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Biomedical subjects

Hiroki Hayashi

Publications and source records attributed to Hiroki Hayashi.

At least 19 recordsLinked to original sources
PubMed2006

Transfection of human hepatocyte growth factor gene ameliorates secondary lymphedema via promotion of lymphangiogenesis.

BACKGROUND: Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return and swelling of the extremities. Treatment for this disabling condition remains limited and largely ineffective. The goal of the present study was to investigate the therapeutic efficacy of hepatocyte growth factor (HGF) in animal models of lymphedema. METHODS AND RESULTS: Immunofluorescent analysis demonstrated that canine primary lymphatic endothelial cells (cLECs) were positive for lymphatic-specific markers (vascular endothelial growth factor receptor-3, LYVE-1, podoplanin, and Prox1) and the HGF receptor c-Met. Treating cLECs with human recombinant HGF resulted in a dose-dependent increase in cell growth and migration and increased activity of extracellular signal-regulated kinase and Akt. In human LECs, c-Met also was expressed, and treatment with HGF increased cell growth and migration in a dose-dependent manner. Transfection of human HGF plasmid DNA in cLECs also increased the c-fos promoter activity. Furthermore, weekly HGF gene transfer in a rat tail lymphedema model by disruption of lymphatic vessels resulted in a decrease in lymphedema thickness. Although expression of the endothelial cell marker PECAM-1 was increased in both HGF- and vascular endothelial growth factor 165-injected groups, expression of LEC markers (LYVE-1 and Prox1) was increased only in the HGF-injected group. CONCLUSIONS: These data demonstrate that expression of HGF via plasmid transfer improves lymphedema via promotion of lymphangiogenesis. Further studies to determine the clinical utility of this approach would be of benefit to patients with lymphedema.

Animals↗
PubMed2006

Electrochemical detection of E. coli 16S rDNA sequence using air-plasma-activated fullerene-impregnated screen printed electrodes.

A new DNA modified electrode for the electrochemical detection of 16S rDNA extracted from Escherichia coli (JCM1649) is proposed. The electrodes were fabricated by screen printing a fullerene-impregnated carbon ink onto a poly(methylmethacrylate) substrate and immobilizing a probe DNA on the surface after activating the electrode with air plasma. The results indicated a dramatic improvement in the surface coverage of the immobilized probe DNA, and of the reduction peak of the redox indicator (Co(phen)(3)(3+)) due to the incorporation of fullerene. By immobilizing the probe onto the fullerene-impregnated screen-printed electrodes, the PCR product of the 16S rDNA extracted from E. coli was directly detected without any pretreatment. A well defined signal difference was observed between the perfectly matching oligonucleotide and the mismatching one, and it was possible to detect the target at the modified electrode. This method enabled us to clearly detect the two base mismatches in the ca. 1500-bases long 16S rDNA sequence.

Air↗
PubMed2006

Humanization of the bispecific epidermal growth factor receptor x CD3 diabody and its efficacy as a potential clinical reagent.

PURPOSE: Bispecific antibodies (BsAb) have been exploited as both cancer immunodiagnostics and cancer therapeutics and show promise in clinical trials of cancer imaging and therapy. For development of BsAbs as clinical reagents, we have focused on construction of small recombinant BsAbs, called bispecific diabodies. Here, we constructed and characterized a humanized bispecific diabody. EXPERIMENTAL DESIGN: We have reported significant antitumor activity of an anti-epidermal growth factor receptor (EGFR) x anti-CD3 bispecific diabody (Ex3) in in vitro cytotoxicity assays and in vivo. We humanized the Ex3 diabody (hEx3) by grafting the complementarity-determining region and compared its biological properties with those of Ex3. We also tested its physiologic stability and ability to alter survival in xenografted mice. RESULTS: The final yield of hEx3 was 10 times that of Ex3, and refolded hEx3 and Ex3 showed identical binding profiles in EGFR-positive cell lines and EGFR-transfected Chinese hamster ovary cells. hEx3 showed dose-dependent cytotoxicity to EGFR-positive cell lines, which could be specifically inhibited by parental monoclonal antibody IgGs against EGFR or CD3 antigens. The heterodimeric structure was retained in PBS for 6 months, and growth inhibition was maintained after incubation under physiologic conditions. Coadministration of hEx3 with T-LAK cells and interleukin-2 prolonged the survival of nude mice with human colon carcinoma. CONCLUSIONS: The humanized diabody hEx3 is an attractive molecule for cancer therapy and may provide important insights into the development of EGFR-based cancer-targeting reagents.

Animals↗
PubMed2006

Mitral valve repair for anterior leaflet prolapse with expanded polytetrafluoroethylene sutures.

BACKGROUND: This study was a long-term Doppler echocardiographic assessment of mitral valve repair for anterior mitral leaflet prolapse using expanded polytetrafluoroethylene sutures. METHODS: Between April 1992 and December 2003, we performed mitral valve repair using expanded polytetrafluoroethylene sutures in 204 patients (mean age, 54.6 years) with severe mitral regurgitation (MR) having anterior mitral leaflet prolapse. The cause of valve disease was degenerative in 181 patients (88.7%). Postoperative serial transthoracic echocardiographic studies were performed in all hospital survivors. Residual MR flow detected by color Doppler echocardiography was classified according to the maximum regurgitant jet area. RESULTS: The 30-day mortality of 204 patients was 1.4% (3 deaths). There were 12 late deaths and 14 reoperations in this series. Kaplan-Meier survival and freedom from reoperation at 12 years were 84.6% +/- 4.0% and 89.9% +/- 2.9%, respectively. Postoperative transthoracic echocardiographic assessment after discharge (mean follow-up, 4.2 +/- 3.0 years) showed less than mild regurgitation (maximum regurgitant jet area < 4.0 cm2) in 80.9% of the patients. Overall, freedom from severe MR (maximum regurgitant jet area > or = 7.0 cm2) estimates at 12 years were 88.1% +/- 3.1%. Freedom from severe MR at 12 years for 114 patients with no MR (maximum regurgitant jet area = 0 cm2) on intraoperative transesophageal echocardiography and 77 patients with MR was 95.3% +/- 2.1% and 82.9% +/- 5.1%, respectively (p = 0.033). CONCLUSIONS: Twelve-year echocardiographic follow-up demonstrates good long-term results of chordal replacement with expanded polytetrafluoroethylene sutures for anterior mitral leaflet prolapse. To avoid recurrence of regurgitation, a significantly high level of competence of the valve is essential in the repair of anterior mitral leaflet prolapse.

Adolescent↗
PubMed2006

[An advanced pulmonary adenocarcinoma case in which TS-1 proved effective].

A sixty-two-year-old female presented with non-small-cell lung carcinoma with multiple bone metastasis. Combination chemotherapy with CBDCA and GEM was implemented, but as no effect was obtained, TS-1 was begun with a single course of oral administration of 100 mg/day continuously for 28 days followed by a 14-day rest. Following the start of TS-1, PR of the primary tumor was obtained, and whole-body bone scintigraphy revealed a reduction in abnormal areas of uptake. The woman continues to receive TS-1 orally as an outpatient.

Adenocarcinoma↗
PubMed2006

[A case of an elderly patient having advanced hepatocellular carcinoma with tumor thrombus in the inferior vena cava treated with chemo-radio-therapy--intraarterial infusion of weekly high dose 5-FU (WHF)].

The patient was an 81-year-old man, diagnosed with advanced huge hepatocellular carcinoma (HCC) with tumor thrombus extending into the inferior vena cava (Vv3), for which resection was judged impossible. The radio therapy (51 Gy) for tumor thrombus was carried out, and he received a weekly hepatic arterial infusion therapy (weekly high-dose 5-fluorouracil (5-FU)) for these legions. After 8 cycles, the CT scan revealed a minor response of the tumor (SD), and,the tumor marker reduced. After 10 months, these legions had markedly regressed (PR), the tumor thrombus in the inferior vena cava was not detectable. There were no severe side effects. Ten months since the start of chemo-radio therapy, the positron emission tomography (PET) revealed a metastatic tumor of the femoral bone in recurrence. In conclusion, some elderly patients of advanced HCC with tumor thrombus may obtain a long term survival through this treatment.

Aged, 80 and over↗
PubMed2005

Domino coupling relay approach to polycyclic pyrrole-2-carboxylates.

CuBr2-catalyzed three-component coupling of N-benzylallylamine, ethyl glyoxalate, and terminal alkynes afforded glycine-tethered 1,6-enynes, which were further transformed into polycyclic pyrrole-2-carboxylates via novel cycloisomerization/Diels-Alder cycloaddition/dehydrogenation sequence under iridium-catalyzed conditions.

Journal Article↗
PubMed2005

[Sutureless technique for oozing type postinfarction left ventricular free wall rupture].

We report our experience using a sutureless technique for oozing type postinfarction left ventricular free wall rupture. Several materials such as fibrin seat, autologous or heterologous pericardial patch, fibrin glue, and geratin-resorcin-formaldehyde (GRF) glue have been used. Nine patients, who developed postinfarction left ventricular free wall rupture, underwent surgical repair using a sutureless technique between 1999 and 2004. All patients survived and discharged our hospital without any postoperative complications. And all are alive an exellent condition in 5 to 44 months. A sutureless technique for the treatment of oozing type postinfarction left ventricular free wall rupture is simple, effective, and associated with a favorable outcome.

Aged↗
PubMed2005

[Low-dose cisplatin plus 5-fluorouracil with radiotherapy for unresectable upper and hilar bile duct carcinoma].

We report herein the value of a combined treatment modality for bile duct cancer (intraluminal brachytherapy: ILBT+extrabeam radiation therapy+chemotherapy+expandable metallic biliary stent), employed for cases in which excision of the upper and the hilar bile duct cancer was not possible. The combined treatment modality group did not attain a two-year survival rate according to the Kaplan Meier method. They were not able to surpass the treatment results of the excision group which ended in radical cure level of C. Moreover, with the combined modality treatment, the addition of radiation therapy significantly improved the cumulative survival rate. In conclusion, the first choice of treatment for the upper and the hilar bile duct cancer is excision, and it is necessary to thoroughly examine the possibility of excision. Additionally, to surpass the treatment results of the excision group that ended in a radical cure level of C, it is necessary to include radiation therapy and chemotherapy using new anti-cancer drugs in a combined treatment modality.

Antimetabolites, Antineoplastic↗
PubMed2004

Participation of singlet oxygen in ultraviolet-a-induced lipid peroxidation in mouse skin and its inhibition by dietary beta-carotene: an ex vivo study.

Dietary beta-carotene acts as a photoprotective agent in the skin, but the exact mechanism of protection is unknown. This ex vivo study is focused on determining the mechanism of action of beta-carotene against UV-A-induced skin damage by characterizing peroxidized phosphatidylcholine (PC) and beta-carotene oxidation products. BALB/c mice were fed with basal or a beta-carotene-supplemented diet, and homogenates from their dorsal skin were prepared after 3 weeks for UV-A irradiation. Analyses revealed that the degree of lipid peroxidation in the beta-carotene group was significantly lower than that in the controls. The isomeric composition of hydroperoxy fatty acids, constituting peroxidized PC, was determined by thin-layer chromatography-blotting followed by gas chromatography/mass spectrometry (MS)/selected ion monitoring analysis. The 9- and 10-isomers of peroxidized PC, resulting from the reaction of singlet molecular oxygen ((1)O(2)) with oleic acid, were elevated in the UV-A-exposed control group compared to the experimental group. Similar results were obtained from methylene-blue-sensitized photooxidation of mouse skin lipids in vitro. Liquid chromatography/MS analysis of the homogenates confirmed the formation of beta-carotene 5,8-endoperoxide, a specific marker for the (1)O(2) reaction. These results indicate that dietary beta-carotene accumulates in the skin and acts as a protective agent against UV-A-induced oxidative damage, by quenching the (1)O(2).

Animals↗
PubMed2003

A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells.

PURPOSE: In the field of cancer immunotherapy research, the targeting of effector cells with specific antibodies is a very promising approach. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems. METHODS: We have produced an anti-epidermal growth-factor receptor (EGFR) x anti-CD3 bispecific diabody (Ex3 diabody) in an Escherichia coli (E. coli) expression system with refolding method. The Ex3 diabody targets lymphokine-activated killer cells with a T-cell phenotype (T-LAK cells) to EGFR positive bile duct carcinoma cells with dramatic enhancement of cytotoxicity in vitro. This specific killing of EGFR-positive cells was completely inhibited by parental mAb IgGs directed to EGFR and the CD3 antigen. RESULTS: When T-LAK cells were cultured with EGFR-positive tumor cells in the presence of Ex3 diabody, they produced much higher levels of IFN-gamma, GM-CSF, and TNF-alpha than in its absence, this being a possible mechanism underlying specific antitumor activity. The Ex3 diabody showed good stability when tested at 37 degrees C for 48 h, and also markedly inhibited tumor growth of bile duct carcinoma xenografts in severe combined immunodeficient (SCID) mice. When Ex3 diabody (20 microg/mouse) was administrated intravenously, together with T-LAK cells and interleukin-2 (IL-2), complete cure of tumors were observed in three of six mice, and the other three showed marked retardation of tumor growth. CONCLUSION: The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.

Animals↗
PubMed2003

A novel adenovirus expressing human 4-1BB ligand enhances antitumor immunity.

4-1BB ligand (4-1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4-1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4-1BB in immune function, but relatively few investigations of human 4-1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4-1BBL (Ad4-1BBL) and its stimulation of antitumor immunity. Ad4-1BBL was able to efficiently infect several human adenocarcinoma cell lines and induce 4-1BBL expression on the cell surface within 24 h, this enhancing the antitumor activity not only of lymphokine-activated killer cells with a T cell phenotype (T-LAK) but also naive peripheral blood mononuclear cells (PBMC). This antitumor activity with T-LAK cells was further enhanced by addition of bispecific antibody (BsAb; anti-MUC1xanti-CD3). Cocultivation of Ad4-1BBL-infected tumor cells with either T-LAK cells or PBMC resulted in significant elevation of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) production. Furthermore, remarkable tumor growth inhibition was observed in cholangiocarcinoma-grafted severe combined immunodeficient (SCID) mice to which Ad4-1BBL and T-LAK cells were administered when tumor size exceeded 5 mm in diameter. These results provide strong evidence in support of the efficacy of adenovirally delivered 4-1BBL for genetic immunotherapy of cancer.

4-1BB Ligand↗
PubMed2003

Prosthetic valve endocarditis with extensive aortic root abscess: full aortic root reconstruction with stentless bioprosthesis, xenopericardium and mitral valve replacement.

Prosthetic valve endocarditis with an extensive aortic root abscess usually has high mortality and morbidity. A 71-year-old male with an extended aortic root abscess following aortic valve replacement survived after full aortic root reconstruction with glutaraldehyde bovine pericardium, mitral valve replacement and full root replacement using stentless bioprosthesis. The patient is well without recurrence of infection, 18 months postoperatively. This procedure might be an alternative treatment for prosthetic valve endocarditis with an extended aortic root abscess.

Abscess↗
PubMed2002

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment.

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins. Two types of refolded Fv were prepared: (i) Heavy and light chains of the immunoglobulin variable regions (VH and VL, respectively) were coexpressed with a dicistronic expression vector (designated Fv(co)); (ii) VH and VL were expressed separately, mixed stoichiometrically, and refolded (designated Fv(mix)). All samples refolded with high efficiency; Fv(co), Fv(mix), scFv, and the bispecific diabody bound to several CEA-positive cell lines, exactly as did soluble Fv fragments secreted by E. coli (Fv(sol)) and the parent IgG. The refolded fragments inhibited binding of the parent IgG to CEA-positive cell lines, indicating that their epitope is identical to that of IgG. The bispecific diabody, which combined variable-region fragments of anti-CEA antibody with variable-region fragments of anti-CD3 antibody, was also prepared using the refolding system. This refolded diabody could bind to lymphokine-activated killer cells. In addition, its cytotoxicity toward human bile duct carcinoma TFK-1 and other several other CEA-positive cell lines was concentration-dependent. Taken together, our results suggest that a refolding procedure can be used to prepare various functional antibody fragments (Fv, scFv, and diabody).

Animals↗