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Biomedical subjects

K Ikuta

Publications and source records attributed to K Ikuta.

At least 253 records · Page 14Linked to original sources
PubMed1993

The AP-1 binding site in the feline immunodeficiency virus long terminal repeat is not required for virus replication in feline T lymphocytes.

Sequences of 31 bp containing putative AP-1 and AP-4 binding sequences in the U3 region of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were deleted and the basal promoter activity of the LTR was measured by the chloramphenicol acetyltransferase (CAT) assay. The activity of the FIV LTR was reduced in Felis catus whole foetus 4 (fcwf-4) cells and Crandell feline kidney cells by this deletion. Cotransfection of murine c-Fos or c-Jun expression plasmids with the FIV LTR-CAT reporter plasmid into fcwf-4 cells revealed that FIV LTR could be activated by c-Fos but not c-Jun in the cells. The mutated LTR was introduced into an infectious molecular clone of FIV and the replication rate and the cytopathogenic activity of the mutant were compared with those of the wild-type in two feline CD4-positive T lymphoblastoid cell lines. It was found that the rate and activity of the mutant were almost the same as those of the wild-type. From these data, we conclude that the 31 bp fragment is important for achieving maximal expression of the FIV genome, but not required for the replication of FIV in feline T lymphocytes.

Animals↗
PubMed1993

Persistently human immunodeficiency virus type 1-infected T cell clone expressing only doubly spliced mRNA exhibits reduced cell surface CD4 expression.

Several cell clones possessing the human immunodeficiency virus type 1 (HIV-1) genome, consisting of an almost full-length DNA sequence, were isolated by limiting dilution of the clonal cell line M10 derived from MT-4 that survived infection with HIV-1 vpr mutant (M10/vpr-). One of the isolated clones (termed Vpr-1) expressed only doubly spliced mRNA, but not unspliced or singly spliced mRNA. Western blots of Vpr-1 revealed the presence of the nef translation product, although no expression of major structural genes such as gag, pol, and env was detected by indirect immunofluorescence and assay of reverse transcriptase activity. These HIV-1 phenotypes differed greatly from those of the original M10/vpr-, most of which expressed major structural HIV-1 proteins. Despite undetectable levels of env expression in Vpr-1, CD4 antigens were greatly down-modulated on the surface without alteration of steady-state levels of CD4 mRNA expression, similar to M10/vpr-. These HIV-1 phenotypes in Vpr-1 did not change after the treatment of the cells with both phorbol 12-myristate 13-acetate and phytohemagglutinin. Therefore, the abnormal HIV-1 life cycle in Vpr-1 seems to be due to some viral factor(s), as well as cellular factors. Thus, Vpr-1 could be a useful model for understanding one HIV-1 latent form.

Base Sequence↗
PubMed1993

[High-dose busulfan, VP-16 and ACNU therapy with stem cell transplantation for the treatment of children with acute leukemia].

In order to strengthen the anti-leukemia effect, we developed a new conditioning regimen with high dose busulfan, VP-16 and ACNU (BVA) for cytoreduction before stem cell transplantation. Fourteen patients with refractory acute leukemia received allogeneic bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT) after conditioning with the BVA regimen, 7; allogeneic BMT, 1; syngeneic BMT, 6; PBSCT. # Seven patients were transplanted in the first complete remission, and 8 patients were in their second or third remission. Although total body irradiation or cyclophosphamide was not included in this regimen, engraftment was obtained in all cases. Two patients suffered relapse, and one patient died of cytomegalovirus interstitial pneumonitis (IP) 64 days after PBSCT. The other 11 patients are alive and free of disease at a median follow up time of 647 days (98-1235 days). Major regimen-related toxicity was pulmonary complications such as IP (3 cases) and pulmonary edema (2 cases). However, all patients recovered rapidly following steroid therapy. The results indicate that this conditioning regimen is highly effective for the treatment of childhood acute leukemia.

Acute Disease↗
PubMed1993

[HIV vaccine trials].

There has been significant progress on several candidate vaccines against HIV infection over the past few years, i.e., live recombinant virus vaccines, inactivated virus vaccines, and purified antigens (subunit vaccines). There have been many phase I studies in healthy volunteers uninfected with HIV, as well as phase II trials with high-risk uninfected volunteers. Most of them focused on productions of neutralizing antibodies and cytotoxic T lymphocytes (CTLs) against HIV Env proteins. In addition, host immune responses, particularly cell-mediated immunity, were shown to be a major immune defense mechanism which may provide resistance to HIV spread leading to AIDS, indicating the possibility to develop prophylactic vaccine which may prevent the onset of AIDS in HIV carriers. However, various studies have implicated the concurrent activities of several HIV proteins, such as Env gp120, Env gp41, Tat, and Nef, in the induction of immunodeficiency. Therefore, the role of HIV-1 proteins in inducing immunodeficiency after HIV vaccination should be considered. Here, I introduce a recombinant BCG (rBCG) vaccine which expresses the immunodominant gp120 epitope. The tuberculosis vaccine strain Mycobacterium bovis BCG is a widely used vaccines with a low rate of serious complications. To develop a vaccine expressing an immunodominant epitope in the gp120 V3-loop, we used our system to express and secrete an epitope which was fused with an extracellular alpha antigen of Mycobacterium kansasii. Balb/c mice inoculated with this rBCG, rapidly induced HIV-specific CTLs. Target cell lysis was restricted to the murine class I major histocompatibility complex, H-2d.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Vaccines↗
PubMed1993

[Serum-free culture for leukemia cells].

Several human and murine acute myeloid leukemia cell lines growing in serum-free and protein-free culture medium are known to be established. In this paper, recent development of serum-free and protein-free culture methods for leukemia cells and the characteristics of the established cell lines are reviewed. Chemically defined culture medium is preferable as compared with serum-containing culture medium because the latter contains various, undefined factors which make it difficult to investigate the interactions between cell proliferation or differentiation and various growth factors. Investigations using leukemia cell lines which are maintained in a serum-free and protein-free chemically defined medium will provide the important information with respect to cell biology of leukemia cells and leukemia therapy.

Animals↗
PubMed1993

[Treatment and prognosis after late relapse in childhood acute lymphoblastic leukemia].

We evaluated the clinical courses and laboratory features in 13 late-relapse cases of 55 children with acute lymphoblastic leukemia who had been in complete remission for longer than their years. In 8 of 13 cases, leukemia relapsed in bone marrow; 2 with testicular, 1 with central nervous system and one with ovarian involvement. Further, extramedullary relapse not involving bone marrow occurred in 5 cases (4 testicular and 1 CNS). Late-relapse was more frequently observed in boys (37.5%) than in girls (4.4%). Initial age and leukocyte counts were of no value in predicting late-relapse. The relapse rate in cases initially treated by the VPL regimen was twice that of those by a multi-drug regimen. A second prolonged remission was achieved in 5 of 10 cases by combinations of intensive chemotherapy (modified HEX) and irradiation to the testes or CNS. On the contrary, all late relapse patients initially treated by the multi-drug chemotherapy had a poor outcome. More intensive chemotherapy, including high-dose chemoradiotherapy and bone marrow transplantation, should be employed in this group of patients.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1992

Development of gamma delta T-cell subsets from fetal hematopoietic stem cells.

Hematopoietic stem cells (HSC) were isolated from mouse fetus, and their developmental potential was compared with adult HSC. Donor-derived V gamma 3+T cells were detected in fetal thymic lobes, repopulated in vitro with fetal liver HSC, but not in those with adult bone marrow HSC. Single clonogenic fetal HSC gave rise to thymic progeny that include V gamma 3+, other gamma delta+, and alpha beta+ T cells. No V gamma 3+ T cells were detected in adult thymus injected intrathymically with either fetal or adult HSC. These results support a hypothesis that only fetal HSC have the capacity to differentiate into V gamma 3+ T cells in the fetal thymic microenvironment, and that the developmental potential of HSC may change during ontogeny.

Aging↗
PubMed1992

Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation.

The interaction of the mouse c-kit receptor, designated Kit receptor, and steel factor promotes the proliferation and differentiation of hematopoietic progenitor cells. Monoclonal antibodies against the extracellular portion of the mouse Kit receptor were established. Five percent to 10% of total bone marrow cells expressed the Kit receptor, and half of them lack the expression of lineage markers. The Kit receptor was expressed on 70-80% of Thy-1.1lo Lin-Sca-1+ cells, which express Thy-1.1 antigen at a low level and constitute approximately 0.05% of adult bone marrow and fetal liver; by previous studies, these cells have been shown to be highly enriched for multipotent hematopoietic stem cells (HSCs) and are the only hematopoietic cell subset with this activity. Spleen colony formation and long-term multilineage reconstitution activities were contained in the Kit+ but not in the Kit- subpopulations of Thy-1lo Lin-Sca-1+ cells from adult bone marrow, suggesting that the Kit receptor is expressed on HSCs from the earliest stage-i.e., pluripotent HSCs. The role of steel factor in the development and self-renewal of HSCs was tested with Sl/Sl homozygote fetuses, which lack genes to encode functional steel factor. They were shown to have 30-40% of the number of HSCs on days 13-15 when compared with normal litermates. However, the absolute number of HSCs increased during fetal development in the Sl/Sl mice. The results suggest that the Kit receptor-steel factor interaction may not be essential for the initiation of hematopoiesis and the self-renewal of (at least) fetal HSCs.

Animals↗
PubMed1992

Determination and validity of critical velocity as an index of swimming performance in the competitive swimmer.

The purpose of this investigation was to test whether the concept of critical power used in previous studies could be applied to the field of competitive swimming as critical swimming velocity (vcrit). The vcrit, defined as the swimming velocity over a very long period of time without exhaustion, was expressed as the slope of a straight line between swimming distance (dlim) at each speed (with six predetermined speeds) and the duration (tlim). Nine trained college swimmers underwent tests in a swimming flume to measure vcrit at those velocities until the onset of fatigue. A regression analysis of dlim on tlim calculated for each swimmer showed linear relationships (r2 greater than 0.998, P less than 0.01), and the slope coefficient signifying vcrit ranged from 1.062 to 1.262 m.s-1 with a mean of 1.166 (SD 0.052) m.s-1. Maximal oxygen consumption (VO2max), oxygen consumption (VO2) at anaerobic threshold, and the swimming also velocity at the onset of blood lactate accumulation (vOBLA) were also determined during the incremental swimming test. The vcrit showed significant positive correlations with VO2 at anaerobic threshold (r = 0.818, P less than 0.01), vOBLA (r = 0.949, P less than 0.01) and mean velocity of 400 m freestyle (r = 0.864, P less than 0.01). These data suggested that vcrit could be adopted as an index of endurance performance in competitive swimmers.

Adolescent↗
PubMed1992

Relationship between superoxide dismutase (SOD) and viral liver diseases.

Immunohistochemical evaluation of Cu, Zn- and Mn-superoxide dismutase (SOD) activity in various viral liver diseases was performed by the peroxidase-conjugated antibody indirect method. Anti-human Cu, Zn-SOD (rabbit) and anti-human Mn-SOD (guinea-pig) derived and purified from SOD of human erythrocytes and placentas were used to determine SOD distribution in liver tissues. SOD in the liver tissues was detected in 68 inpatients of our unit. They consisted of 23 cases with chronic hepatitis caused by hepatitis B virus (13) and hepatitis C virus (10), 24 with liver cirrhosis caused by hepatitis B virus (5) and hepatitis C virus (19) (15: compensatory, 9: decompensatory) and 21 with hepatocellular carcinoma caused by hepatitis B virus (2) and hepatitis C virus (18) complicated of liver cirrhosis. In viral liver diseases, SODs in the liver tissues were distributed to hepatocytes mainly in the pattern of cytoplasmic diffusion. The incidence of immunohistochemical Cu, Zn-SOD and Mn-SOD were 47.8% and 56.5% in chronic hepatitis, 93.3% and 86.7% in compensated liver cirrhosis, 11.1% and 22.2% in decompensated liver cirrhosis, respectively. The aggression of viral liver disease was accompanied with the decrease of SOD concentration in the liver tissues. Hepatocellular carcinoma cells were negative for Mn-SOD in all cases, and weakly positive for Cu, Zn-SOD in 2 out of 21 cases. Comparatively strongly positive SOD findings were obtained from normal regions neighboring carcinomas. A close relationship between the depletion of SOD in liver tissues and carcinogenesis in viral liver diseases was observed.

Carcinoma, Hepatocellular↗
PubMed1992

Monoclonal antibodies to a CD4 peptide derivative which includes the region corresponding to an immunoglobulin CDR3: evidence of the involvement of pre-CDR3-related region in HIV-1 and host cell interaction.

A CD4 peptide of amino acid residues 68-130 [CD4(68-130)], which had the capacities to inhibit HIV-1 replication and HIV-1-induced syncytium formation, was used as an immunogen for the preparation of mAb. The mAbs prepared were classified into at least five types (I-V) in terms of their recognition sites by ELISA using various kinds of smaller CD4 peptides. Among them, the type I mAb no. 35 recognizing amino acid residues 72-84, which lies just before the region corresponding to an immunoglobulin third complementarity-determining region (CDR3), showed the strongest effects in reducing both HIV-1 infection and HIV-1-induced syncytium formation, although a large amount of no. 35 mAb was necessary to reduce such HIV-1 activities compared with those of anti-Leu-3a and OKT4A mAbs which recognize CD4 epitopes near a portion corresponding to an immunoglobulin CDR2. Western blot analysis showed that the reactivities of CD4 molecule in CD4-positive cells or sCD4 molecule with types I-V mAbs were stronger than that with anti-Leu-3a mAb. Flow cytometry showed that no. 35 mAb was faintly reactive with native CD4 molecule on cell surface at the concn showing the inhibitory effects on HIV-1 infection and syncytium formation. In addition, a smaller peptide CD4(66-92), one of the good epitope peptides for no. 35 mAb, also showed strong inhibitory effect on HIV-1 infection as well as a weaker inhibitory effect on syncytium formation. These results suggest that, in addition to the CD4 CDR2-related region, the pre-CDR3-related region is also involved in the early events of the interactions between the host cell and HIV-1.

Amino Acid Sequence↗
PubMed1992

Major core proteins, p24s, of human, simian, and feline immunodeficiency viruses are partly expressed on the surface of the virus-infected cells.

We have previously shown the expression of human immunodeficiency virus type 1 (HIV-1) major gag protein, p24, on the surface of persistently HIV-1-infected cells by using murine monoclonal antibodies (mAb). We now report that the cell surface gag p24 antigen expression is a universal phenomenon among HIV-1, simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). The mAbs prepared by immunization with purified HIV-1 particles were used as antibodies cross-reactive to HIV-1 and SIVagmp24 antigens. The mAbs to FIV p24 were raised against the gag precursor 50 kDa protein of FIV, which was expressed by Baculovirus vector. The p24 antigen expression on the cell surface was detectable in certain combinations of virus-host cell systems in all of these viruses. Since these p24 regions of the animal viruses seem to play as important a role in cell-mediated immunity as that of HIV-1, the p24 applicability as a candidate epitope for vaccine development could be evaluated in those animals.

Animals↗
PubMed1992

Cells surviving infection by human immunodeficiency virus type 1: vif or vpu mutants produce non-infectious or markedly less cytopathic viruses.

Under conditions in which a clonal cell line (M10) isolated from a human T cell lymphotrophic virus type I-transformed MT-4 cell line was completely killed by infection with wild-type human immunodeficiency virus type 1 (HIV-1), equivalent M10 cells survived infection with HIV-1 vif, vpr or vpu mutant virus after transient cytopathic effects. Several cell clones, which were isolated from the proliferating M10 cells after infection with vif and vpu mutant viruses (M10/vif- and M10/vpu-), had heterogeneous HIV-1 phenotypes in terms of HIV-1 antigen expression, their syncytium forming capacity, reverse transcriptase activity and the infectivity of HIV-1 particles produced. When the replication kinetics of the HIV-1 particles produced were assayed in M10 cells, the clones could be classified into three types, i.e. type I producing non-infectious HIV-1, type II producing infectious HIV-1 with low replicative ability and type III producing infectious HIV-1 with a replicative ability similar to that of wild-type HIV-1. HIV-1 major viral cell proteins and virus particle fractions were almost typical in types II and III but not in type I. Electron microscopic examination of particles released by I, II and III clones revealed rare defective, predominantly defective and essentially normal virions, respectively. Northern and Southern blot analyses revealed no apparent deletion in the proviral DNA and mRNA prepared from these clones, except in the case of type I and II clones isolated from M10/vpu- which contained large deletions in the mRNAs for gag and gag-pol proteins. Thus, M10 cells surviving infection with HIV-1 vif or vpu mutants are heterogeneous, persistently expressing HIV-1 antigens and producing non-infectious or less cytopathic virus.

Blotting, Northern↗
PubMed1992

Human immunodeficiency virus type 1 (HIV-1) superinfection of a cell clone converting it from production of defective to infectious HIV-1 is mediated predominantly by CD4 regions other than the major binding site for HIV-1 glycoproteins.

A cell clone, L-2, which produces non-infectious doughnut-shaped human immunodeficiency virus type 1 (HIV-1) particles, was permissive for HIV-1 superinfection, which resulted in the production of infectious particles. The superinfection showed slow kinetics compared with primary HIV-1 infection of M10 cells, the parent of the L-2 cell clone. Inhibition studies on the superinfection of L-2 cells using several CD4-related reagents showed that the CD4 molecule was an essential component of the receptor for superinfection. Strong inhibitory effects were obtained using CD4 peptides such as CD4(68-130), which includes a portion homologous to the immunoglobulin third complementarity-determining region (CDR3), as well as recombinant soluble CD4. In contrast, a CD4(45-60) peptide, which includes most of the CDR2-related region, was not effective, although the Leu-3a monoclonal antibody (MAb), which recognizes a site near the CDR2-related region, did slightly, but significantly, delay the superinfection kinetics. Comparative flow cytometry of L-2 and M10 cells revealed that the cell surface of L-2 cells despite expressing HIV-1 env protein, reacted slightly with OKT4 or anti-CD4(68-130) MAb, but not with Leu-3a or OKT4A MAb. In contrast, no reaction was detected with any of these anti-CD4 MAbs on the surface of another HIV-1 superinfection-resistant cell clone, MOLT-#8IIIB-14, which expresses HIV-1 env proteins but does not produce infectious HIV-1 particles. These results strongly suggest that expression of the CD4 major receptor site for primary HIV-1 infection is preferentially decreased on the surface of L-2 cells, but that the OKT4 epitope and the nearby region corresponding to immunoglobulin CDR3 remain exposed on the cell surface. Consequently, the CD4 CDR3-related region could play a major role as the receptor for the superinfection reported here.

Binding Sites↗
PubMed1992

Highly conserved epitope domain in major core protein p24 is structurally similar among human, simian and feline immunodeficiency viruses.

Linear B cell epitopes were mapped on the major core protein p24 of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIVAGM) and feline immunodeficiency virus (FIV) using a fusion protein-based method and murine monoclonal antibodies reactive against the p24 antigens expressed on the surface of HIV-1- and FIV-infected cells. The results suggest that the sites identified here are encoded at similar positions in the three virus genomes and consist of highly conserved epitopes, which could exhibit immunodominance.

Amino Acid Sequence↗
PubMed1992

A unique monoclonal antibody that recognizes mature p17 of HIV-1 but not its precursor.

The entire and partial gag regions of human immunodeficiency virus type 1 (HIV-1) were overproduced in Escherichia coli and used for epitope mapping of antibodies against p17. We found that a mouse monoclonal antibody to p17, V17 recognizes the mature p17 but not the unprocessed Gag proteins containing the entire p17 moiety. Further analysis revealed that V17 recognizes the C-terminal 12-amino-acid region of p17 having free C-terminus. This monoclonal antibody may be useful for monitoring the maturation of virus particles.

Antibodies, Monoclonal↗