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Biomedical subjects

K Ozawa

Publications and source records attributed to K Ozawa.

At least 37 records · Page 2Linked to original sources
PubMed2003

Congestive heart failure is associated with the rate of bone loss.

AIMS: To characterize relationships between mineral homeostasis, bone turnover, bone mass, and congestive heart failure (CHF), we evaluated 75 women with mild to moderate CHF. METHODS AND RESULTS: We examined the association in annual rate of change in spinal bone mineral density (BMD) with polymorphism of the vitamin D receptor (VDR) gene. Compared with the control group, the CHF group had reduced left ventricular ejection fraction (LVEF: 68.2 +/- 7.5% vs. 60.2 +/- 12.9%; P = 0.0249), human atrial natriuretic peptide (hANP) was elevated (hANP: 10.7 +/- 4.7 pmol L-1 vs. 25.8 +/- 24.2 pmol L-1; P = 0.001) and had lower peak VO2 (22.3 +/- 7.5 mL kg-1 min-1 vs. 15.8 +/- 7.4 mL kg-1 min-1; P = 0.0429). The CHF patients with the VDR FF genotype had a significantly high annual rate of decrease in BMD. In the CHF patients with the VDR FF genotype, urinary calcium excretion (FECa) was elevated (1.40 +/- 0.91% vs. 2.39 +/- 1.40%; P = 0.028), and serum bone-type alkaline phosphatase (B-ALP) was reduced (62.6 +/- 13.7 IU L-1 vs. 47.0 +/- 18.6 IU L-1; P = 0.0123). Also, FECa was correlated positively with furosemide dose (R = 0.881; P = 0.0087) and hANP concentrations (R = 0.635; P = 0.0147) and negatively with DeltaBMD (R = 0.72; P = 0.044) in the CHF patients with the VDR FF genotype. CONCLUSION: The CHF patients with the VDR FF genotype have higher rates of bone loss. These patients may need to increase their calcium intake and BMD may need to be followed more carefully over time.

Absorptiometry, Photon↗
PubMed2002

Relationship between urinary symptoms and disease-related parameters in multiple sclerosis.

OBJECTIVE: To find the prevalence of voiding dysfunction in patients with multiple sclerosis and to examine the relationship between the voiding dysfunction and various parameters of the disease (disease severity, disease duration, lesion site, age and sex). METHODS: Using the International prostate symptom score, lower urinary tract symptoms were quantitatively evaluated in all patients with multiple sclerosis who had visited our neurological department during three months. The lesion site in the central nervous system was determined by a combination of neurological and magnetic resonance imaging findings. RESULTS: Of the 47 patients who had completed the questionnaire, 25 (53 %) were considered to have symptomatic voiding dysfunction. Even at early stage of the disability, 6 of 19 (32 %) patients were symptomatic. Eight (17 %) patients had irritative urinary symptoms alone, whereas 9 (19 %) patients had obstructive urinary symptoms alone. The irritative and obstructive symptoms were concomitant in 10 (21 %) patients. Compared with reports from Western countries, the ratio of obstructive symptoms to irritative symptoms was high in Japan. The degree of irritative symptoms was well correlated with the disease severity, whereas the correlation of obstructive symptoms with the disease severity was less significant. Irritative symptoms also showed a weak correlation with the disease duration, but obstructive symptoms did not. Among the lesion sites in the central nervous system, only the presence of the spinal cord lesion was related to the degree of urinary symptoms. Urinary symptoms were not significantly influenced by the age or the sex. Quality of life was disturbed by urinary symptoms, and this disturbance paralleled the disease severity. CONCLUSION: Urinary symptoms, especially irritative symptoms, reflect the condition of the disease. Thus, the quantified urinary symptoms may assist neurological diagnosis.

Adolescent↗
PubMed2002

Selective growth advantage of wild-type lymphocytes in X-linked SCID recipients.

The cytokine receptor common gamma chain (gamma c) plays a pivotal role in multiple interleukin signaling, and gamma c gene mutations cause an X-linked form of SCID (X-SCID). Recently, gamma c gene transfer into the autologous X-SCID BM achieved appreciable lymphocyte reconstitution, contrasting with the limited success in previous gene therapy trials targeting hematopoietic stem cells. To understand the mechanisms underlying this success, we examined the repopulating potential of the wild-type (WT) BM cells using an X-SCID mouse model. Limited numbers of WT cells were infused into non-ablated WT and X-SCID hosts. Whereas no appreciable engraftment was observed in WT recipients, donor-derived lymphocytes repopulated well in X-SCID, reaching 37% (10(6)cells given) and 53% (10(7) cells given) of the normal control value 5 months post BMT. A lineage analysis showed a predominance of the donor-derived lymphocytes (CD4(+) T, CD8(+) T, B and NK cells) in X-SCID while the donor-derived granulocytes and monocytes engrafted poorly. These results showed a selective advantage of WT cells in X-SCID, and that the advantage was restricted to lymphocytes. In human gene therapy for X-SCID, an analogous growth advantage would greatly enhance the repopulation of lymphocytes derived from a very small number of gamma c gene-supplemented precursors.

Animals↗
PubMed2002

Delayed delivery of AAV-GDNF prevents nigral neurodegeneration and promotes functional recovery in a rat model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is a strong candidate agent in the neuroprotective treatment of Parkinson's disease (PD). We investigated whether adeno-associated viral (AAV) vector-mediated delivery of a GDNF gene in a delayed manner could prevent progressive degeneration of dopaminergic (DA) neurons, while preserving a functional nigrostriatal pathway. Four weeks after a unilateral intrastriatal injection of 6-hydroxydopamine (6-OHDA), rats received injection of AAV vectors expressing GDNF tagged with FLAG peptide (AAV-GDNFflag) or beta-galactosidase (AAV-LacZ) into the lesioned striatum. Immunostaining for FLAG demonstrated retrograde transport of GDNFflag to the substantia nigra (SN). The density of tyrosine hydroxylase (TH)-positive DA fibers in the striatum and the number of TH-positive or cholera toxin subunit B (CTB, neuronal tracer)-labeled neurons in the SN were significantly greater in the AAV-GDNFflag group than in the AAV-LacZ group. Dopamine levels and those of its metabolites in the striatum were remarkably higher in the AAV-GDNFflag group compared with the control group. Consistent with anatomical and biochemical changes, significant behavioral recovery was observed from 4-20 weeks following AAV-GDNFflag injection. These data indicate that a delayed delivery of GDNF gene using AAV vector is efficacious even 4 weeks after the onset of progressive degeneration in a rat model of PD.

Animals↗
PubMed2002

In vivo selective expansion of gene-modified hematopoietic cells in a nonhuman primate model.

A major problem limiting hematopoietic stem cell (HSC) gene therapy is the low efficiency of gene transfer into human HSCs using retroviral vectors. Strategies, which would allow in vivo expansion of gene-modified hematopoietic cells, could circumvent the problem. To this end, we developed a selective amplifier gene (SAG) consisting of a chimeric gene composed of the granulocyte colony-stimulating factor (G-CSF) receptor gene and the estrogen receptor gene hormone-binding domain. We have previously demonstrated that primary bone marrow progenitor cells transduced with the SAG could be expanded in response to estrogen in vitro. In the present study, we evaluated the efficacy of the SAG in the setting of a clinically applicable cynomolgus monkey transplantation protocol. Cynomolgus bone marrow CD34(+) cells were transduced with retroviral vectors encoding the SAG and reinfused into each myeloablated monkey. Three of the six monkeys that received SAG transduced HSCs showed an increase in the levels of circulating progeny containing the provirus in vivo following administration of estrogen or tamoxifen without any serious adverse effects. In one monkey examined in detail, transduced hematopoietic progenitor cells were increased by several-fold (from 5% to 30%). Retroviral integration site analysis revealed that this observed increase was polyclonal and no outgrowth of a dominant single clonal population was observed. These results demonstrate that the inclusion of our SAG in the retroviral construct allows selective in vivo expansion of genetically modified cells by a non-toxic hormone treatment.

Animals↗
PubMed2002

Enhanced Abeta40 deposition was associated with increased Abeta42-43 in cerebral vasculature with Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D).

Cerebrovascular deposition of the amyloid beta-protein (Abeta) is a common pathologic event in patients with Alzheimer's disease (AD) and certain related disorders. Such an Abeta vascular deposition occurs primarily in the medial layer of the cerebral vessel wall in an assembled fibrillar state. These deposits are associated with several pathological responses, including degeneration of the smooth muscle cells in the cerebral vessel wall. Severe cases of cerebrovascular Abeta deposition are also accompanied by loss of vessel wall integrity and hemorrhagic stroke. Although the reasons for this pathological consequence are unclear, altered proteolytic mechanisms within the cerebral vessel wall may be involved. We analyzed cerebral Abeta deposition in brains with AD and Dutch-type hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) on the basis of two amyloid species of Abeta(40) and Abeta(42/43) using specific monoclonal antibodies. Compared to Abeta deposition in senile plaques, the molecular composition of Abeta was distinguishable, indicating that the Abeta(40) species is the main component for vascular amyloid. Furthermore, we found Abeta(42/43) immunoreactivity was also much increased in amyloid angiopathy of all cases with HCHWA-D. Taken together, amyloid angiopathy in HCHWA-D may share an Abeta(42)-driven deposition mechanism with plaque amyloid, resulting in enhanced Abeta(40) deposition.

Amyloid beta-Peptides↗
PubMed2002

[Surgery for thymomas and thymic carcinomas; treatment results in terms of WHO histologic typing, Masaoka staging system, and p53 expression].

Forty thymomas and thymic carcinomas were classified in terms of WHO histologic typing, Masaoka staging system, and p53 expression. In WHO histologic typing, type A, AB, B1, B2, B3, and C were 1, 10, 16, 5, 4, and 4 cases, respectively. In Masaoka staging system, I, II, III, and IV were 15, 9, 10, and 6 cases, respectively. Thirteen thymomas exhibited positive p53 expression and 27 did not. Type A and AB thymomas had more favorite prognosis than type B3 and C thymomas, and prognosis of type B1 and B2 was middle. Staging by the Masaoka system also correlated with survival rates. Patients who had p53-negative thymomas survived longer than those who had p53-positive thymomas. A treatment strategy for thymomas and thymic carcinomas should be made on the basis of WHO histologic typing, Masaoka staging system, and p53 expression.

Adult↗
PubMed2001

Chronic effects of the nucleus pulposus applied to nerve roots on ectopic firing and conduction velocity.

STUDY DESIGN: This study analyzed the effects of the autologous nucleus pulposus (NP) on nerve root function using electrophysiologic methods. OBJECTIVES: To investigate the chronic effects of the NP applied to nerve roots and dorsal root ganglions (DRGs) on ectopic firing and nerve conduction velocity. SUMMARY OF BACKGROUND DATA: It was reported that ectopic firing in DRGs is involved in abnormal sensation such as sciatica and that ectopic firing was increased shortly after application of the NP to nerve roots. It was also reported that various inflammatory reactions occur around herniated NP clinically, which may affect nerve roots. However, the chronic action of NP on ectopic firing in the stage of inflammatory reaction is unknown. METHODS: Forty-six rats were divided into three groups: nucleus pulposus (NP) group, sham group, and control group. In the NP group the autologous NP was applied to L4 and L5 nerve roots. At 1, 2, 4, and 8 weeks after treatment, antidromically propagated ectopic firing from nerve roots was recorded from the sural nerve, and the nerve conduction velocity between the cauda equina and sciatic nerve was analyzed. The frequencies of ectopic firing in the three groups were compared. The ratios of the nerve conduction velocities on the treatment side and opposite side were compared between the NP and sham groups. RESULTS: The frequency was significantly higher in the NP group than in the sham group at 4 weeks. There were no significant differences among the three groups at 8 weeks. There was no significant difference in the treatment side/opposite side ratio of nerve conduction velocity between the NP and sham groups at any time point. CONCLUSIONS: Increased ectopic firing in the stage of inflammatory reaction to NP may be related to abnormal sensation.

Animals↗
PubMed2001

Characterization of stage progression in chronic myeloid leukemia by DNA microarray with purified hematopoietic stem cells.

Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells (HSCs). Without effective treatment, individuals in the indolent, chronic phase (CP) of CML undergo blast crisis (BC), the prognosis for which is poor. It is therefore important to clarify the mechanism underlying stage progression in CML. DNA microarray is a versatile tool for such a purpose. However, simple comparison of bone marrow mononuclear cells from individuals at different disease stages is likely to result in the identification of pseudo-positive genes whose change in expression only reflects the different proportions of leukemic blasts in bone marrow. We have therefore compared with DNA microarray the expression profiles of 3456 genes in the purified HSC-like fractions that had been isolated from 13 CML patients and healthy volunteers. Interestingly, expression of the gene for PIASy, a potential inhibitor of STAT (signal transducer and activator of transcription) proteins, was down-regulated in association with stage progression in CML. Furthermore, forced expression of PIASy has induced apoptosis in a CML cell line. These data suggest that microarray analysis with background-matched samples is an efficient approach to identify molecular events underlying the stage progression in CML.

AC133 Antigen↗
PubMed2001

Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex.

Fanconi anemia (FA) is a genetic disorder that predisposes to hematopoietic failure, birth defects and cancer. We identified an interaction between the FA protein, FANCA and brm-related gene 1 (BRG1) product. BRG1 is a subunit of the SWI/SNF complex, which remodels chromatin structure through a DNA-dependent ATPase activity. FANCA was demonstrated to associate with the endogenous SWI/SNF complex. We also found a significant increase in the molecular chaperone, glucose-regulated protein 94 (GRP94) among BRG1-associated factors isolated from a FANCA-mutant cell line, which was not seen in either a normal control cell line or the mutant line complemented by wild-type FANCA. Despite this specific difference, FANCA did not appear to be absolutely required for in vitro chromatin remodeling. Finally, we demonstrated co-localization in the nucleus between transfected FANCA and BRG1. The physiological action of FANCA on the SWI/SNF complex remains to be clarified, but our work suggests that FANCA may recruit the SWI/SNF complex to target genes, thereby enabling coupled nuclear functions such as transcription and DNA repair.

Cell Line↗
PubMed2001

Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production.

One of the limitations of recombinant adeno-associated virus (rAAV) vector systems for gene therapy applications has been the difficulty in producing the vector in sufficient quantity for adequate evaluation. Since the AAV Rep proteins are cytotoxic, it is not easy to establish stable cell lines that express them constitutively. We describe a novel 293-derived prepackaging cell line which constitutively expresses the antisense rep/cap driven by a loxP-flanked CMV promoter. This cell line was converted into a packaging cell line expressing Rep/Cap for rAAV vector production through adenovirus-mediated introduction of a Cre recombinase gene. Without the introduction of the Cre recombinase gene, the cell line was shown to produce neither Rep nor Cap. rAAV vector was produced (1 x 10(9) genome copies/3.5-cm dish) 4 days after the transduction with Cre-expression adenovirus vector together with transfection of AAV vector plasmid. We further showed that the addition of Cap-expression adenovirus vector caused a 10-fold increase in the yield of rAAV vector. This system is also capable of producing rAAV as a transfection-free system by using a small amount of rAAV instead of vector plasmid.

Adenoviridae↗
PubMed2001

Sak serine-threonine kinase acts as an effector of Tec tyrosine kinase.

The murine sak gene encodes a putative serine-threonine kinase which is homologous to the members of the Plk/Polo family. Although Sak protein is presumed to be involved in cell growth mechanism, efforts have failed to demonstrate its kinase activity. Little has been, therefore, elucidated how Sak is regulated and how Sak contributes to cell proliferation. Tec is a cytoplasmic protein-tyrosine kinase (PTK) which becomes activated by the stimulation of cytokine receptors, lymphocyte surface antigens, heterotrimeric G protein-linked receptors, and integrins. To clarify the in vivo function of Tec, we have tried to isolate the second messengers of Tec by using the yeast two-hybrid screening. One of such Tec-binding proteins turned out to be Sak. In human kidney 293 cells, Sak became tyrosine-phosphorylated by Tec, and the serine-threonine kinase activity of Sak was detected only under the presence of Tec, suggesting Sak to be an effector molecule of Tec. In addition, Tec activity efficiently protects Sak from the "PEST" sequence-dependent proteolysis. Internal deletion of the PEST sequences led to the stabilization of Sak proteins, and expression of these mutants acted suppressive to cell growth. Our data collectively supports a novel role of Sak acting in the PTK-mediated signaling pathway.

Amino Acid Sequence↗
PubMed2001

Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells.

Since hematopoietic stem cells (HSCs) differentiate readily ex vivo resulting in the loss of self-renewal and engraftment abilities, the transient block of differentiation is essential to maintain those abilities during their ex vivo expansion culture. To this end, we developed a method of reversible integration of the dominant negative retinoic acid receptor (DN-RAR) gene, a differentiation-blocking gene, into cells utilizing the Cre/loxP-dependent gene recombination system. The murine immature hematopoietic 32D cells differentiate into mature neutrophils upon G-CSF treatment. However, 32D cells transduced with a retroviral vector expressing the DN-RAR gene put between two loxP sites continued to proliferate without showing differentiation even in the presence of G-CSF. After the cells were fully amplified, the cells were transduced with the Cre recombinase gene. The cells then restored the ability to differentiate into mature neutrophils upon G-CSF treatment. PCR analysis showed that the DN-RAR gene was efficiently removed from the genome by introduction of the Cre gene. This system may eventually be applicable to the ex vivo expansion of HSCs.

Animals↗
PubMed2001

Identification of myelodysplastic syndrome-specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction.

Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.

DNA↗
PubMed2001

A novel recombinant adeno-associated virus vaccine induces a long-term humoral immune response to human immunodeficiency virus.

Recombinant adeno-associated virus (AAV) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HIV-1 vaccine, using an AAV vector expressing HIV-1 env, tat, and rev genes (AAV-HIV vector). A single injection of the AAV-HIV vector induced strong production of HIV-1-specific serum IgG and fecal secretory IgA antibodies as well as MHC class I-restricted CTL activity in BALB/c mice. The titer of HIV-1-specific serum IgG remained stable for 10 months. When AAV-HIV vector was coadministered with AAV-IL2 vector, the HIV-specific cell-mediated immunity (CMI) was significantly enhanced. Boosting with AAV-HIV vector strongly enhanced the humoral response. Furthermore, the mouse antisera neutralized an HIV-1 homologous strain, and BALB/c mice immunized via the intranasal route with an AAV vector expressing the influenza virus hemagglutinin (HA) gene showed protective immunity against homologous influenza virus challenge. These results demonstrate that AAV-HIV vector immunization may provide a novel and promising HIV vaccination strategy.

AIDS Vaccines↗
PubMed2001

Morphological and physiological restorations of hereditary form of dilated cardiomyopathy by somatic gene therapy.

TO-2 strain hamsters with dilated cardiomyopathy, gene deletion of delta-sarcoglycan (SG) and no expression of alpha-, beta-, gamma-, and delta-SG proteins are useful for developing the potential gene therapy of intractable heart failure. We prepared recombinant adeno-associated virus vector including normal delta-SG gene driven by CMV promoter and intramurally administered in vivo. The transfected myocardium induced robust expression of both transcript and transgene for 2/3 period of the animal's life expectancy. Immunostaining demonstrated reexpression of not only delta-SG but also other three SGs in 40% cells in the transfected region and normalization of the diameter of transduced cardiomyocytes. Hemodynamic study revealed preferential amelioration of the diastolic indices (LVEDP, the dP/dt(min) and CVP). These results provide the first evidence that supplementation of a specific gene with efficient and sustained transfection capability restores the genetic, morphological, and functional deteriorations.

Animals↗
PubMed2001

Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB.

We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.

Animals↗