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Hiroyuki Okamoto

Publications and source records attributed to Hiroyuki Okamoto.

At least 19 recordsLinked to original sources
PubMed2006

A simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence.

Degradation of heparan sulfate proteoglycan by heparanase is an important process in tissue invasion by malignant tumor cells and inflammatory cells. Several heparanase assays have been reported previously, but all of them are cumbersome and time-consuming for separating degradative products from uncleaved substrates and detecting these products. This paper describes the development of a simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence based on non-radiative energy transfer. In this assay, heparan sulfate proteoglycan labeled with biotin and europium cryptate (fluorescence donor) was used as a substrate for heparanase. Degradation of the substrate by incubation with murine melanoma cell extract was detected by measuring the time-resolved fluorescence after addition of XL665 (cross-linked allophycocyanin, fluorescence acceptor)-labeled streptavidin. As heparanase activity can be simply measured by successive addition of substrate, enzyme and detection reagent onto one assay plate, our heparanase assay allows rapid processing of large numbers of samples.

Animals↗
PubMed2006

Ultraviolet B radiation suppresses Langerhans cell migration in the dermis by down-regulation of alpha4 integrin.

BACKGROUND/PURPOSE: Ultraviolet B (UVB) radiation affects the migration and function of epidermal Langerhans cells (LC) and causes immunosuppression of contact hypersensitivity. It is known that LC leaves the epidermis after exposure to UVB. To know the behavior of LC in the dermis after UVB radiation, we studied the effect of UVB radiation on the expression of integrin families on freshly isolated or cultured murine LC. We also examined whether UVB radiation affects the migration of LC to secondary lymphoid tissue chemokine (SLC/6Ckine). METHODS: Integrin expressions of murine LC cultured in epidermal cell suspension were analyzed using flowcytometry. We used murine LC sorted flowcytometrically for binding assay to extracellular matrix and for migration assay to chemokine. Skin explant assay and immnohistochemical staining for 'cords formation' were performed as previously described. RESULTS: Twenty and 40 mJ/cm2 of UVB radiation down-regulated the expression of alpha4 integrin on 24 h-cultured LC, but not that of alpha6, beta1, or beta4 integrin. The number of cultured LC adhered to fibronectin, a ligand for alpha4 integrin, was decreased after UVB irradiation, while that to laminin, a ligand for alpha6 integrin, was not influenced. UVB radiation reduced the number of migrating LC to SLC. Furthermore, skin sheet explant experiments showed that UVB radiation inhibited the 'cords' formation in dermal vessels of the 48 h-cultured skin. CONCLUSIONS: These data suggest that UVB radiation may suppress the migration of LC from the dermis to lymphatic vessels. UVB radiation may downregulate the adherence of LC to dermal fibronectin and migration to SLC, and consequently suppress the migration of LC from the UVB-irradiated dermis to lymphatics.

Animals↗
PubMed2006

Microdosimetric measurements and estimation of human cell survival for heavy-ion beams.

The microdosimetric spectra for high-energy beams of photons and proton, helium, carbon, neon, silicon and iron ions (LET = 0.5-880 keV/microm) were measured with a spherical-walled tissue-equivalent proportional counter at various depths in a plastic phantom. Survival curves for human tumor cells were also obtained under the same conditions. Then the survival curves were compared with those estimated by a microdosimetric model based on the spectra and the biological parameters for each cell line. The estimated alpha terms of the liner-quadratic model with a fixed beta value reproduced the experimental results for cell irradiation for ion beams with LETs of less than 450 keV/microm, except in the region near the distal peak.

Cell Line↗
PubMed2005

DNA damage initiates photobiologic reactions in the skin.

Ultraviolet (UV) radiation can induce acute and chronic photobiologic reactions in the absence of exogenous chromophores. Nuclear DNA is a major chromophore to initiate UV-induced physiologic reactions. The XPA-gene deficient mouse, an animal model of xeroderma pigmentosum, develops increased photobiologic reactions including acute inflammation, immunosuppression and skin cancers, because of the defect in the excision repair of UV-induced DNA lesions.

Animals↗
PubMed2005

Effects of UVB on fascin expression in dendritic cells and Langerhans cells.

BACKGROUND: Fascin is an actin-binding protein that regulates the rearrangement of cytoskeletal elements and their interactions with the cell membrane. Previous studies have indicated that fascin expression is enhanced in DC upon maturation and plays a critical role in T cell activation. Ultraviolet irradiation exerts immunosuppressive effects. OBJECTIVE: We examined the effects of UVB irradiation on the interaction of DC/LC with T cells through fascin. METHOD: Murine bone marrow-derived DC (BM-DC) were induced by recombinant murine GM-CSF and LPS, and UVB irradiation was applied prior to supplementation with LPS. I-A(+) cells (Langerhans cells (LC)) in the epidermal cell suspensions were exposed to UVB irradiation at the beginning of the 24-h culture. BM-DC and LC were analysed by immunohistochemical staining and flow cytometric analyses. To evaluate the effects of UVB irradiation on DC-T cell binding, we examined the clustering of BM-DC with allogeneic CD4(+) T cells under a confocal microscope. RESULTS: Fascin expression in BM-DC and LC was decreased by UVB irradiation. Furthermore, UVB irradiation reduced the ability of BM-DC to cluster with allogeneic CD4(+) T cells. Polarization of fascin and filamentous actin (F-actin) at the point of contact of BM-DC with T cells was also disturbed by UVB irradiation. CONCLUSION: These results suggest that the suppression of fascin expression by UVB irradiation down-regulates the rearrangement of the cytoskeleton and, thereby, antigen presentation in DC/LC.

Animals↗
PubMed2005

Oligoclonal expansion of T lymphocytes with multiple second-site mutations leads to Omenn syndrome in a patient with RAG1-deficient severe combined immunodeficiency.

Omenn syndrome (OS) is a rare primary immunodeficiency characterized by the presence of activated/oligoclonal T cells, eosinophilia, and the absence of circulating B cells. OS patients carry leaky mutations of recombination activating genes (RAG1 or RAG2) resulting in partial V(D)J recombination activity, whereas null mutations cause severe combined immunodeficiency with absence of mature T and B cells (T-B- SCID). Here we describe somatic mosaicism due to multiple second-site mutations in a patient with RAG1 deficiency. We found that he is homozygous for a single base deletion in the RAG1 gene, which results in frameshift and likely abrogates the protein function. However, the patient showed typical OS features. Molecular analysis revealed that several second-site mutations, all of which restored the RAG1 reading frame and resulted in missense mutations, were demonstrated in his T cells. These findings suggest that his revertant T-cell mosaicism is responsible for OS phenotype switched from T-B- SCID.

Cell Proliferation↗
PubMed2005

Primary bypass surgery from the descending aorta to the iliac arteries for a severely calcified aorta: report of two cases.

We herein report two patients with end-stage renal failure who complained of disabling claudication due to abdominal aortic atherosclerosis. Both were on chronic hemodialysis for more than 15 years, and their abdominal aorta was densely calcified. We elected to perform a descending aorta to iliac artery bypass in order to avoid hazardous clamping of the calcified aorta. Hemodialysis was able to be resumed on the first postoperative day. They tolerated the operation well and their symptoms disappeared. This operative procedure is therefore considered to be a useful option for patients with a porcelain aorta who are on chronic hemodialysis.

Aorta, Abdominal↗
PubMed2005

Long-term outcome after surgical treatment of arterial lesions in Behçet disease.

OBJECTIVE: Surgical treatment of arterial lesions associated with Behçet disease (BD) is often complicated by graft occlusion and recurrence of aneurysms. The purpose of this study was to clarify the long-term outcome of surgical intervention for arterial involvement in BD. METHODS: Ten patients with BD (9 men, 1 woman) who underwent surgical treatment for arterial aneurysms between 1980 and 2004 were included in the study. The age of patients at the first operation ranged from 36 to 69 years (mean, 50.4 +/- 9.0 years). The mean period between the onset of BD and that of arterial manifestations was 8.0 +/- 5.0 years. We retrospectively reviewed their postoperative courses, including survival, graft occlusion, formation of anastomotic false aneurysms, and the development of aneurysms at different sites. The Kaplan-Meier method was used to calculate the chronologic incidence of complications after surgery. RESULTS: The mean follow-up period was 133 +/- 92 months, ranging from 5 to 285 months. One patient died of rupture of a dissecting aortic aneurysm after undergoing several surgical interventions for multiple aneurysms. There were five graft occlusions among 21 grafts. The cumulative primary graft patency rate in the infrainguinal region was 83.9% at 3 years. Five anastomotic false aneurysms formed among 49 anastomoses between grafts and host arteries. The overall cumulative incidence of formation of anastomotic pseudoaneurysm was 12.9% at 5 and 10 years. All of them formed within 18 months after surgery. Development of new aneurysms in different arteries was observed in two patients. CONCLUSIONS: Early occurrence of anastomotic false aneurysm is characteristic of BD. Further investigation is necessary to establish effective postoperative treatment.

Anastomosis, Surgical↗
PubMed2005

Clinical implication of plasma level of soluble fibrin monomer-fibrinogen complex in patients with abdominal aortic aneurysm.

OBJECTIVE: We prospectively studied the clinical implication of plasma level of soluble fibrin monomer (FM)-fibrinogen complex, a recently established molecular marker reflecting thrombin activity, in patients with abdominal aortic aneurysm (AAA) undergoing elective aortic repair. METHODS: The study included 49 patients who underwent elective aneurysm repair using a gelatin-sealed or nonimpregnated Dacron prosthesis. Plasma level of soluble FM-fibrinogen complex was measured before surgery and on days 1, 3, 5, 7, and 10 postoperatively by latex agglutination assay utilizing monoclonal antibody IF-43. Plasma levels of thrombin-antithrombin complex (TAT), D-dimer, alpha2-plasmin inhibitor-plasmin complex (PIC), and fibrinogen were also evaluated. RESULTS: The preoperative level of soluble FM-fibrinogen complex showed variation in the degree of hemostatic activation, with fair correlations with TAT (r = 0.509, P < .001), D-dimer (r = 0.521, P < .001), and PIC (r = 0.579, P < .001). The patients with greater intraoperative blood loss (> or = 800 mL) showed a significantly elevated plasma level of soluble FM-fibrinogen complex preoperatively compared with those with less intraoperative blood loss (P = .009). Its postoperative fluctuation showed a similar pattern to that of TAT, reflecting the time course of coagulation activity. Gelatin impregnation of the Dacron vascular graft did not seem to influence the postoperative systemic coagulation mechanism. CONCLUSIONS: The results indicated that soluble FM-fibrinogen complex appears to be a useful diagnostic molecular marker to assess the activity of the coagulation system, and that its preoperative level may serve as a potential risk factor for intraoperative hemorrhagic diathesis in patients undergoing elective AAA repair.

Aged↗
PubMed2005

M(2) and M(3) muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice.

Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M(2) or M(3) muscarinic receptors, in order to characterize the mechanisms involved in M(2) and M(3) receptor-mediated contractile responses. Single applications of carbachol (0.1-100 microM) produced concentration-dependent contractions in preparations from M(2)-knockout (KO) and M(3)-KO mice, mediated via M(3) and M(2) receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M(2)-preferring antagonist methoctramine (300 nM) or the M(3)-preferring antagonist 4-DAMP (30 nM). The M(2)-mediated contractions were mimicked in shape by submaximal stimulation with high K(+) concentrations (up to 35 mM), almost abolished by voltage-dependent Ca(2+) channel (VDCC) antagonists or depolarization with 140 mM K(+) medium, and greatly reduced by pertussis toxin (PTX) treatment. The M(3)-mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K(+)-depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K(+) depolarization consisted of different components, either sensitive or insensitive to extracellular Ca(2+). The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M(2) receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca(2+) entry, especially associated with action potential discharge, and that the M(3) receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca(2+) entry and intracellular Ca(2+) release. In intact tissues coexpressing M(2) and M(3) receptors, M(2) receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

Animals↗
PubMed2005

Development of a new mouse model (xeroderma pigmentosum a-deficient, stem cell factor-transgenic) of ultraviolet B-induced melanoma.

It is well established that exposure to sunlight or ultraviolet radiation (UVR) is the major environmental risk factor for the development of skin neoplasms. To date, however, there have been few appropriate mouse models available for studying the role of UVR in melanoma carcinogenesis, mainly because of the murine lack of the epidermal melanocyte, which is a major source of origin of human melanoma. In this study, we established xeroderma pigmentosum group A gene-deficient, stem cell factor-transgenic mice, which are defective in the repair of damaged DNA and do have epidermal melanocytes. The mice were exposed to UVR three times a week for 10 wk. More than 30% of the irradiated mice developed tumors of melanocyte origin that metastasized to the lymph nodes. Histologically, proliferated cells exhibited lentigo maligna melanoma or nodular melanoma. Immunohistochemistry confirmed that the tumor cells were characteristic of melanoma. Non-irradiated mice did not develop skin tumors spontaneously. The newly generated model mouse might be useful for studying the photobiological aspects of human melanoma, because the mice developed melanoma from epidermal melanocytes only after UVR exposures.

Animals↗
PubMed2005

A common variable immunodeficient patient who developed acute disseminated encephalomyelitis followed by the Lennox-Gastaut syndrome.

Common variable immunodeficiency (CVID) is a primary disorder characterized by impaired antibody production. CVID patients may develop recurrent infections, autoimmune disorders, and malignant lymphomas, but to our knowledge, there is no report on CVID patients who develop acute disseminated encephalomyelitis (ADEM) or the Lennox-Gastaut syndrome. We describe a 1-yr-old female CVID patient with ADEM who evolutionally manifested the Lennox-Gastaut syndrome. She was admitted with convulsions and T2-weighted magnetic resonance imaging (MRI) revealed high-intensity areas in the right temporal lobe and the left fronto-parietal region but she became conscious soon. Her serum findings showed severe hypogammaglobulinemia and a follow up MRI revealed that these areas had diminished. Consequently, she was diagnosed as having CVID with ADEM. After 5 months, she fell to having tonic and absence seizures and we diagnosed her as having the Lennox-Gastaut syndrome from electroencephalograms (EEG) and the seizure pattern. She is now 7 yr old and her tonic seizures are controlled with valproic acid, clobazam, and immunoglobulin replacement therapy which is administrated every 2 wk. It is well known that the immune and neurologic systems have a close relationship. We suspect that a genetic defect in the immune system of our patient might also be associated with the neurologic disorders of ADEM and the Lennox-Gastaut syndrome.

Common Variable Immunodeficiency↗
PubMed2005

Expansion of activated eosinophils in infants with severe atopic dermatitis.

BACKGROUND: There is increasing concern in Japan over infants with atopic dermatitis (AD) who present with severe systemic complications, such as hypoproteinemia, electrolyte disturbances, and delayed growth and development. They are often associated with extremely increased numbers of circulating eosinophils. However, the clinical significance of eosinophil expansion has not been thoroughly investigated. METHODS: This study attempted to determine the significance of eosinophilia and eosinophil activation in infant cases of AD by comparing multiple clinical parameters, indexes of eosinophil activation, and levels of serum cytokines. CD69 expression was determined by flow cytometry. The clinical severity of AD was graded by the severity SCORing of atopic dermatitis (SCORAD) method. Patients were classified into two groups, with and without CD69 on eosinophils. Nuclear lobes were evaluated under a microscopy. Serum levels of eosinophil-derived neurotoxin (EDN), interleukin (IL)-12, IL-18, IL-4, IL-5 and interferon (IFN)-gamma were determined by enzyme-linked immunosorbent assay. RESULTS: Patients with CD69-positive eosinophils had significantly higher numbers of eosinophils and platelets, total IgE, and eosinophil nuclear lobes. They also showed growth failure, developmental delay, low serum albumin, and electrolyte disturbances. EDN and IL-18 levels were significantly increased in this group, but the levels of IL-4, IL-5 and IL-12 were not significantly different between the two groups. IFN-gamma was not detectable in all patients with AD. Surface expression of CD69 indicates intense systemic allergic inflammation induced in severe cases of AD. CONCLUSIONS: Evaluation of eosinophil activation and early therapeutic intervention is mandatory for the treatment of severe AD during infancy.

Antigens, CD↗
PubMed2005

Effects of calcium concentration on the SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes.

BACKGROUND/PURPOSE: Cellular differentiation due to the extracellular calcium (Ca(2+)) concentration affects the level of several antioxidant enzymes in cultured human keratinocytes. Because the epidermis includes well- and un-differentiated keratinocytes, we expected that keratinocytes possess different antioxidant capacity and sensitivity to damaging effects of ultraviolet-B (UVB) depending on the differentiation. We examined the effects of Ca(2+) concentration of culture medium (DMEM (Dulbecco's modified Eagle's medium)) on the superoxide dismutase (SOD) activity and UVB-induced cytotoxicity in cultured human keratinocytes in order to investigate the relationship between cell differentiation and antioxidant defense. METHODS: Human keratinocytes (HaCaT cells) were incubated in high Ca(2+) (>1 mM) or low Ca(2+) (<0.1 mM) concentration DMEM for 24 h at 37 degrees C in 5% CO(2). Then, we measured total SOD activity and also individual Cu,Zn- and Mn-SOD activities in keratinocytes. Furthermore, after incubation in high or low Ca(2+) concentration DMEM, human keratinocytes were irradiated with 10, 20 or 30 mJ/cm(2) UVB. The quantity of lactate dehydrogenase (LDH) leaked in the supernatant from damaged keratinocytes, cell viability and TdT-mediated dUTP nick end labelings (TUNEL) positive keratinocytes were measured at 24 h after UVB irradiation. RESULTS: Total SOD activity and Cu,Zn-SOD activity in human keratinocytes cultured in low Ca(2+) were significantly lower than in keratinocytes cultured in high Ca(2+) concentration DMEM. In contrast, Mn-SOD activity was not affected. LDH leakage in the supernatant from keratinocytes cultured in low Ca(2+) concentration was significantly higher than that from keratinocytes cultured in high Ca(2+) concentration DMEM after UVB irradiation. The cell viability of keratinocytes cultured in low Ca(2+) concentration DMEM was significantly decreased compared to that of keratinocytes cultured in high Ca(2+) concentration DMEM after UVB irradiation. Furthermore, UVB-induced apoptosis was increased in keratinocytes cultured in low Ca(2+) concentration DMEM by the TUNEL method. CONCLUSIONS: These results suggest that cellular differentiation due to the change of Ca(2+) concentration of culture medium affects the Cu,Zn-SOD activity and UVB-induced cytotoxicity in cultured human keratinocytes.

Apoptosis↗
PubMed2005

Therapeutic and prophylactic effects of PUVA photochemotherapy on atopic dermatitis-like lesions in NC/Nga mice.

BACKGROUND: Psoralens and ultraviolet A radiation (PUVA) photochemotherapy has been used for severe cases of atopic dermatitis (AD). To understand the mechanisms of action is important for the choice of treatments. AD-like lesions can be induced experimentally in NC/Nga mice. OBJECTIVES: To evaluate clinically and histologically the therapeutic and prophylactic effects of PUVA on AD-like dermatitis using NC/Nga mice. METHODS: PUVA therapy was performed with intraperitoneal injection of 4 mg/kg of 8-methoxypsoralen (8-MOP) and 4 J/cm(2)-UVA irradiation before and after development of AD-like lesions in NC/Nga mice which had been maintained in a conventional room (Conv-NC/Nga mice). Clinical skin conditions were evaluated periodically by a clinical severity score defined. Lesions were histologically examined in haematoxylin-eosin or toluidine blue-stained sections. Plasma levels of total IgE were measured at various time points. RESULTS: In Conv-NC/Nga mice infested with mite, AD-like lesions started to develop at 8 week of age and thereafter increased in severity score. PUVA therapy at lower does than minimal phototoxic dose suppressed the development of dermatitis and was also therapeutically effective against established lesions. Proliferation of dermal mast cells in AD-like lesions was suppressed, but IgE hyperproduction was not changed after PUVA. CONCLUSIONS: These observations suggest that PUVA photochemotherapy reveals not only therapeutic but also prophylactic effects on human AD.

Animals↗
PubMed2005

Targeted deletion or pharmacological inhibition of MMP-2 prevents cardiac rupture after myocardial infarction in mice.

MMPs are implicated in LV remodeling after acute myocardial infarction (MI). To analyze the role of MMP-2, we generated MI by ligating the left coronary artery of MMP-2-KO and WT mice, the latter of which were administered orally an MMP-2-selective inhibitor or vehicle (TISAM). The survival rate was significantly higher in MMP-2-KO and TISAM-treated mice than in control WT mice. The main cause of mortality in control WT mice was cardiac rupture, which was not observed in MMP-2-KO or TISAM-treated mice. Control WT mice, but not MMP-2-KO or TISAM-treated mice, showed activation of the zymogen of MMP-2, strong gelatinolytic activity, and degradation of ECM components, including laminin and fibronectin, in the infarcted myocardium. Although infarcted cardiomyocytes in control WT mice were rapidly removed by macrophages, the removal was suppressed in MMP-2-KO and TISAM-treated mice. Macrophage migration was induced by the infarcted myocardial tissue from control WT mice and was inhibited by treatment of macrophages with laminin or fibronectin peptides prior to migration assay. These data suggest that inhibition of MMP-2 activity improves the survival rate after acute MI by preventing cardiac rupture and delays post-MI remodeling through a reduction in macrophage infiltration.

Animals↗