia.

Ganglioside expression on cytotoxic T lymphocytes induced against the mouse erythroleukaemia line FBL-3N, was analysed, compared with that of naive T lymphocytes in the thymus, lymph nodes and spleen. Although normal uncultured and cultured T lymphocytes expressed no GD2, GD3 or GM2 gangliosides, cytotoxic T cells with CD4+CD8-, CD4-CD8+, and CD4-CD8- phenotypes reacted with anti-GD2 monoclonal antibody with various intensities. The reactivity with anti-GD2 was associated with the intensity of cytotoxic activity as analysed using cytotoxic T lymphocyte (CTL) clones established from the bulk CTLs of each phenotype. An increase of the mRNA expression of GM2/GD2 synthase gene was demonstrated by Northern blot hybridization using RNA extracted from thymocytes, spleen cells, Con A-treated spleen cells and various types of CTL cells. These results indicated that GD2 ganglioside expression might be associated with the functional differentiation of murine T lymphocytes.

Animals↗
PubMed1994

Clonal expansion of CD8+ cytotoxic T lymphocytes against human T cell lymphotropic virus type I (HTLV-I) genome products in HTLV-I-associated myelopathy/tropical spastic paraparesis patients.

Short-term culture of peripheral blood mononuclear cells (PBMC) derived from patients with human T cell lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis resulted in dominance by DR+ activated CD8+ T cells. Variations in the T cell receptor (TCR) V alpha and V beta chains in these cells were analyzed, and in all 10 patients examined, 2-3 V gene families were dominant in both TCR V alpha and V beta. In five patients we examined, cultured lymphocytes contained cytotoxic lymphocytes for p40tax (patients HAM2, 3, 7, and 8) or env protein (patient HAM4) of human T lymphotropic virus type I. In patients HAM2 and HAM8, cultured lymphocytes contained a large proportion of V beta 8+ CD8+ and/or V beta 12+ CD8+ cells. The sequence of V beta 8+ and V beta 12+ cDNA revealed that they were oligoclonal with identical or similar sequences in each patient. Elimination experiments with monoclonal antibodies for TCR V beta 8 and V beta 12 showed that they were CD8+ cytotoxic T lymphocytes (CTL) for p40tax. In addition, flow cytometry and sequencing analysis of uncultured PBMC revealed that in HAM2, V beta 8+ CTL and their precursors account for 7% and V beta 12+ CTL and their precursors account for 18% of total CD8+ cells. This indicates the presence of two markedly expanded clones in vivo. No common dominant TCR V alpha or V beta were observed among 10 HAM patients analyzed.

Amino Acid Sequence↗
PubMed1994

erbB-2 expression in well-differentiated adenocarcinoma of the stomach predicts shorter survival after curative resection.

BACKGROUND: Correlation between erbB-2 expression, histologic type of gastric carcinoma, and survival after curative resection was evaluated. METHODS: Paraffin-embedded specimens from 120 patients who underwent curative resection of gastric carcinoma were analyzed immunohistochemically for the expression of erbB-2. RESULTS: Enhanced erbB-2 expression correlated with tumor stage and depth of invasion. Well-differentiated adenocarcinomas had a higher incidence of erbB-2 expression than did poorly differentiated carcinomas. Survival rates of 33 patients with erbB-2-positive carcinomas were significantly lower than those of 87 with erbB-2-negative carcinomas (p < 0.001). Survival rates of patients with well-differentiated adenocarcinomas that were erbB-2 positive were significantly lower than those that were erbB-2 negative (p < 0.001). However, the presence of erbB-2 was not associated with altered survival in 46 patients with poorly differentiated carcinomas. Multivariate analysis of all 120 patients revealed that independent predictors of recurrent disease include nodal involvement (p = 0.003) and erbB-2 expression (p = 0.0051). CONCLUSIONS: Our results suggest that erbB-2 expression is a new marker associated with poor prognosis in well-differentiated gastric adenocarcinomas.

Adenocarcinoma↗
PubMed1994

Alteration of nm23 gene expression during the induced differentiation of human leukemia cell lines.

The nm23-H1 gene is regarded as a human homologue of the mouse nm23 gene, which was expressed in a non-metastatic subline of mouse melanoma K-1735. The expression levels of nm23-H1 mRNA and the levels of protein during induced differentiation of human leukemia cell lines were analysed. mRNA levels of the megakaryoblastic leukemia line MEG-01, which were induced to differentiate into megakaryocyte by TPA, decreased rapidly from 2 days after the start of treatment and became almost undetectable at day 4. Similar down-regulation of nm23-H1 mRNA was also observed in the induced differentiation of the promyelocytic leukemia line HL-60 by TPA, or DMSO into monocyte-macrophage lineage or granulocytes, respectively. The amount of Nm23-H1 protein was analysed by Western immuno-blot analysis using mouse antiserum raised against a recombinant fusion protein with glutathione S-transferase. The amount of Nm23-H1 protein also decreased during the induced differentiation of these leukemia cell lines. On the other hand, in the differentiation of the erythroleukemia line K562 by hemin, levels of both mRNA and protein of Nm23-H1 elevated transiently, then reduced to the original level. When MEG-01 and K562 were stably transfected with nm23-H1 cDNA, MEG-01 transfectants showed reduced sensitivity to the induction of differentiation, whereas K562 transfectants were better induced to synthesize hemoglobin than controls. These findings suggest the possibility that Nm23-H1 protein plays an important role to maintain the proliferation of immature leukemic cells in MEG-01 and HL-60, but it may also play a role in the early stage of K562 differentiation, possibly in the different manner.

Animals↗
PubMed1993

Epoc-1: a POU-domain gene expressed in murine epidermal basal cells and thymic stromal cells.

POU-domain transcription factors are known as developmental regulators which control organ development and cell phenotypes. In order to clarify the roles of POU-domain transcription factors in cell differentiation, we cloned a novel POU family gene, Epoc-1, from a murine thymus cDNA library. The amino acid (aa) sequence of the POU-specific domain of Epoc-1 is almost identical to those of Oct-1 and Oct-2. However, within the POU-homeodomain, 13 out of 60 aa differ between Epoc-1 and Oct-2. Recombinant Epoc-1 products were found to bind specifically to the octamer sequence. Epoc-1 was found to be expressed in skin, thymus, stomach and testis. In situ hybridization experiments and RNase protection assays indicated that Epoc-1 is expressed in the epidermal basal cells of the skin, which contain stem cells unipotent for keratinocyte differentiation and in thymic stromal elements. These results suggest that Epoc-1 might be one of the developmental regulators which controls epidermal development and thymic organogenesis.

Amino Acid Sequence↗
PubMed1993

Genetic and enzymatic basis for the differential expression of GM2 and GD2 gangliosides in human cancer cell lines.

Using beta 1,4-N-acetylgalactosaminyltransferase (EC 2.4.1.92) complementary DNA, the correlation of gene expression, enzyme activity, and expression of ganglioside antigens was analyzed in 20 human tumor cell lines. In many lines, GM2 and/or GD2 were the most complex structures examined. Northern blot analysis revealed 5.2- and 3.0-kilobase mRNAs in almost all cell lines expressing GD2 and/or GM2. Some melanoma lines, however, showed no bands although they expressed fairly high levels of GD2. These cell lines expressed very high levels of alpha 2,8-sialyltransferase and the resulting product, GD3. Semiquantitative RT-PCR demonstrated that even cell lines with no bands in Northern blot contained 0.4-2.5% of mRNA level in the highest expressing cell line. These results indicate that GD2 expression on individual cell lines is regulated not only by the expression level of the N-acetylgalactosaminyl transferase but also by the amount of its precursor structure (GD3) and alpha 2,8-sialyltransferase present in the cells. beta 1,4-N-acetylgalactosaminyltransferase activities and mRNA levels generally correlated quite closely. A few lines, however, showed lower enzyme activities than expected from their mRNA levels, indicating the possibility that the enzyme is being regulated by translational or posttranslational modification such as phosphorylation and glycosylation as well as by transcriptional regulation. Depending on their patterns of ganglioside synthesis and expression, the lines examined were classified into 6 groups which were characteristic of different tumor cell types.

Antibodies, Monoclonal↗
PubMed1993

Reduced expression of nm23-H1, but not of nm23-H2, is concordant with the frequency of lymph-node metastasis of human breast cancer.

The nm23 gene is a potential metastasis-suppressor gene originally identified in a murine melanoma line. Several investigators have reported the probable inverse association of nm23 expression with disease prognosis and/or metastasis. Since there are now 2 known isotypes of human nm23, namely nm23-HI and -H2, we immunohistochemically examined expression of these isotypes in human breast-cancer tissues using monoclonal antibodies (MAbs) specific for each isotype protein. We also analyzed expression of c-erbB-2 in the same collection of cancer tissues, in order to examine the significance of nm23 expression in comparison with c-erbB-2 expression. Of 130 tumors from breast-cancer patients, 73 (56%) and 69 (53%) positively expressed nm23-HI and -H2 respectively. Expression of c-erbB-2 was positive in 36 (28%). Expression of nm23-HI, but not nm23-H2, was inversely associated with lymph-node metastasis (p < 0.01). Expression of c-erbB-2 was associated with Tnm stage, tumor size and lymph-node metastasis (p < 0.01, p < 0.05 and p < 0.05 respectively). Overall survival was better (p = 0.014) in patients in whom expression of nm23-HI was positive than in those in whom it was negative. In multivariate analyses using a Cox's proportional-hazards regression model with 9 variables, nm23-HI showed the fourth greatest contribution to patient survival following lymph-node metastasis, Tnm stage and menopausal status. No significant contribution was shown for c-erbB-2 expression. nm23-HI, but not nm23-H2, may perform a role in disease prognosis in addition to its participation in cancer metastasis. It may have value for predicting long-term survival of human breast-cancer patients.

Adult↗
PubMed1993

Rejection of an IA+ variant line of FBL-3 leukemia by cytotoxic T lymphocytes with CD4+ and CD4-CD8- T cell receptor-alpha beta phenotypes generated in CD8-depleted C57BL/6 mice.

FBL-3N is an MHC class II Ag+ variant line that was obtained spontaneously during maintenance of Friend virus-induced leukemia FBL-3 in an athymic C57BL/6 (B6) mouse. Inocula of FBL-3N, but not the parental FBL-3 tumor, regressed after initial growth in CD8-depleted, syngeneic B6 mice. The cellular mechanisms by which FBL-3N was rejected in these mice were investigated in this study. We demonstrated that CTL with both CD4+ and CD4-CD8-TCR-alpha beta phenotypes were generated in mixed lymphocyte tumor cell culture spleen cells obtained from CD8-depleted B6 mice that had rejected FBL-3N by in vitro stimulation with mitomycin C-treated FBL-3N. After adoptive transfer of these CTL that were generated in vitro into athymic B6 mice, challenge with the FBL-3N tumor resulted in tumor regression after its initial growth. Thus, CD4+ and CD4-CD8-TCR-alpha beta CTL mediated rejection of the FBL-3N tumor in CD8-depleted B6 mice. Furthermore, the findings that depletion of B6 mice of CD4+ cells in addition to CD8+ cells abrogated the rejection of FBL-3N and generation of CTL in mixed lymphocyte tumor cell culture spleen cells suggest that CD4+ cells were required not only as a source of CD4+ CTL, but also as helper cells for generation of CD4-CD8-TCR-alpha beta CTL. Tumor Ag recognition of CD4-CD8-TCR-alpha beta CTL was restricted to Db, like that of classical CD8+ CTL, but the restriction appeared to be less obligatory than that of CD8+ CTL.

Animals↗
PubMed1993

Relationship between the anti-HTLV-1 antibody level, the number of abnormal lymphocytes and the viral-genome dose in HTLV-1-infected individuals.

Two distinct diseases, adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), develop in a minor population of HTLV-1 carriers. We examined the relationship between the viral genome dose in the peripheral-blood mononuclear cells and the serological response in HTLV-1 carriers and patients with HAM/TSP. The antibody titer to HTLV-1 gag and env proteins, as well as the frequency of an antibody response to viral protein p40tax and the titer, increased with increasing viral genome dose. However, the number of abnormal lymphocytes was not directly related to the host viral load. Patients with HAM/TSP generally showed a higher genome dose than healthy carriers and also had higher antibody titers than healthy carriers with the same HTLV-1 load, supporting the existence of an augmented immune response in these patients. These findings suggest that the antibody titer to HTLV-1 genome products, and not the number of abnormal lymphocytes, intimately reflects the approximate viral load in HTLV-1-infected individuals.

Adolescent↗