Search PubMed⌕ Search

Biomedical subjects

Y Sakiyama

Publications and source records attributed to Y Sakiyama.

At least 55 records · Page 3Linked to original sources
PubMed1998

Visible integration of the adenosine deaminase (ADA) gene into the recipient genome after gene therapy.

Gene therapy for patients with adenosine deaminase (ADA) deficiency has become practical in the 1990s, and the exogenous gene has been reported to survive for several years in the recipient genome. To evaluate the integration efficiency of the ADA gene (ADA) into peripheral blood lymphocytes (PBL) of a patient with ADA deficiency who is receiving gene therapy, we performed two-color interphase fluorescence in situ hybridization (FISH) analysis by using digoxigenin-labeled ADA-cDNA and the biotin-labeled lambda-genomic ADA clone as probes. After each of 9 sequential series of gene therapy, interphase nuclei of 100 mononuclear cells from the patient were analyzed, and those of a LASN-producing cell line were used as a control. FISH signals were detected with rhodamine and FITC for the cDNA and the genomic DNA, respectively. The number of PBL giving a transgene signal grew after the sequential gene therapies, and the proportion of signal-positive cells reached about 10%. Our results indicate that the two-color FISH system can be used as a potential aid to monitor the efficiency of the ADA gene therapy.

3T3 Cells↗
PubMed1998

Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection.

The B-cell defect in X-linked agammaglobulinemia (XLA) is caused by mutations in the gene for Bruton's tyrosine kinase (BTK). Using the anti-BTK monoclonal antibody (48-2H), a flow cytometric analysis of intracytoplasmic BTK protein expressed in monocytes was successfully performed. To examine the possible identification of XLA patients and female carriers by this assay, we studied 41 unrelated XLA families with (35) or without (6) known BTK mutations. A flow cytometric assay showed deficient expression of the BTK protein in 40 of 41 patients, complete BTK deficiency in 35, and partial BTK deficiency in 5. One patient exhibited a normal level of BTK expression. All 6 patients with partial BTK deficiency or normal BTK expression had missense BTK mutations. The cellular mosaicism of BTK expression in monocytes from obligate carriers was clearly shown in 35 of 41 families. The results suggested that most BTK mutations in XLA might result in deficient expression of the BTK protein. We conclude that deficient expression of BTK protein can be evaluated by a flow cytometric assay, and the clinical usefulness and limitations in diagnosis of XLA patients and carriers are discussed.

Adolescent↗
PubMed1998

Successful peripheral T-lymphocyte-directed gene transfer for a patient with severe combined immune deficiency caused by adenosine deaminase deficiency.

Ten patients with adenosine deaminase deficiency (ADA-) have been enrolled in gene therapy clinical trials since the first patient was treated in September 1990. We describe a Japanese ADA- severe combined immune deficiency (SCID) patient who has received periodic infusions of genetically modified autologous T lymphocytes transduced with the human ADA cDNA containing retroviral vector LASN. The percentage of peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% during the 12 months since the fourth infusion. ADA enzyme activity in the patient's circulating T cells, which was only marginally detected before gene transfer, increased to levels comparable to those of a heterozygous carrier individual and was associated with increased T-lymphocyte counts and improvement of the patient's immune function. The results obtained in this trial are in agreement with previously published observations and support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA-SCID.

Adenosine Deaminase↗
PubMed1998

Positron emission tomography reveals changes in global and regional cerebral blood flow during noxious stimulation of normal and inflamed elbow joints in anesthetized cats.

In cats the global (gCBF) as well as the regional cerebral blood flow (rCBF) and blood pressure were measured before, during, and after noxious inward and outward rotations of normal and inflamed elbow joints. The animals were anesthetized with halothane and immobilized by gallamine triethiodide. The gCBF as well as the rCBF were measured using positron emission tomography (PET) with a camera specifically designed for use in small animals. Slow intravenous bolus injections of 15O-labeled water were followed by 3-min acquisition of regional radioactivity starting at the time of injection. In all experiments the gCBF as well as the blood pressure were increased by noxious inward-outward rotations of the normal and of the inflamed joint, whereas the blood pressure and the rCBF remained unchanged during bolus injections under control conditions (without any joint movement). Movements of the inflamed joint evoked significantly greater increases in blood pressure and gCBF than corresponding ones of the normal joint. These increases in gCBF were paralleled by increases in rCBF along the complete anterior to posterior axis of the brain. Again, the increases in rCBF were larger, more extensive and more uniform following the stimulation of the inflamed joint relative to the results obtained with stimulation of the normal joint. No significant laterality was seen, but when an atlas-based region of interest (ROI) analysis was carried out and when the individual variations in rCBF were removed with two-way ANOVA, significant differences were disclosed in rCBF between the stimulated condition and the resting condition in a large number of brain regions. In particular, noxious rotation of the normal (right) elbow joint induced a significant increase in rCBF over the cerebral cortex and in the right thalamus and hippocampus. The same stimulation of the (left) inflamed joint induced a significant increase in rCBF throughout the brain; the biggest increase being over the right posterior cortex. It is concluded that under the conditions of the present experiments the generally accepted autoregulation of the cerebral blood flow is not fully functioning, and various factors that may be responsible for this failure (which obscures rCBF differences) are discussed. The more pronounced increases in rCBF when moving inflamed joints instead of normal ones is thought to be a direct consequence of the peripheral sensitization of the articular nociceptors and the consequent central hyperexcitability induced in the articular nociceptive pathways.

Anesthesia↗
PubMed1998

Elevation of serum IgE level and peripheral eosinophil count during T lymphocyte-directed gene therapy for ADA deficiency: implication of Tc2-like cells after gene transduction procedure.

We have successfully carried out T-cell-directed gene therapy for a boy with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA SCID) and unexpectedly found an elevation of serum IgE level and peripheral eosinophil count during the course. More than 90% of transduced cells cultured for 7-11 days before infusion into the patient were positive for CD8 and expressed Th2-type cytokine genes such as IL-4, IL-5 and IL-13. Furthermore, CD4(+) T-depleted PBMC (peripheral blood mononuclear cells) from the patient synthesized IgE in vitro by stimulation with IL-4. Collectively, these results suggested that Tc2-like cells in the transduced cells have distinct immunological functions to help IgE synthesis and activate eosinophils.

Adenosine Deaminase↗
PubMed1998

Genetic analysis of 13 families with X-linked chronic granulomatous disease reveals a low proportion of sporadic patients and a high proportion of sporadic carriers.

X-linked chronic granulomatous disease (X-CGD) is the most common type of CGD, whose responsible gene has been identified and termed as CYBB, according to the gp91-phox, a subunit of cytochrome b558. Although approximately 200 different mutations of the gp91-phox gene have been reported, no precise study of the proportion of sporadic cases in X-CGD, based on molecular genetic analysis, has been reported. We made a genetic analysis of six newly identified X-CGD patients together with that of eight previously reported X-CGD patients. The mutations newly detected were three missense mutations, two splice mutations, and one insertion of 2 bases. All of the mutations were novel. Twelve mothers (two of them came from the same family) and four maternal grandmothers from 13 different X-CGD families were available for further genetic studies. It was revealed that a proportion of sporadic patients was low and that of sporadic carriers was high. These results suggest that the mutation for the disease originates mainly from male gametes.

Alternative Splicing↗
PubMed1998

[Intravenous immunoglobulin (GB-0998) for prophylaxis of recurrent acute otitis media and lower respiratory tract infection in infancy with IgG 2 deficiency].

The prophylactic effect of intravenous immunoglobulin (GB-0998) on the recurrent acute otitis media, bronchitis and bronchopneumonia in IgG 2 deficient infants was investigated in a multicenter trial. GB-0998 was administered 6 times every 4 weeks. The doses were 300 mg/kg wt. during the first treatment, followed by 5 doses of 200 mg/kg wt. The results indicated that GB-0998 was effective in the prophylaxis of the recurrent infection of acute otitis media, bronchitis and bronchopneumonia in infancy with IgG 2 deficiency and/or IgG 2 antibody deficiency specific for Streptococcus pneumoniae.

Acute Disease↗
PubMed1998

[Study of T cell-directed gene transfer for a patient with ADA deficiency].

We have been undertaking the gene therapy for a 6 year-old boy with ADA deficiency and performed 11 cycles of the infusion of the peripheral T cells transduced with retroviral vector LASN since August 8th 1995. The percentage of the peripheral blood lymphocytes carrying the transduced ADA gene has remained stable at 10% to 20% since the 4th infusion. ADA enzyme activity in his circulating T cells increased to levels comparable to 1/3 of a heterozygous carrier individual and was associated with increased T lymphocytes counts and improvement in both humoral and cellular immune function. The results obtained in this clinical study support the usefulness of T lymphocyte-directed gene transfer in the treatment of ADA deficiency.

Adenosine Deaminase↗
PubMed1997

Inhibition of apoptosis by the actin-regulatory protein gelsolin.

Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti-Fas antibody, C2-ceramide or dexamethasone, without changing the F-actin morphology or the levels of Fas or Bcl-2 family proteins. Upon the induction of apoptosis, an increase in CPP32(-like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro-CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti-Fas treatment in the gelsolin transfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(-like) proteases strongly inhibited Fas-mediated apoptosis, but only partially suppressed both C2-ceramide- and dexamethasone-induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(-like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

Actins↗
PubMed1997

Characterization of a deletion mutation involving exons 3-7 of the WASP gene detected in a patient with Wiskott-Aldrich syndrome.

A case of Wiskott-Aldrich syndrome (WAS) suspected to have a deletion mutation in the WAS protein (WASP) gene had previously been reported (Ariga et al., 1997). Genomic polymerase chain reaction (PCR) suggested that exons 3-7 of the WASP gene were included in the deletion. Present Southern blot studies confirm that the deletion is approximately 2.0 kb in length, involving exons 3-7 and seemed to have been created by the fusion of introns 2 and 7. To characterize the deletion mutation in detail, we analyzed the PCR-amplified fragments of introns 2 and 7 from normal individuals and the fragment suspected of including the deletion junction from the patient. Sequencing of the patient fragment revealed that the deletion mutation involving exons 3-7 of the WASP gene did, indeed, result from the fusion of introns 2 and 7.

Base Sequence↗
PubMed1997

Myocardial adenosine A2a receptor imaging of rabbit by PET with [11C]KF17837.

Adenosine A2a receptors are found in the endothelia, vascular smooth muscle cells and cardiac myocytes. The properties of a carbon-11 labeled A2a antagonist [11C]KF17837 ([7-methyl-11C](E)-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methy lxa nthine) for myocardial imaging were evaluated by dynamic PET scanning of the myocardium in rabbits. Myocardial uptake of [11C]KF17837 was clearly visualized by PET. The tracer was taken up at a high level by the myocardium immediately after the injection, and the myocardial level of radioactivity gradually decreased. On the other hand, an inactive [11C]Z-isomer of [11C]KF17837 showed a very low myocardial uptake and the myocardium was not visualized with a selective A1 antagonist [11C]KF15372. By co-injection with carrier KF17837 or a xanthine type A2a antagonist 7-chlorostyrylcaffeine (CSC), the myocardial uptake of [11C]KF17837 was completely blocked. The effect of non-xanthine A2a antagonists ZM 241,385 and SCH 58,261, which have a higher affinity than CSC, was smaller than that of the CSC. The effect of weak antagonists caffeine and alloxazine or a xanthine type A1 antagonist KF15372 on the radioactivity level was small. It is concluded that PET with [11C]KF17837 can image myocardial adenosine A2a receptors.

Animals↗
PubMed1997

A stereotaxic method of anatomical localization by means of H(2)15O positron emission tomography applicable to the brain activation study in cats: registration of images of cerebral blood flow to brain atlas.

In the neuronal activation study of normal animals, precise anatomical correlation, preferentially to a detailed brain atlas, is required for the activation foci co-registration. To obtain precise regional correlation between H(2)15O-PET images and the brain atlas, a method of stereotaxic image reorientation was applied to an activation study with vibrotactile stimulation. Cats anesthetized with halothane underwent repeated measurements of regional cerebral blood flow (rCBF) in the resting condition and during vibration of the right forepaw. The image set was adjusted three-dimensionally to the atlas. The postmortem brain was sectioned according to the atlas planes. The activated areas were determined by the stimulus-minus-resting subtraction images, and the areas were projected to the atlas. The PET images of the cat brain were compatible both to the postmortem brain slices and to the brain atlas. The activation foci obtained from the subtraction images corresponded to the area around the coronal sulcus, which is electrophysiologically known as the primary sensory area as described in the atlas. There were precise regional correlations between the PET image and anatomy in a PET activation study of the cat by means of stereotaxic image reorientation.

Animals↗
PubMed1997

Extremely high serum level of IgE during immunosuppressive therapy: paradoxical effect of cyclosporine A and tacrolimus.

A case of X-linked autoimmune enteropathy was successfully treated with cyclosporine A (CsA) or tacrolimus (FK506) and developed extremely high serum levels of IgE during the immunosuppressive therapy. Serum IgE levels increased from 190 to 1,000-2,500 IU/ml with CsA therapy and as high as 80,000 IU/ml with subsequent FK506 therapy. Serum IgG2 and IgG4 levels were slightly elevated compared to serum IgE levels. Thereafter, serum IgE levels progressively decreased in parallel with a reduced dosage of FK506. Total serum IgG levels and peripheral eosinophil counts, however, showed no significant changes during the course. These observations suggest that both CsA and FK506, potent immunosuppressants, could paradoxically enhance some immune responses, possibly through the action of CsA-/FK506-resistant immune systems.

Anemia, Hemolytic↗
PubMed1997

Mutation analysis of five Japanese families with Wiskott-Aldrich syndrome and determination of the family members' carrier status using three different methods.

Mutation analysis for five families with Wiskottt-Aldrich syndrome was performed. The mutations found were two missense mutations, two one-base deletion mutations, and a large deletion mutation in the WASP gene. The three mutations had been reported before, but the remaining two were new. We used the mutation information to determine the carrier status of the female relatives of the patients. Three different approaches were taken depending on the type of mutation, and the carrier determination was successfully performed.

Adult↗
PubMed1997

[The gene therapy for a patient with ADA deficiency; report of the first gene therapy trial in Japan].

Since the first gene therapy clinical trial for an ADA deficient patient was performed in September 1990, 10 ADA deficient patients have been enrolled in gene therapy clinical trial. We have been performing the first gene therapy trial in Japan for a 5 year boy with ADA deficiency since August 1995. Activated T cells from the patient's peripheral mononuclear cells were transduced by a retrovirus vector, LASN, which contained cDNA of human ADA gene, and re-infused to him intravenously after 7-11 days. We have already performed 10 cycles of the therapy for the patient. Here, we report the successful results of the gene therapy with laboratory and clinical evaluation. Furthermore, we overview the results of gene therapy for ADA deficient patients which were recently reported from 4 other groups.

Adenosine Deaminase↗