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Biomedical subjects

H Hamada

Publications and source records attributed to H Hamada.

At least 109 records · Page 6Linked to original sources
PubMed2000

In vivo transfer of hepatocyte growth factor gene accelerates proliferation of hepatic oval cells in a 2-acetylaminofluorene/partial hepatectomy model in rats.

To clarify the effect of hepatocyte growth factor (HGF) on proliferation of hepatic oval cells, we transferred HGF gene into liver of the Solt-Farber rat model. Male Fisher 344 rats were infected with a recombinant adenovirus carrying the cDNA for HGF (pAxCAHGF) from tail vein. HGF mRNA showed its peak at 4 days, and diminished thereafter. The total and proliferating cell nuclear antigen-positive hepatic oval cells were significantly elevated in HGF-transferred rats, in which stem cell factor and c-kit mRNA increased at each time point. Our results suggest that in vivo transfer of the HGF gene into liver accelerates proliferation of hepatic oval cells in the Solt-Farber model in rats.

2-Acetylaminofluorene↗
PubMed2000

Beryllium-stimulated production of tumor necrosis factor-alpha by a mouse hybrid macrophage cell line.

Chronic beryllium disease (CBD) results from exposure to the light-weight metal beryllium (Be). In vitro stimulation of bronchoalveolar lavage cells from CBD subjects causes the production of high levels of TNF-alpha, IFN-gamma and IL-6. We tested the hypothesis that Be-stimulation might induce the production of TNF-alpha by macrophage cell lines. We observed that H36.12j cells (12j), a mouse hybrid macrophage cell line, but not other mouse and human macrophage cell lines, produced TNF-alpha upon Be-stimulation. The response was maximal at 100 microM BeSO4 and did not occur when 12j cells were stimulated with either aluminum sulfate or cobalt sulfate. Beryllium-stimulated the production of 725+/-25 pg/ml (mean +/- SEM) TNF-alpha protein by 12j cells as measured by ELISA of culture supernatants after 24 h. As measured by RT-PCR, Be-stimulated 12j cell TNF-alpha protein production was accompanied by an increased intracellular TNF-alpha mRNA at 3 and 24 h. The addition of 10U or 100U of rMu-IFN-gamma to Be-stimulated 12j cells further increased TNF-alpha production 1.5-4 fold (1.6+/-0.1 ng/ml) respectively. Bacterial lipopolysaccharide (LPS, 1 microg/ml) stimulated production of TNF-alpha in 12j culture supernatants after 6 h (515+/-151 pg/ml). This early versus late TNF-alpha production suggests that LPS and Be both stimulate 12j cell TNF-alpha synthesis, but through different pathways. We report for the first time, the direct effects of Be stimulation on the ability of 12j cells to produce TNF-alpha. The 12j cell line, contrasted with other macrophage hybrids that do not respond to Be-stimulation, may provide a useful tool to evaluate the mechanisms by which Be stimulates macrophage cytokine production, and by which T cell derived IFN-gamma amplifies TNF-alpha production in granulomatous diseases.

Animals↗
PubMed2000

Beryllium-stimulation does not activate transcription factors in a mouse hybrid macrophage cell line.

We tested the hypothesis that beryllium (Be) could stimulate H36.12j cell (12j) TNF-alpha production by transcription factor-mediated pathways similar to those induced by either LPS- or IFN-gamma stimulation. Unstimulated 12j cells produce constitutive levels of TNF-alpha (175+/-18 pg/ml, mean +/- SEM) detected by ELISA of culture supernatants after 24 h. Beryllium-stimulated (100 microM BeSO4) 12j cell TNF-alpha (724+/-47 pg/ml) was observed after 24 h while LPS-stimulated (1 microg/ml) TNF-alpha (515+/-151 pg/ml) after 6 h. Recombinant-Mu-IFN-gamma (10 U) stimulated 12j cell TNF-alpha at lower levels (284+/-31 pg/ml) while rMu-IFN-gamma + Be-stimulated 12j cells produced 1195+/-225 pg/ml TNF-alpha. Constitutive levels of transcription factors were observed in unstimulated 12j cell nuclei. In LPS-stimulated 12j cells IkappaBalpha was degraded in the cytoplasm and increased levels of NF-kappaB were found in nuclei after 30 min. After 3 h there were increased levels of AP-1 and CREB, with increased amounts of Fos family, Jun B and Jun D transcription factors. In contrast, Be-stimulation failed to increase the levels of any transcription factor tested, NF-kappaB, AP-1, AP-2, CREB, C/EBP, Sp-1, Egr-1, Ets, NF-Y or Oct-1, in 12j cells. A pattern of increased transcription factors, similar to that observed for LPS-stimulation, was found in 12j cell nuclei after stimulation with rMu-IFN-gamma. However, NF-kappaB was increased at 3 h while AP-1 (Jun B and Jun D) and CREB were increased at 15 h. Co-stimulation of 12j cells with rMu-IFN-gamma + Be increased the levels of NF-KB in 12j cell nuclei at 3 h, and the levels of AP-1 and CREB at 15 h, however, only Jun B was increased. Our data show 12j cell TNF-alpha production was associated with increased levels of transcription factors present in nuclei with disparate kinetics and patterns of expression depending on the trigger. We reject our initial hypothesis and conclude that Be-stimulation signals 12j cell TNF-alpha synthesis via a transcription factor-independent pathway. Beryllium may induce novel pathways of macrophage cytokine gene regulation.

Animals↗
PubMed2000

Adenovirus-mediated transfer of Bcl-X(L) protects neuronal cells from Bax-induced apoptosis.

Bax-mediated apoptosis in neurons is involved in many pathologic conditions affecting the central nervous system, including degenerative diseases, stroke, and trauma. Two molecules belonging to the Bcl-2 family, Bcl-2 and Bcl-X(L), protect cells from Bax-induced apoptosis and show distinct expression patterns in adult neurons, with downregulated Bcl-2 and highly upregulated Bcl-X(L) expression. To investigate the biological functions of these two molecules in Bax-mediated apoptosis in neurons, we transduced various levels of Bcl-X(L) or Bcl-2 via adenoviral vectors into nerve growth factor (NGF)-treated PC12 cells. Overexpression of Bax induced drastic apoptosis in NGF-treated PC12 cells. Bcl-X(L) expressed at a wide range of levels conferred a high level of protection against Bax-mediated apoptosis. In contrast, Bcl-2 at various levels conferred far less protection against apoptosis. Moreover, Bcl-X(L) protected PC12 cells from apoptosis induced by NGF withdrawal. These data indicate that Bcl-X(L)-mediated protection is the major pathway that suppresses apoptosis in NGF-treated PC12 cells and that Bcl-X(L) would be a more relevant target of manipulation in future treatment strategies, including gene therapies.

Adenoviridae↗
PubMed2000

Effective gene therapy for pancreatic cancer by cytokines mediated by restricted replication-competent adenovirus.

Pancreatic cancer has a poor prognosis even when surgical treatment can be accomplished. Studies have demonstrated that pancreatic cancer is associated with various genetic abnormalities in oncogenes and tumor suppressor genes including p53. New therapeutic approaches for pancreatic cancer can be developed by targeting these genetic alterations. Adenovirus (Adv) lacking the 55-kDa E1B protein (E1B55K) replicates preferentially in p53-deficient cancer cells. We constructed E1B55K-deleted Adv (AxE1AdB), and studied its replication and cytopathic effect on pancreatic cancer cells. AxE1AdB replicated in and caused cell death of the p53-deficient pancreatic cancer cell lines tested (e.g., PANC-1, MIAPaCa-2, SU.86.86, BxPC-3, and PK-1). To enhance its therapeutic effect, we examined the combination of coinfecting this restricted replication-competent adenovirus (RRCA) with other Adv. Coinfection of E1-deficient Adv expressing the reporter lacZ gene (AxCAlacZ) together with AxE1AdB resulted in the replication of both viruses and a marked increase in reporter gene expression. PANC-1 cells coinfected with AxE1AdB and the Adv for human IL-2 (AxCAhIL2), produced 110 times more IL-2 than those infected with AxCAhIL2 alone. Similarly, coinfection of AxE1AdB and Adv for human IL-12 augmented the IL-12 production by 370-fold. Injecting AxE1AdB into the PANC-1 tumor of severe combined immunodeficient mice (SCID mice) resulted in marked reduction of the volume of the tumor. Moreover, injecting AxE1AdB with AxCAhIL2 into the PANC-1 tumor resulted in complete regression of the established tumors. These data suggest that RRCA, which augments the antitumor effect of a viral transgene (i.e., cytokines), may be a powerful tool for treating p53-deficient pancreatic cancer.

Adenoviridae↗
PubMed2000

Experimental gene therapy for brain tumors using adenovirus-mediated transfer of cytosine deaminase gene and uracil phosphoribosyltransferase gene with 5-fluorocytosine.

Transduction of the cytosine deaminase (CD) gene into tumor cells followed by administration of 5-fluorocytosine (5-FC), called 5-FC/CD gene therapy, was created as suicide gene therapy for various cancers. The uracil phosphoribosyltransferase (UPRT) gene, which is absent from mammalian cells, directly converts 5-fluorouracil (5-FU) to 5-fluorouridine 5'-monophosphate. We evaluated whether the coexpression of CD and UPRT genes could generate a synergistic antitumor effect on experimental brain tumors. In vitro study showed that 9L cells, transduced with the UPRT gene by an adenovirus, were 16 times more sensitive to 5-FU, and CD + UPRT-transduced cells were 6,000 times more sensitive to 5-FC than parent cells, indicating that the acquisition of CD and UPRT further increased the 5-FC sensitivity of 9L cells compared with cells transduced with CD alone. In a rat brain tumor model, decreased amounts of CD and UPRT vectors were inoculated into the tumors to detect any additional effect of UPRT. CD and UPRT coexpression followed by 5-FC administration showed an antitumor effect as detected by sequential magnetic resonance imaging. This therapy significantly prolonged animal survival. These results suggest that 5-FC/CD + UPRT gene therapy can enhance the antitumor effect of 5-FC/CD gene therapy. Consequently, this approach might be a more feasible modality for the treatment of malignant brain tumors.

Adenoviridae↗
PubMed2000

Proteasome inhibitors induce cytochrome c-caspase-3-like protease-mediated apoptosis in cultured cortical neurons.

The ubiquitin-proteasome protein degradation pathway is crucial in controlling intracellular levels of a variety of short-lived proteins and maintaining cellular growth and metabolism. In a previous study, we showed the accumulation of conjugated ubiquitin in CA1 neurons of the gerbil after 5 min of forebrain ischemia (; ). The accumulation of conjugated ubiquitin may reflect proteasome malfunction. In the present study, we investigated the effects of proteasome inhibitors on primary neuronal cultures to determine whether proteasomal malfunction induces neuronal death. When carbobenzoxy-Leu-Leu-Leu-aldehyde or lactacystin, two different types of proteasome inhibitors, were separately used to suppress proteasome activity, we observed induction of apoptotic neuronal cell death in both cases. During the apoptotic process, mitochondrial membrane potential was disrupted, cytochrome-c was released from mitochondria into the cytosol, and caspase-3-like proteases were activated. Apoptosis was inhibited by pretreatment with acetyl-aspartyl-glutamyl-valyl-aspart-1-aldehyde or overexpression of Bcl-x/(L). These results demonstrated that suppression of proteasome function induces neuronal apoptosis via the release of cytochrome c from mitochondria and activation of caspase-3-like proteases.

Animals↗
PubMed2000

High rate of TTV infection in multitransfused patients with pediatric malignancy and hematological disorders.

The prevalence of transfusion-transmitted virus (TTV) infection has not been known in patients suffering from pediatric malignancies and hematological disorders who receive blood transfusion and/or blood products during treatment. Blood samples were taken from 75 patients. TTV infection was identified when TTV DNA was detected in serum by a polymerase chain reaction (PCR) assay. Hepatitis C virus (HCV) and hepatitis G virus (HGV) RNA were also assayed by PCR. TTV DNA was detected in 38 of 75 patients (51%). In 4 of 38 patients, the amount of blood transfused was less than 3 units. By time since last transfusion, TTV DNA was detected in 12 of 35 patients after more than 4 years, 12 of 21 between 1 and 4 years, and 14 of 19 within 1 year. Six patients had mixed infection of TTV and HCV, and 12 patients had mixed infection of TTV and HGV. Three different kinds of virus were found simultaneously in serum from 3 patients. Eight out of 75 patients showed abnormal levels of alanine aminotransferase (ALT) (>40 IU/liter), and 3 of them had TTV DNA. All patients who had TTV DNA and elevated ALT levels also were positive for HCV RNA and HGV RNA. The prevalence of TTV infection is high in patients with pediatric malignancies and hematological disorders after episodes of blood transfusion. Transfusion is one of the most important risk factors for TTV infection regardless of the amount of blood transfused.

Adolescent↗
PubMed2000

Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor alpha and basic fibroblast growth factor is associated with the expression of apoptosis-related molecules.

OBJECTIVE: Fas-mediated apoptosis is associated with the pathophysiology of rheumatoid arthritis (RA). However, the molecular mechanisms of this process remain to be elucidated in rheumatoid synovium. We investigated the behavior of intracellular signaling molecules that regulate Fas-mediated apoptosis in RA synoviocytes. METHODS: Anti-Fas monoclonal antibody (mAb) was added to RA synoviocytes after treatment with tumor necrosis factor alpha (TNFalpha) or basic fibroblast growth factor (bFGF) for 5 days. The cytotoxic activity was measured using a lactate dehydrogenase-release assay. The expression of apoptosis-related molecules in RA synoviocytes was examined by immunoblot analysis. The enzymatic activities of caspases 3 and 8 under Fas ligation were examined. Transfer of the FADD (Fas-associated death domain) protein and the FLIP(L) (long form of the FLICE [FADD-like interleukin-1beta-converting enzyme]-inhibitory protein) gene into RA synoviocytes was performed using adenoviral vectors. RESULTS: Following a 5-day treatment with TNFalpha or bFGF, Fas ligation with its agonistic mAb induced apoptosis of almost all TNFalpha-treated RA synoviocytes but only showed a weak apoptotic activity in bFGF-treated synoviocytes. Although there was no correlation between the induction of Fas-mediated apoptosis and the expression of apoptosis-related molecules among these cells, a high enzymatic activity of caspases 3 and 8 was observed only in the TNFalpha-treated RA synoviocytes after Fas ligation. The bFGF-treated RA synoviocytes were relatively resistant to apoptosis induced by FADD gene transfection, as compared with the TNFalpha-treated synoviocytes. In addition, the expression of FLIP(L), an inhibitory molecule of Fas-mediated apoptosis, was reduced in TNFalpha-treated RA synoviocytes, and the expression of FLIP43 was augmented in bFGF-treated RA synoviocytes. Moreover, Fas-mediated apoptosis in TNFalpha-treated RA synoviocytes was partially inhibited by FLIP(L) gene transfection. CONCLUSION: Our results suggest that Fas-mediated apoptosis of RA synoviocytes is differentially regulated by TNFalpha and bFGF. In addition, the regulatory mechanisms of apoptosis involve the formation of the death-inducing signaling complex, especially at the level of caspase 8 activation, and this process may be partly associated with FLIP expression.

Antibodies, Monoclonal↗
PubMed2000

Adenovirus-mediated gene therapy for mucopolysaccharidosis VII: involvement of cross-correction in wide-spread distribution of the gene products and long-term effects of CTLA-4Ig coexpression.

Recombinant adenoviruses expressing human beta-glucuronidase (AxCAhGUS) and CTLA-4Ig (AxCACTLA-4Ig) were generated and therapeutic efficacy was investigated using a murine model of mucopolysaccharidosis type VII (MPSVII). Seven days after the intravenous administration of AxCAhGUS, high levels of beta-glucuronidase (GUSB) activity were observed in the liver, spleen, heart, lung, kidney, and serum, while viral DNA was predominantly detected in the liver. To investigate the contribution of in vivo cross-correction of GUSB between the liver and other organs, we injected the serum obtained from the transduced mice into untreated MPSVII mice. Similar distributions of GUSB activity were observed in the serum-injected mice, suggesting that GUSB activities detected in the extrahepatic organs were due to the cross-correction rather than the direct gene transduction. This result also suggested that maintaining high levels of GUSB in the systemic circulation was essential for the effective treatment of MPSVII. To achieve this, we injected AxCAhGUS and AxCACTLA-4Ig into MPSVII mice. Serum GUSB activity was sustained at high levels for more than 200 days and morphological normalization of the liver and spleen was observed for a year. This suggests that long-term therapeutic efficacy in visceral organs of MPSVII is achievable by coexpression of CTLA-4Ig through an in vivo cross-correction pathway.

Abatacept↗
PubMed2000

Usefulness of niravoline, an arginine vasopressin inhibitor, on tumour-origin brain oedema.

Niravoline is a selective agonist of kappa-opioid receptors having potent aquaretic activity which may peripherally be mediated in animals. This effect of niravoline is thought to be due to inhibition of arginine vasopressin secretion. Niravoline does not cause an overt excretion of electrolytes that is seen with conventional diuretics, and it is anticipated that niravoline may prove useful in the treatment of oedema. In this study, effect of niravoline on tumour-origin brain oedema was investigated in a brain tumour model created by implantation of C6 glioma cells into the brains of Wistar rats. Five weeks after the tumour cell implantation, niravoline was administered i.v. at 1 mg/kg a total of four times at one-hour intervals. In the control group (i.e., saline or vehicle-treated rats), this brain tumour model was found to result in a statistically significant increase in the water content in selected brain regions remote from the tumour. Administration of niravoline inhibited the increase in the water content in such selected brain regions. Niravoline might be useful on tumour-origin brain oedema clinically.

Animals↗
PubMed2000

Progress reports on immune gene therapy for stage IV renal cell cancer using lethally irradiated granulocyte-macrophage colony-stimulating factor-transduced autologous renal cancer cells.

There is no effective treatment for patients with stage IV renal cell cancer (RCC), although the introduction of new therapy is imminent. Cancer gene therapy is currently considered to be one of the most promising therapeutic modalities in the field of cancer treatment. Based on the results of animal studies, vaccination using autologous granulocyte-macrophage colony-stimulating factor-transduced renal cancer cells appears promising. Before initiating a clinical study using an ex vivo gene-transduced autologous cell vaccine-based immunogene therapy for RCC in Japan, in 1992 we initially planned a Japanese version of a clinical protocol in collaboration with a US group. In 1993, the original protocol was refined. We performed five preclinical qualification studies using RCC nephrectomy specimens from patients in 1997, and the results showed that preparation of RCC cells for autologous vaccines at the Clinical Cell Technology Facility, Research Hospital of the Institute of Medical Science, University of Tokyo, was feasible. Subsequently in August 1998, the Ministry of Health and Welfare and the Ministry of Education, Science, Culture, and Sport approved our clinical protocol. We have recruited two patients with stage IV RCC to our study so far. Here we report the background to the initiation of cancer gene therapy in Japan.

Adult↗
PubMed2000

Adenovirus-mediated gene transfer of CTLA4Ig gene results in prolonged survival of heart allograft.

It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection. The purpose of this study is to determine the effect of adenovirus-mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats. Adenoviral vectors with beta galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed. These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection. Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats. Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506. The transgene expression after adenovirus-mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein. By the administration of 1 x 10(9) PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 x 10(8) PFU of Adex/hCTLA4Ig. In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable. The combination therapy using a low titer Adex/hCTLA4Ig and low-dose of FK506 was synergistically effective on this cardiac transplantation model. In conclusion, adenovirus-mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival.

Abatacept↗
PubMed2000

Heterotopic thyroid tissue at the porta hepatis in a fetus with trisomy 18.

Heterotopic thyroid tissue at the porta hepatis in a fetus with trisomy 18 is described. The fetus had an eutopic thyroid gland without any abnormalities. The heterotopic thyroid was found at the porta hepatis and showed histological features similar to the eutopic thyroid. Immunohistochemically, the heterotopic follicles were positive for thyroglobulin, but no calcitonin-positive cells were found. Intra-abdominal heterotopic thyroid is exceedingly rare in locations other than the ovary, and to our knowledge, this is the first report of a fetal case. The present case provides clear evidence that abdominal heterotopic thyroid can occur as a congenital anomaly. Migration abnormality of the median anlage of the thyroid is the most likely histogenesis of heterotopic thyroid at the porta hepatis.

Abnormalities, Multiple↗
PubMed2000

The 4G/5G polymorphism of the plasminogen activator inhibitor-1 gene is associated with severe preeclampsia.

Preeclampsia is associated with thrombosis of the intervillous or spiral artery. A deletion/insertion polymorphism (4G or 5G) in the promoter of the plasminogen activator inhibitor type 1 (PAI-1) gene is suggested to be involved in regulating the synthesis of the inhibitor, 4G allele, being associated with the enhanced gene expression and plasma PAI-1 levels. We assessed the association between preeclampsia and the 4G/5G polymorphism of the PAI-1 gene in 115 preeclamptic patients, 210 pregnant controls, and 298 healthy volunteer controls. The frequency of the homozygotes for the 4G allele was significantly higher in the patients than in the control pregnant women (P = 0.04) or in the healthy volunteers (P = 0.02). The 4G allele frequency was also significantly higher in the patients than in the control group of pregnant women (P = 0.03) and in the healthy volunteers (P = 0.02). These results suggest that the presence of the 4G/4G genotype of the PAI-1 gene is one of the risk factors for preeclampsia.

Alleles↗
PubMed2000

Dominant expansion of human T cells in non-obese diabetic/severe combined immunodeficiency mice implanted with human bone fragments.

OBJECTIVE: To establish an in vivo animal model in which human T cells develop and function normally, a step toward developing new vaccines or chemical compounds that modulate immune functions and toward understanding T-cell immunity in humans. MATERIALS AND METHODS: Human bone fragments were implanted into non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice. The presence of human blood cells in the peripheral blood of these mice was monitored periodically by immunostaining and fluorescence-activated cell sorting. RESULTS: After implantation of bone fragments, dominant expansion of human T lymphocytes, rather than myeloid and B cells, was observed over a 3-month period. In some cases, the proportion of human T cells rose to 40% of the peripheral blood mononuclear cells. These T cells showed CD4/CD8 ratios similar to those observed in human peripheral blood lymphocytes and had a broad repertoire of rearranged T-cell receptor genes. Graft-versus-host reaction was not noted in any organ analyzed. To assess the suitability of NOD/SCID mice implanted with human bone fragments (hu-bone-NOD/SCID mice) as an in vivo model for HIV infection, the mice were infected with a T-lymphotropic strain of HIV-1 (NL4-3) at 7 weeks posttransplant. Serum p24 gag was detected at 2 weeks after inoculation, after which total CD4-positive cell numbers declined, as seen clinically in patients infected with HIV. CONCLUSION: Although the precise mechanism is yet to be determined by which predominant expansion of human T cells occurs in hu-bone-NOD/SCID mice, such mice appear likely to serve as a useful and versatile model for studies involving human T-cell immunity.

Animals↗