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Biomedical subjects

Hiroaki Mitsuya

Publications and source records attributed to Hiroaki Mitsuya.

68 records · Page 4Linked to original sources
PubMed2002

Tryptophanyl phosphoramidates as prodrugs of synadenol and its E-isomer: synthesis and biological activity.

Phosphorotryptophanates 2c and 3c were synthesized and investigated as prodrugs of synadenol (2a) and its E-isomer 3a. The antiviral activity of 2c corresponds to parent analogue 2a but it is lower than that of phenylphosphoralaninate 2b. This may indicate an enzymatic cleavage of phosphorotryptophanate 2c to 2a before or after entering the host cells. The E-isomer 3c was effective only against EBV with parameters suggesting intracellular delivery of the respective phosphate. Compound 2c has a moderate but selective activity against solid tumors.

Adenine↗
PubMed2002

Decreased susceptibility of leukemic cells with PIG-A mutation to natural killer cells in vitro.

The cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNH clone expands. Many reports have suggested that an immune mechanism operates to cause bone marrow failure in some patients with PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow diseases, one hypothesis is that the PNH clone escapes immune attack, producing a survival advantage by immunoselection. To test this hypothesis, we examined the sensitivity of blood cells, with or without PIG-A mutations, to killing by natural killer (NK) cells, using 51Cr-release assay in vitro. To both peripheral blood and cultured NK cells, PIG-A mutant cells prepared from myeloid and lymphoid leukemic cell lines were less susceptible than their control counterparts (reverted from the mutant cells by transfection with a PIG-A cDNA). NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was perforin-dependent. There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between PIG-A mutant and control cells. From these results, we infer that PIG-A mutant cells lack molecules needed for NK activation or to trigger perforin-mediated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes.

Cell Survival↗
PubMed2002

Cross-linking HLA-DR molecules on Th1 cells induces anergy in association with increased level of cyclin-dependent kinase inhibitor p27(Kip1).

HLA class II molecules play pivotal roles in antigen presentation to CD4+ T cells. We investigated signaling via HLA-DR molecules expressed on CD4+ T cells. When HLA-DR or CD3 molecules on cloned CD4+ T cells were cross-linked by solid-phase mAbs, T cells proliferated, and this resulted in anergy. Whereas cross-linking of HLA-DR and CD3 resulted in secretion of the same levels of IFN-gamma and IL-8, secretion of IL-10 induced by cross-linking of HLA-DR was less than that induced by cross-linking of CD3 on CD4+ T cells. Interestingly, expression of p27(Kip1) but not p21(Cip1) increased after stimulation by either anti-HLA-DR or anti-CD3 mAb. This was indeed the case, when T cells were rendered anergic using a soluble form of antigenic peptide. In contrast, T cells stimulated by peptide-pulsed PBMC expressed little p27(Kip1). We propose that signaling via HLA-DR molecules on CD4+ T cells at least in part contributes to the induction of T cell anergy, through the upregulated expression of the p27(Kip1). The implication of our finding is that HLA-DR molecules play a role in human T cell anergy induced by a soluble form of antigenic peptide.

Amino Acid Sequence↗
PubMed2002

Genetic evidence of transmission of human T cell lymphotropic virus type 1 between spouses.

Sexual transmission of human T cell lymphotropic virus type 1 (HTLV-1) is considered to be an important route of infection in adults. However, no direct evidence has been reported that supports this observation. To address this issue, sequence variations of the gp46 (envelope)-coding region of HTLV-1 were determined in 13 patients infected with HTLV-1 who experienced seroconversion and in their spouses. Twenty-two nucleotide changes that were different from the reference sequence of lambdaATK-1 were identified. However, the gp46 sequences found were identical within each married couple. HTLV-1 proviral DNA loads measured in 11 of these couples varied from 10 to 3430 copies per 10(5) PBMC, and the proviral DNA loads of spouses often differed. This study provides the first genetic confirmation of the transmission of HTLV-1 from a carrier spouse to his/her partner. The findings also suggest that host-related factors play a more important role than do virus-specific factors in determining HTLV-1 proviral DNA load.

Base Sequence↗
PubMed2002

Mechanism of hypercalcemia in adult T-cell leukemia: overexpression of receptor activator of nuclear factor kappaB ligand on adult T-cell leukemia cells.

Hypercalcemia is one of the most frequent and serious complications in patients with adult T-cell leukemia (ATL) and is due to marked bone resorption by accumulation of osteoclasts (OCLs). Although several cytokines such as interleukin 1 and parathyroid hormone-related protein are thought to be involved in the development of high serum Ca(++) levels, its precise underlying mechanism remains unknown. This study analyzed the expression of various genes that are thought to regulate serum Ca(++) levels in ATL and showed that the overexpression of the receptor activator of nuclear factor kappaB (RANK) ligand gene correlated with hypercalcemia. ATL cells from patients with hypercalcemia, which highly expressed the transcripts of the RANK ligand (RANKL) gene, induced the differentiation of human hematopoietic precursor cells (HPCs) into OCLs in vitro in the presence of macrophage colony-stimulating factor (M-CSF). In contrast, ATL cells from patients without hypercalcemia did not induce such differentiation, suggesting that the induction of the differentiation correlated with the expression of the RANKL gene in ATL cells. Cell differentiation was suppressed by osteoprotegerin/Fc, an inhibitor of RANKL, indicating that such differentiation occurred through the RANK-RANKL pathway. In addition, direct contact between ATL cells and HPCs was essential for the differentiation, suggesting that not the soluble form but membrane-bound RANKL played a role in this process. These results strongly suggested that ATL cells induce the differentiation of HPCs to OCLs through RANKL expressed on their surface, in cooperation with M-CSF, and ultimately cause hypercalcemia.

Adult↗
PubMed2002

Frequent detection of T cells with mutations of the hypoxanthine-guanine phosphoribosyl transferase gene in patients with paroxysmal nocturnal hemoglobinuria.

Acquired mutations of the PIG-A gene result in the hemolysis characteristic of paroxysmal nocturnal hemoglobinuria (PNH). Although the etiology of the mutation(s) is unclear, mutable conditions have been suggested by the coexistence of multiple clones with different mutations of PIG-A and by the appearance of leukemic clones in patients with PNH. This study sought to test this hypothesis by examining the frequency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene mutations, identified by both resistance to 6-thioguanine (6-TG) and gene analysis. T-cell colonies resistant to 6-TG formed in methylcellulose culture were found in 8 (67%) of 12 PNH patients and 3 (18%) of 17 age-matched healthy volunteers (P <.02, Fisher exact probability test). The incidence of resistant colonies ranged from 40 to 367 (mean 149, x 10(-7)) in the 8 patients and from 1 to 16 (mean 7, x 10(-7)) in the 3 healthy donors. Thus, the HRPT gene mutated more frequently in patients with PNH than in healthy controls (P <.02, Mann-Whitney test). Analysis of bone marrow cells supported these findings. Like the PIG-A mutations in PNH, the HPRT mutations were widely distributed in the coding regions and consisted primarily of base deletions. Unlike PNH cells, 6-TG-resistant cells expressed CD59, indicating that the HPRT mutations did not occur in PNH clones. No correlation was noted between HPRT mutation frequency and content of therapy received by the patients. It is concluded that in PNH patients, conditions exist that favor the occurrence of diverse somatic mutations in blood cells.

Adult↗
PubMed2002

The absence of the p15INK4B gene alterations in adult patients with precursor B-cell acute lymphoblastic leukaemia is a favourable prognostic factor.

We examined deletion and methylation of the p15INK4B (p15) and p16INK4A (p16) genes, using Southern blotting and methylation-specific polymerase chain reaction (PCR), in 70 untreated adult patients with precursor B-cell acute lymphoblastic leukaemia (PBC-ALL) and analysed the relationship between their genetic changes and clinical outcome. Methylation and homozygous deletion of the p15 gene were detected in 30 (43%) and 18 (26%) patients, while those of the p16 gene were found in 16 (23%) and 11 (16%) patients respectively. Thirteen out of 17 patients with wild-type p15 gene showed expression of p15 mRNA, whereas 31 out of 39 patients with alteration (deletion and methylation) of the p15 gene showed no p15 mRNA expression by reverse transcription-PCR, suggesting that alterations of the p15 gene are highly associated with loss of p15 mRNA expression. Disease-free survival (DFS) at 4 years in patients with wild-type p15 gene is 33%, compared with 4% of those with p15 gene alterations (P = 0.049). Multivariate analysis showed that the absence of p15 gene alterations was an independent significant favourable prognostic factor for longer DFS (P = 0.0001). These results suggest that alterations in the p15 but not p16 gene can be used as a genetic prognostic indicator in PBC-ALL.

Adolescent↗
PubMed2002

A potent human immunodeficiency virus type 1 protease inhibitor, UIC-94003 (TMC-126), and selection of a novel (A28S) mutation in the protease active site.

We identified UIC-94003, a nonpeptidic human immunodeficiency virus (HIV) protease inhibitor (PI), containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranyl urethane (bis-THF) and a sulfonamide isostere, which is extremely potent against a wide spectrum of HIV (50% inhibitory concentration, 0.0003 to 0.0005 microM). UIC-94003 was also potent against multi-PI-resistant HIV-1 strains isolated from patients who had no response to any existing antiviral regimens after having received a variety of antiviral agents (50% inhibitory concentration, 0.0005 to 0.0055 microM). Upon selection of HIV-1 in the presence of UIC-94003, mutants carrying a novel active-site mutation, A28S, in the presence of L10F, M46I, I50V, A71V, and N88D appeared. Modeling analysis revealed that the close contact of UIC-94003 with the main chains of the protease active-site amino acids (Asp29 and Asp30) differed from that of other PIs and may be important for its potency and wide-spectrum activity against a variety of drug-resistant HIV-1 variants. Thus, introduction of inhibitor interactions with the main chains of key amino acids and seeking a unique inhibitor-enzyme contact profile should provide a framework for developing novel PIs for treating patients harboring multi-PI-resistant HIV-1.

Alanine↗
PubMed2002

[The current status of structured treatment interruption].

There is considerable interest in structured treatment interruption(STI), which has been stimulated by certain evidence that treatment interruption may boost the infected individual's immune response to HIV-1. There are several distinct settings where STI may be instituted: (i) STI for those with acute HIV-1 infection, (ii) STI for those with chronic infection, and (iii) STI before switching drugs. A number of STI protocols have been evaluated and certain promising results have been reported in certain groups of HIV-1-infected individuals while viral rebound as the consequence of interruption has been seen in other patients and drug failure to re-suppress HIV-1 upon re-starting therapy, STI-associated CD4+ cell decline and viral drug resistance emergence have been serious concern. Currently, STI cannot be recommended in clinical settings. Randomized, controlled trials to determine the applicability of STI for therapy of HIV-1 infection are required.

Anti-HIV Agents↗
PubMed2001

Amino acid substitutions in Gag protein at non-cleavage sites are indispensable for the development of a high multitude of HIV-1 resistance against protease inhibitors.

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.

Amino Acid Sequence↗
PubMed2001

Identification of amino acid residues critical for LD78beta, a variant of human macrophage inflammatory protein-1alpha, binding to CCR5 and inhibition of R5 human immunodeficiency virus type 1 replication.

In an attempt to determine which amino acid(s) of LD78beta, a variant of human macrophage inflammatory protein-1alpha, plays a critical role in the interaction with CCR5, we generated six LD78beta variants with an amino acid substituted to Ala at the NH(2) terminus of LD78beta. There was no significant difference in eliciting Ca(2+) flux and chemotaxis among the variants with the exception of LD78beta(T9A) showing a substantially reduced activity. The comparative order for human immunodeficiency virus type 1 (HIV-1) replication inhibition was: LD78beta(P8A) > LD78beta(D6A) > LD78beta(WT), LD78beta(L3A) > LD78beta(T7A), LD78beta(P2A) > LD78beta(T9A). In binding inhibition assays of LD78beta variants using 2D7 monoclonal antibody and (125)I-labeled macrophage inflammatory protein-1alpha, the comparative order was: LD78beta(P8A), LD78beta(D6A) > LD78beta(WT) > LD78beta(L3A) > LD78beta(T7A) > LD78beta(T9A), LD78betaP2A). The order for CCR5 down-regulation induction was comparable to that for binding inhibition. The present data suggest that Pro-2, Asp-6, Pro-8, and Thr-9 are critical for LD78beta binding to CCR5 and HIV-1 replication inhibition, and that LD78beta binding to CCR5, regardless of affinity, is sufficient for the initial signal transduction of LD78beta, whereas the greater anti-HIV-1 activity requires the greater magnitude of binding. The data also suggest that LD78beta variants with appropriate amino acid substitution(s) such as LD78beta(D6A) and LD78beta(P8A) may represent effective chemokine-based anti-HIV-1 therapeutics while preserving LD78beta-CCR5 interactions.

Amino Acid Sequence↗