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Biomedical subjects

S Izumo

Publications and source records attributed to S Izumo.

At least 73 records · Page 4Linked to original sources
PubMed1999

A rat model of human T lymphocyte virus type I (HTLV-I) infection: in situ detection of HTLV-I provirus DNA in microglia/macrophages in affected spinal cords of rats with HTLV-I-induced chronic progressive myeloneuropathy.

To investigate the pathogenetic role of human T lymphocyte virus type I (HTLV-I) in central nervous system disease, a rat model for HTLV-I-associated myelopathy/tropical spastic paraparesis, designated as HAM rat disease, has been established. Wistar-King-Aptekman-Hokudai strain rats with induced HTLV-I infection develop a chronic progressive myeloneuropathy with paraparesis of hind limbs after an incubation period of 15 months. In the affected spinal cord in these rats, white matter degeneration, demyelination and vacuolar change with microglia/macrophage infiltration are present as are the provirus DNA and the virus mRNA. To identify infected cells in the affected lesions, we carried out in situ hybridization of amplified fragments of the provirus DNA by polymerase chain reaction on thin sections, plus immunohistochemistry on the same sections. The provirus DNA was localized in some microglia/macrophages in the spinal cord lesion. In addition, the HTLV-I provirus was clearly evident not only in ED-1-negative lymphoid cells but also in ED-1-positive macrophages from lymph nodes. These observations suggest that cells of microglia/macrophage lineage may be one of dominant viral reservoirs in the spinal cords and lymph nodes in HAM rat disease. These infected microglia/macrophages may relate to cause the myeloneuropathy through neurotoxic cytokine synthesis.

Animals↗
PubMed1999

Activation of macrophages/microglia with the calcium-binding proteins MRP14 and MRP8 is related to the lesional activities in the spinal cord of HTLV-I associated myelopathy.

Macrophages and microglia may play an important role in the pathogenesis of chronic inflammatory process in HTLV-I associated myelopathy (HAM) and tropical spastic paraparesis (TSP). However, the etiology and cellular mechanism of chronic inflammation are poorly understood in HAM/TSP. To help to define the roles of macrophages and microglia we analyzed the various patterns of macrophage and microglia activation in the central nervous system (CNS) of HAM/TSP using several monoclonal antibodies recognizing the different states of activation. The results indicate that a large number of macrophages and microglia express both MRP14 and MRP8 in active-chronic inflammatory lesions of the patients with a short duration of illness (2.5 years). In the patient whose duration of illness was 4.5 years, perivascular and parenchymal macrophages and microglia were reactive for MRP8 but not for MRP14. In contrast, MRP14 and MRP8 were negative on the perivascular and parenchymal macrophages and microglia in inactive-chronic lesions and in controls. This study suggests that (a) activated macrophages and microglia as well as CD4+ T lymphocytes and CD8+ cytotoxic T lymphocytes are main components of the inflammatory process in the CNS in HAM/TSP, (b) activation of macrophages and microglia is related to the amount of HTLV-I proviral DNA in situ.

Aged↗
PubMed1999

Expression of the Nkx3.1 homobox gene during pre and postnatal development.

We report here the expression pattern of Nkx3.1, a murine homolog of Drosophila bagpipe, in different stages of embryos and in neonates. Nkx3.1 was expressed in paraxial mesoderm at day 7.5 p.c., in the somite by day 8.0 p.c. and in a subset of vascular smooth muscle cells by day 9.5 p.c. In later stage embryos and neonates, Nkx3.1 was expressed in subpopulations of epithelial cells, particularly in epithelial outgrowths. Nkx3.1 was also expressed in restricted regions of the central nervous system.

Animals↗
PubMed1999

Germline mosaicism of MPZ gene in Dejerine-Sottas syndrome (HMSN III) associated with hereditary stomatocytosis.

We report on two sisters with Dejerine-Sottas syndrome (DSS) who had a heterozygous Gly 167 Arg mutation in the myelin protein zero (MPZ) gene and hereditary stomatocytosis (HSt). Genetic haplotype analysis suggested that the allele with the MPZ gene mutation originated from maternal lineage. However, the parents, who were normal clinically and electrophysiologically, had no mutation in the MPZ gene. Therefore, the MPZ gene mutation in these sisters was due to germline mosaicism of the MPZ gene in their mother. Stomatocytosis was detected in their mother and a sister who had no neurological symptoms, and therefore autosomal dominant HSt was suspected in this family. As stomatocytosis is very severe in our patients with DDS, we speculate that the association of DSS with stomatocytosis is coincidental but may have additively affected erythrocyte morphology. To our knowledge, these are the first familial cases of DSS with a mutation due to germline mosaicism of the MPZ gene to be reported.

Adult↗
PubMed1999

Modulation by pathophysiological stimuli of the shear stress-induced up-regulation of endothelial nitric oxide synthase expression in endothelial cells.

OBJECTIVE: Fluid shear stress (the frictional force resulting from blood flow) is a principal regulator of endothelial nitric oxide synthase (eNOS) expression. We examined the responses of eNOS messenger ribonucleic acid (mRNA) levels to dynamic shear stimuli in the presence of pathological risk modifiers. METHODS: Confluent bovine aortic endothelial cells were subjected in vitro to shear stress (using a cone-plate viscometer) and to hydrostatic pressure (using a custom-built pressure chamber device). eNOS mRNA levels were quantitated by densitometric analysis of Northern blots. RESULTS: In contrast to steady laminar shear stress, which elevated eNOS mRNA levels in a time- and dose-dependent manner (2.9- and 3.6-fold after 6 h at 4 and 20 dyn/cm2, respectively), steady hydrostatic pressure of 150 mm Hg decreased eNOS mRNA levels by 46%. eNOS mRNA up-regulation by shear stress was reversible after cessation of flow, although it was not influenced by previous shear exposure, and it was not mediated by a stable transferable factor. eNOS mRNA up-regulation by sinusoidal shear stress was frequency-dependent, with a moderate response at 1-Hz oscillating shear and no change at 0.3 Hz. Hypoxia (3% O2) suppressed eNOS mRNA expression by 78% under static conditions and by 72% under shear conditions but did not alter the fold induction by shear. Elevated glucose concentrations reduced eNOS mRNA levels in both resting and shear stress-exposed cells but did not reduce the fold induction by shear; the protein kinase C inhibitor calphostin C was without effect. Shear-induced up-regulation of eNOS mRNA was unaffected by changes in the medium partial pressure of CO2/pH, by the Na+/H+-exchanger inhibitor HOE694, or by aspirin. In contrast, the shear response was potentiated by homocysteine. CONCLUSION: Both physical and chemical stimuli regulate eNOS mRNA levels in endothelial cells. Although eNOS mRNA expression is increased by shear stress, it is decreased by hydrostatic pressure, hypoxia, and elevated glucose levels. The effect of shear on eNOS mRNA expression involves a reversible, frequency-dependent process. These in vitro findings suggest possible contributions of the eNOS flow response to atherosclerosis, in the presence of systemic risk factors.

Animals↗
PubMed1999

Marked suppression of T cells by a benzothiophene derivative in patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis.

In a search for new anti-autoimmune agents that selectively suppress activation of autoreactive T cells, one such agent, 5-methyl-3-(1-methylethoxy)benzo[b]thiophene-2-carboxamide (CI-959-A), was found to be effective. This compound, which is known to suppress tumor necrosis factor alpha (TNF-alpha)-induced CD54 expression, inhibited the primary proliferative response of the T cell to antigen (Ag)-presenting cells (APCs) including allogenic dendritic cells (DCs), autologous Epstein-Barr virus-infected B cells, and human T lymphotropic virus type I (HTLV-I)-infected T cells. Autoreactive T cells from patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) spontaneously proliferate in vitro, and their activation is reported to be associated with CD54 expression. The spontaneous proliferation of T cells from patients with HAM/TSP was entirely blocked by CI-959-A. However, in this study, the T-cell proliferation in 15 patients with HAM/TSP was found to depend more extensively on major histocompatibility complex (MHC) class II and CD86 than on CD54 Ags. Since most important APCs for the development of HAM/TSP are DCs and HTLV-I-infected T cells, the effect of CI-959-A on DC generation and on the expression of surface molecules on activated T cells is examined. CI-959-A suppressed recombinant granulocyte-macrophage colony stimulating factor (GM-CSF)- and recombinant interleukin-4-dependent differentiation of DCs from monocytes and inhibited the expression of CD54 and, more extensively, MHC class II and CD86 Ags. CI-959-A showed little toxicity toward lymphoma or HTLV-I-infected T-cell lines or toward monocytes and cultured DCs. These results suggest that CI-959-A might be a potent anti-HAM/TSP agent.

Adult↗
PubMed1999

The role of human T-lymphotropic virus type 1 (HTLV-1)-infected dendritic cells in the development of HTLV-1-associated myelopathy/tropical spastic paraparesis.

The development of human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is closely associated with the activation of T cells which are HTLV-1 specific but may cross-react with neural antigens (Ags). Immature dendritic cells (DCs), differentiated from normal donor monocytes by using recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4, were pulsed with HTLV-1 in vitro. The pulsed DCs contained HTLV-1 proviral DNA and expressed HTLV-1 Gag Ag on their surface 6 days after infection. The DCs matured by lipopolysaccharides stimulated autologous CD4(+) T cells and CD8(+) T cells in a viral dose-dependent manner. However, the proliferation level of CD4(+) T cells was five- to sixfold higher than that of CD8(+) T cells. In contrast to virus-infected DCs, DCs pulsed with heat-inactivated virions activated only CD4(+) T cells. To clarify the role of DCs in HAM/TSP development, monocytes from patients were cultured for 4 days in the presence of the cytokines. The expression of CD86 Ag on DCs was higher and that of CD1a Ag was more down-regulated than in DCs generated from normal monocytes. DCs from two of five patients expressed HTLV-1 Gag Ag. Furthermore, both CD4(+) and CD8(+) T cells from the patients were greatly stimulated by contact with autologous DCs pulsed with inactivated viral Ag as well as HTLV-1-infected DCs. These results suggest that DCs are susceptible to HTLV-1 infection and that their cognate interaction with T cells may contribute to the development of HAM/TSP.

Antigen-Presenting Cells↗
PubMed1999

Identification of the in vivo casein kinase II phosphorylation site within the homeodomain of the cardiac tisue-specifying homeobox gene product Csx/Nkx2.5.

Csx/Nkx2.5, a member of the homeodomain-containing transcription factors, serves critical developmental functions in heart formation in vertebrates and nonvertebrates. In this study the putative nuclear localization signal (NLS) of Csx/Nkx2.5 was identified by site-directed mutagenesis to the amino terminus of the homeodomain, which is conserved in almost all homeodomain proteins. When the putative NLS of Csx/Nkx2.5 was mutated a significant amount of the cytoplasmically localized Csx/Nkx2.5 was unphosphorylated, in contrast to the nuclearly localized Csx/Nkx2.5, which is serine- and threonine-phosphorylated, suggesting that Csx/Nkx2.5 phosphorylation is regulated, at least in part, by intracellular localization. Tryptic phosphopeptide mapping indicated that Csx/Nkx2.5 has at least five phosphorylation sites. Using in-gel kinase assays, we detected a Csx/Nkx2.5 kinase whose molecular mass is approximately 40 kDa in both cytoplasmic and nuclear extracts. Mutational analysis and in vitro kinase assays suggested that this 40-kDa Csx/Nkx2.5 kinase is a catalytic subunit of casein kinase II (CKII) that phosphorylates the serine residue between the first and second helix of the homeodomain. This CKII site is phosphorylated in vivo. CKII-dependent phosphorylation of the homeodomain increased Csx/Nkx2. 5 DNA binding. Serine-to-alanine mutation at the CKII phosphorylation site reduced transcriptional activity when the carboxyl-terminal repressor domain was deleted. Although the precise biological function of Csx/Nkx2.5 phosphorylation by CKII remains to be determined, it may play an important role, as this CKII phosphorylation site within the homeodomain is fully conserved in all known members of the NK2 family of the homeobox genes.

Binding Sites↗
PubMed1999

The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development.

Csx/Nkx2.5 is a vertebrate homeobox gene with a sequence homology to the Drosophila tinman, which is required for the dorsal mesoderm specification. Recently, heterozygous mutations of this gene were found to cause human congenital heart disease (Schott, J.-J., Benson, D. W., Basson, C. T., Pease, W., Silberbach, G. M., Moak, J. P., Maron, B. J., Seidman, C. E. and Seidman, J. G. (1998) Science 281, 108-111). To investigate the functions of Csx/Nkx2.5 in cardiac and extracardiac development in the vertebrate, we have generated and analyzed mutant mice completely null for Csx/Nkx2.5. Homozygous null embryos showed arrest of cardiac development after looping and poor development of blood vessels. Moreover, there were severe defects in vascular formation and hematopoiesis in the mutant yolk sac. Interestingly, TUNEL staining and PCNA staining showed neither enhanced apoptosis nor reduced cell proliferation in the mutant myocardium. In situ hybridization studies demonstrated that, among 20 candidate genes examined, expression of ANF, BNP, MLC2V, N-myc, MEF2C, HAND1 and Msx2 was disturbed in the mutant heart. Moreover, in the heart of adult chimeric mice generated from Csx/Nkx2.5 null ES cells, there were almost no ES cell-derived cardiac myocytes, while there were substantial contributions of Csx /Nkx2.5-deficient cells in other organs. Whole-mount &bgr;-gal staining of chimeric embryos showed that more than 20% contribution of Csx/Nkx2. 5-deficient cells in the heart arrested cardiac development. These results indicate that (1) the complete null mutation of Csx/Nkx2.5 did not abolish initial heart looping, (2) there was no enhanced apoptosis or defective cell cycle entry in Csx/Nkx2.5 null cardiac myocytes, (3) Csx/Nkx2.5 regulates expression of several essential transcription factors in the developing heart, (4) Csx/Nkx2.5 is required for later differentiation of cardiac myocytes, (5) Csx/Nkx2. 5 null cells exert dominant interfering effects on cardiac development, and (6) there were severe defects in yolk sac angiogenesis and hematopoiesis in the Csx/Nkx2.5 null embryos.

Animals↗
PubMed1999

Complex modular cis-acting elements regulate expression of the cardiac specifying homeobox gene Csx/Nkx2.5.

The murine homeobox gene Csx/Nkx2.5 is an evolutionarily highly conserved gene related to the Drosophila tinman gene, which specifies cardiac and visceral mesoderm. Since Csx/Nkx2.5 plays an essential role in heart development, studying its regulation is essential for the better understanding of molecular mechanisms of cardiogenesis and the pathogenesis of congenital heart disease in humans. In this study, we characterized the murine Csx/Nkx2.5 gene and identified two novel untranslated exons, 1a, and 1b, resulting in three different Csx/Nkx2.5 transcripts. To examine the tissue-specific transcriptional regulation in vivo, we analyzed a total of 23 kb of Csx/Nkx2.5 upstream and downstream sequences by generating transgenic embryos carrying lacZ reporter constructs containing various lengths of flanking sequence. With 14 kb of 5' flanking sequence, lacZ expression was observed in the cardiac crescent at E7.5, and in the outflow tract, the interatrial groove, the atrioventricular canal and right and left ventricles, as well as in pharyngeal floor, thyroid primordia, and stomach at E10.5. In adult animals, lacZ expression of the transgene was limited to the atrioventricular junction and the subendocardium of the ventricular septum. Reducing the size of flanking sequence to 3.3 kb of intron 2 restricted lacZ expression to the outflow tract and the basal part of the right ventricle in E10.5 embryos. In contrast, the addition of 6 kb of 3' flanking sequence caused strong expression of the reporter gene in the entire right ventricle. Interestingly, Csx/Nkx2. 5 seems to be negatively regulated by its own gene product, because when lacZ was "knocked-in" to replace the entire coding exons, lacZ expression was much higher in the heart of homozygous embryos than that in the heterozygote. These results indicate that the transcriptional regulatory elements of Csx/Nkx 2.5 seems unexpectedly highly modular, and is temporally regulated in a dynamic manner by different enhancer regions. Since Csx/Nkx2.5-like genes are expressed in all species having a heart, their complex modular organization with multiple enhancers probably reflects progressive addition of regulatory elements during the evolution from a simple heart tube to a complex four-chambered organ.

Alternative Splicing↗
PubMed1999

Detection of human T-lymphotropic virus type I p40tax protein in cerebrospinal fluid cells from patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis.

We investigated the role of viral transcripts of human T-lymphotropic virus type I (HTLV-I) in the cerebrospinal fluid (CSF) cells and peripheral blood mononuclear cells (PBMCs) of patients with human T-lymphotropic virus type I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). To detect the HTLV-I p40tax protein, we developed a new sensitive method of immunohistochemistry combined with tyramide signal amplification and quantitative analysis. Seven patients with HAM/TSP were examined. As controls, four patients with other neurological diseases were examined; two of these patients were infected with HTLV-I and the other two were not. Both the CSF cells and PBMCs were reacted with a monoclonal antibody, Lt-4, for p40tax protein, followed by secondary antibody labeled with horseradish peroxidase. This was visualized by fluorescein directly labeled with tyramide and the number of positive cells was quantified with a Laser Scanning Cytometer. In the samples from patients with HAM/TSP, the HTLV-I p40tax protein was successfully detected by tyramide signal amplification, but not without it. In HAM/TSP patients, 0.04-1.16% of the CSF cells and 0.02-0.54% of PBMCs were positive for the HTLV-I p40tax protein, respectively. The expression of the HTLV-I p40tax protein in the CSF cells was more frequent than that in PBMCs in both HAM/TSP patients and HTLV-I carriers, and was also more frequent in the patients with HAM/TSP of shorter duration of illness. This technique could be a powerful tool to investigate the pathogenic mechanism of diseases associated with HTLV-I.

Adult↗
PubMed1999

[A study of clinical symptoms, muscle pathology, therapeutic response, and prognosis of myositis in patients with HTLV-1 associated myelopathy (HAM)].

Myositis is one of well-known complications of HTLV-1 associated myelopathy (HAM). On twelve HAM patients, 10 women and 2 men complicated with myositis, we examined their clinical symptoms, muscle pathology, and therapeutic response. Clinical examination revealed gait disturbance in 10, weakness of upper limbs in 6, muscle atrophy in 4, and myalgia in 3 patients. Blood test disclosed elevated serum CK level in 6 patients. Muscle biopsy showed marked inflammatory changes with many necrotic and degenerating fibers in 7 patients, and the rest showed only focal invasion of inflammatory cells. Eight patients received corticosteroid or immunosuppressant therapy. The neurological symptoms were improved in 5 of 8 patients 3 months after the treatment. And only one out of 5 patients whom we followed up 3 years after the treatment showed improvement.

Adult↗
PubMed1998

Molecular cloning of the human HAND2 gene.

We have cloned and characterized the coding sequence of the human HAND2 basic helix-loop-helix transcription factor. The amino acid sequence includes an amino-terminal polyalanine repeat which is precisely conserved in the rat HAND2 gene. Northern analysis indicates that the HAND2 transcript is 2.3 kb in length and strongly expressed in the human heart.

Amino Acid Sequence↗
PubMed1998

Differential rescue of visceral and cardiac defects in Drosophila by vertebrate tinman-related genes.

tinman, a mesodermal NK2-type homeobox gene, is absolutely required for the subdivision of the early Drosophila mesoderm and for the formation of the heart as well as the visceral muscle primordia. Several vertebrate relatives of tinman, many of which are predominately expressed in the very early cardiac progenitors (and pharyngeal endoderm), also seem to promote heart development. Here, we show that most of these vertebrate tinman-related genes can readily substitute for Drosophila tinman function in promoting visceral mesoderm-specific marker gene expression, but much less in promoting cardiac-specific gene expression indicative of heart development. In addition, another mesodermal NK2-type gene from Drosophila, bagpipe, which is normally only needed for visceral mesoderm but not heart development, cannot substitute for tinman at all. These data indicate that the functional equivalence of the tinman-related subclass of NK2-type genes (in activating markers of visceral mesoderm development in Drosophila) is specific to this subclass and distinct from other homeobox genes. Despite the apparent overall conservation of heart development between vertebrates and invertebrates, the differential rescue of visceral mesoderm versus heart development suggests that some of the molecular mechanisms of organ formation may have diverged during evolution.

Animals↗
PubMed1998

Quantitative in situ PCR assay of HTLV-1 infected cells in peripheral blood lymphocytes of patients with ATL, HAM/TSP and asymptomatic carriers.

We established an in situ PCR (IS-PCR) method that amplified the pX region of human T cell lymphotropic virus type 1 (HTLV-1) proviral DNA. The procedure was highly sensitive in accurately detecting the number of cells infected with HTLV-1. We estimated the number of HTLV-1 infected cells in peripheral blood lymphocytes (PBL) from patients with HAM/TSP, ATL and asymptomatic carriers. ATL patients (n = 5) had 8-93% IS-PCR positive cells for HTLV-1 and these percentages correlated with the clinical stages. Asymptomatic carriers (n = 3) had 0.8-3.8% (mean 1.1%, S.D. 1.7) positive cells. HAM/TSP patients (n = 10) had 3.1-8.5% (5.8% (5.8%, 2.7) positive cells. Patients with shorter duration of illness showed larger percentages compared with patients with longer duration. In one HAM/TSP patient, the number of IS-PCR positive cells decreased from 5.1% to 1.5% coincident with the times of lymphocytapheresis treatment. Our studies may suggest that an increased viral load initiates the pathogenic process of HAM/TSP and the estimation of HTLV-1 proviral load by IS-PCR method is useful to understand the clinical state of HAM/TSP.

Adult↗
PubMed1998

Cardiac and extracardiac expression of Csx/Nkx2.5 homeodomain protein.

Csx/Nkx2.5 is an evolutionary conserved homeobox gene related to the Drosophila tinman gene, which is essential for the dorsal mesoderm formation. Expression of Csx/Nkx2.5 mRNA is the earliest marker for heart precursor cells in all vertebrates so far examined. Previous studies have demonstrated that Csx/Nkx2.5 mRNA is highly expressed in the heart and at lower levels in the spleen, tongue, stomach, and thyroid in the murine embryo. Since some developmental genes are regulated by posttranscriptional mechanisms, we analyzed the developmental pattern of Csx protein expression at the single-cell level using Csx-specific antibodies. Immunohistochemical analysis of murine embryos at 7.8 days post coitum revealed that Csx protein is strongly expressed in the nucleus of endodermal and mesodermal cells in the cardiogenic plate. Subsequently, in the heart, Csx protein was detected only in the nucleus of myocytes of the atrium and the ventricle through the adult stage. During the fetal period, Csx protein expression in the nucleus was also noted in the spleen, stomach, liver, tongue, and anterior larynx. Unexpectedly, confocal microscopy revealed that Csx immunoreactivity was detected only in the cytoplasm of a subset of cranial skeletal muscles. Csx protein was not detected in the thyroid glands. The expression of Csx protein in all organs was markedly downregulated after birth except in the heart. These results raise the possibility that Csx/Nkx2.5 may play a role in the early developmental process of multiple tissues in addition to its role in early heart development.

Animals↗