[Gene therapy vector: retroviral vector].
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Genetic Therapy↗
Biomedical subjects
H Hamada
Publications and source records attributed to H Hamada.
[Chronic eosinophilic pneumonia successfully treated with suplatast tosilate].
The patient was a 45-year-old woman in whom a chest X-ray film obtained on admission showed bilateral infiltrative shadows. A chest CT scan revealed areas of consolidation and ground-glass opacities. Examination of bronchoalveolar lavage fluid revealed many eosinophils, and specimens obtained by transbronchial lung biopsy showed thickened alveolar septa with infiltration of eosinophils. The numbers of eosinophils and the level of IgE in blood were low. The radiographic appearance improved after administration of suplatast tosilate. The level of interleukin 5 was high both in plasma and in bronchoalveolar lavage fluid before treatment, but the level of IL-5 in plasma was low after suplatast tosilate was given. Suplatast tosilate, which suppresses the production of cytokines, may be useful in patients with chronic eosinophilic pneumonia.
Anti-Allergic Agents↗
[An operative case of congenital fistula of the right coronary artery to the left ventricle with congestive heart failure].
We reported an extremely rare case of congenital fistula of the right coronary artery to the left ventricle in a 71-year-old man who suffered from difficulty in breathing and palpitation. The diagnosis was confirmed by the right coronary angiography. The fistula was closed by Symbas's operation under cardiopulmonary bypass. Postoperative recovery was uneventful. Coronary angiography performed one year after the operation revealed that the right coronary artery was completely occluded by thrombus. But there were no signs of myocardial ischemia or infarction. And so we suspected that the dilated right coronary artery had not work as functional vessel.
Aged↗
[Evaluation of intra-arterial infusion chemotherapy for liver metastasis from gastric cancer].
We evaluated the result of intra-arterial infusion chemotherapy on liver metastasis from gastric cancer. Of 92 cases of metastatic liver tumor, 17 cases received intra-arterial infusion chemotherapy after primary resection. For comparison, we assigned the 17 cases to two groups according to the infused agents. One group was treated with the combination therapy of 5-FU, epirubicin and MMC (FEM group: n = 7), and another with other antineoplastic agents (non-FEM group: n = 10). In the FEM group, the response rate, 1-year survival rate and 50% survival period were 33.3%, 51.4%, 430 days, respectively, while those of the non-FEM group were 10.0%, 10.0%. 147 days. Although there was no significant difference (p = 0.0951), improvements in survival rate and survival period were observed. This implies the possibility that intra-arterial infusion chemotherapy, especially the combination therapy of FEM, is an effective treatment for liver metastasis from gastric cancer.
Aged↗
[Complication due to arterial infusion chemotherapy for liver metastasis from colorectal cancer].
OBJECTIVES: Arterial infusion chemotherapy is considered to be an extremely effective treatment for liver metastasis from colorectal cancer in terms of its tumor reduction and preventing recurrence in residual liver after resection. However, there still remain some unclear points as to the influence on hepatic artery and bile duct when this treatment is used over the long term. We report some conclusions obtained by examining cases of hepatic arterial occlusion (stenosis) and biliary complication who received this treatment. MATERIALS AND METHODS: Thirty-six cases who received this treatment over 3 months were the objects of this study, with the aim of direct effect against metastatic focus (21 cases) and prevention of recurrence in residual liver (15 cases). The ages were from 27 to 81; 22 cases were male and 14 were female. Indwelling routes of catheter were gastroduodenal artery (GDA) in 28 cases and femoral artery (FA) in 8 cases. Intermittent high-dose infusion (WHF: 5-FU 1,000 mg/m2/5 hrs qw) was adopted as the method. RESULTS: Hepatic arterial occlusion or stenosis was observed in 12 cases (GDA: 10; FA: 2). There seemed to be no correlation with the total dosage of 5-FU or the number of administrations. Even when hepatic arterial occlusion or stenosis occurred, no change was observed in liver function, and there no death was caused by this. However, CT showed a low-density area followed by atrophy in the right lobe in one case with right hepatic arterial stenosis, despite normal portal blood flow. Of the 6 cases which developed obstructive jaundice, 4 were due to the increase of metastatic focus or lymph nodes, and 1 case without dilatation of bile duct died from suspected sclerosing cholangitis. In this case, ALP had been increasing since 1 month before the onset of jaundice. Another case which developed biloma accompanied by the increase of serum bilirubin improved by discontinuance of chemotherapy. CONCLUSION: Since arterial infusion chemotherapy for liver metastasis from colorectal cancer causes hepatic arterial occlusion (stenosis) at a high rate, early detection of abnormalities by liver function test and imaging diagnosis which leads to early treatment is important.
Adult↗
[Effect of arginine vasopressin (AVP) inhibitor in the inhibition of the peritumoral edema].
We investigated the effect of RU599 Arginine Vasopressin (AVP) inhibitor on peritumoral edema of Wistar rats implanted with C6 glioma. C6 glioma was implanted into the right caudoputamen of 3 week-old rat's brain. Five weeks after the implantation, the changes of the brain water contents were measured by the dry-weight method. RU599 (1 mg/ml/kg body weight) was administered into Wistar rats via the tail vein every one hour (total 4 times), and the same dose of saline was used as control. Rats were sacrificed 1 hour after the last administration, and the brain of each rat was divided into 3 parts such as the anterior part without a tumor, the middle part with a tumor and the posterior part without a tumor. After that each part was separated into the right and left parts. The tumor was removed from the brain. The water contents of the brain were increased in the area around the tumor and the contralateral cerebral hemisphere excluding the anterior part. RU599 could reduce the increased water contents significantly in all areas except in the tumor itself and the area surrounding the tumor. Tumor-origin brain edema includes not only localized one but also diffuse one in the brain, and this study suggests RU599 has an effect of inhibiting the diffuse brain edema.
Animals↗
[High levels of elastolytic activity in bronchoalveolar lavage fluid from a patient with idiopathic interstitial pneumonia].
A 63-year-old female with acure exacerbution of idiopathic interstitial pneumonia (IIP) showed high levels of elastolytic activity in bronchoalveolar lavage fluid (BALF). She was admitted to our hospital with progressive dyspnea. Arterial blood gas analysis and pulmonary function tests were abnormal. Examination of the bronchoalveolar lavage fluid revealed an increased numbers of neutrophils. Despite the administration of corticosteroids, the patient died of respiratory failure. A high level of elastolytic activity was present in the BALF. Western immunoblot analysis, using an anti-alpha 1-protease inhibitor (PI) antibody, revealed a truncated alpha 1-PI in the BALF. These findings suggest that an imbalance between protease and PI in the lower respiratory tract contribute to lung tissue damage in patients with IIP.
Bronchoalveolar Lavage Fluid↗
Regulation of striatal D1A dopamine receptor gene transcription by Brn-4.
Brn-4 is a member of the POU transcription factor family and is expressed in the central nervous system. In this study, we addressed whether Brn-4 regulates expression of the D1A dopamine receptor gene. We found a functional Brn-4 responsive element in the intron of this gene by means of cotransfection chloramphenical acetyltransferase assays. This region contains two consensus sequences for binding of POU factors. Gel mobility-shift assays using glutathione S-transferase-Brn-4 fusion protein indicated that Brn-4 binds to these sequences. Both these sites are essential for transactivation by Brn-4 because deletion of either significantly reduced this enhancer activity. In situ hybridization revealed colocalization of Brn-4 and D1A mRNAs at the level of a single neuron in the rat striatum where this dopamine receptor is most abundantly expressed. Gel mobility-supershift assay using rat striatal nuclear extract and Brn-4 antibody confirmed the presence of Brn-4 in this brain region and its ability to bind to its consensus sequences in the D1A gene. These data suggest a functional role for Brn-4 in the expression of the D1A dopamine receptor gene both in vitro and in vivo.
Animals↗
Augmented antitumor effects of killer cells induced by tumor necrosis factor gene-transduced autologous tumor cells from gastrointestinal cancer patients.
The purpose of this study was to determine the feasibility of a vaccine therapy using tumor necrosis factor (TNF) gene-transduced autologous tumor cells for the treatment of human gastrointestinal cancers, which tend to have lower immunogenicity than other cancers such as melanoma and renal cell carcinoma. We succeeded in establishing primary cultured tumor cells from 12/54 carcinomatous effusions (4 liver cancer patients, 5 gastric cancer patients, 1 pancreatic cancer patient, and 2 colon cancer patients) and in transducing the TNF gene to the tumor cells by using the retrovirus vector MFG-TNF. Even after irradiation, TNF production (0.3-3.5 U/ml per 10(6) cells per 72 hr) was confirmed for 10 of 12 transfectants, and the other two transduced cells were found to have approximately one TNF gene copy. In 7 of the 12 patients, the cytotoxic activity of killer cells to nontransduced autologous tumor cells incubated with these TNF gene transfectants was augmented. This activity was blocked with anti-HLA class I antibody or BrefeldinA (BFA), suggesting that the killer cells were cytotoxic T lymphocytes (CTL) and tumor antigens are presented with HLA class I molecules. Indeed, enhanced expression of HLA class I and/or ICAM-1 molecules on the surface of the TNF gene-transduced tumor cells were observed by fluorescence-activated cell sorting (FACS) analysis. Furthermore, natural killer (NK) and/or lymphokine-activated killer (LAK) activities determined by using K562 or Daudi cells as targets were also enhanced in some of these cases when they were incubated with TNF gene-transduced tumor cells. These findings indicate the feasibility of using TNF gene-transduced tumor cells as a vaccine in gastrointestinal cancer patients.
Carcinoma, Hepatocellular↗
Local production of the p40 subunit of interleukin 12 suppresses T-helper 1-mediated immune responses and prevents allogeneic myoblast rejection.
The p40 subunit of interleukin 12 (IL-12p40) has been known to act as an IL-12 antagonist in vitro. We here describe the immunosuppressive effect of IL-12p40 in vivo. A murine myoblast cell line, C2C12, was transduced with retro-virus vectors carrying the lacZ gene as a marker and the IL-12p40 gene. IL-12p40 secreted from the transfectant inhibited the IL-12-induced interferon gamma (IFN-gamma) production by splenocytes in vitro. Survival of C2C12 transplanted into allogeneic recipients was substantially prolonged when transduced with IL-12p40. Cytokine (IL-2 and IFN-gamma) production and cytotoxic T lymphocyte induction against allogeneic C2C12 were impaired in the recipients transplanted with the IL-12p40 transfectant. Delayed-type hypersensitivity response against C2C12 was also diminished in the IL-12p40 recipients. Furthermore, serum antibodies against beta-galactosidase of the T-helper 1-dependent isotypes (IgG2 and IgG3) were decreased in the IL-12p40 recipients. These results indicate that locally produced IL-12p40 exerts a potent immunosuppressive effect on T-helper 1-mediated immune responses that lead to allograft rejection. Therefore, IL-12p40 gene transduction would be useful for preventing the rejection of allografts and genetically modified own cells that are transduced with potentially antigenic molecules in gene therapy.
Animals↗
Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.
Hematopoietic stem cells (HSCs) supply all blood cells throughout life by making use of their self-renewal and multilineage differentiation capabilities. A monoclonal antibody raised to the mouse homolog of CD34 (mCD34) was used to purify mouse HSCs to near homogeneity. Unlike in humans, primitive adult mouse bone marrow HSCs were detected in the mCD34 low to negative fraction. Injection of a single mCD34(lo/-), c-Kit+, Sca-1(+), lineage markers negative (Lin-) cell resulted in long-term reconstitution of the lymphohematopoietic system in 21 percent of recipients. Thus, the purified HSC population should enable analysis of the self-renewal and multilineage differentiation of individual HSCs.
Animals↗
Left-right asymmetric expression of the TGF beta-family member lefty in mouse embryos.
Examples of lateral asymmetry are often found in vertebrates, such as the heart being on the left side, but the molecular mechanism governing the establishment of this left-right (L-R) handedness is unknown. A diffusible morphogen may determine L-R polarity, but a likely molecule has not so far been identified. Here we report on the gene lefty, a member of the transforming growth factor-beta family, which may encode a morphogen for L-R determination. Lefty protein contains the cysteine-knot motif characteristic of this superfamily and is secreted as a processed form of relative molecular mass 25K-32K. Surprisingly, lefty is expressed in the left half of gastrulating mouse embryos. This asymmetric expression is very transient and occurs just before the first sign of lateral asymmetry appears. In the mouse mutants iv and inv, which cause situs inversus, the sites of lefty expression are inverted, indicating that lefty is downstream of iv and inv. These results suggest that lefty may be involved in setting up L-R asymmetry in the organ systems of mammals.
3T3 Cells↗
Intensified antitumor immunity by a cancer vaccine that produces granulocyte-macrophage colony-stimulating factor plus interleukin 4.
Vaccination with irradiated tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF tumor vaccine) induces a potent systemic antitumor immunity. To develop a protocol for cancer therapy to further augment the host immune response, we examined the effects of the GM-CSF tumor vaccines simultaneously producing additional cytokines. We prepared cancer vaccines expressing double cytokines by sequential recombinant retrovirus-mediated genetic transductions. We then used a murine intracerebral tumor model in which the GM-CSF tumor vaccine was less effective in immunopotentiation and evaluated tumor vaccines producing various cytokines in conjunction with GM-CSF. The cytokine combination of GM-CSF and interleukin 4 induced more potent antitumor immunity than GM-CSF alone. An in vivo depletion test showed that CD4+, CD8+, and asialoGM1+ cells were required for the optimum function of the GM-CSF plus interleukin 4 tumor vaccine. Histological examinations revealed infiltration of inflammatory cells at the site of tumor cell challenge as well as at the site of vaccination, indicating the induction of a systemic antitumor immune response which reached the central nervous system. Our findings suggest the feasibility of applying the intensified vaccination strategy to treat human cancers including malignant brain tumors.
3T3 Cells↗
Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro.
We analyzed the ability of a recombinant replication-defective adenovirus vector with the carcinoembryonic antigen (CEA) promotor to transfer the thymidine kinase gene of herpes simplex virus (HSVtk) into gastric cancer cells to confer sensitivity to ganciclovir (GCV). CEA-producing gastric cancer cell lines (MKN28 and MKN45), a CEA-nonproducing gastric cancer cell line (MKN1), and a human uterine cervical cancer cell line (HeLa) were infected with a recombinant adenovirus carrying lacZ reporter gene coupled to the CEA promoter (AdCEAlacZ). The efficiency of AdCEAlacZ-mediated gene transfer was correlated with the amount of CEA produced by each cell line. Furthermore, the 50% growth inhibitory concentrations (IC50) of GCV were 21 and 5.8 microm for MKN28 and MKN45, respectively, when infected with a recombinant adenovirus carrying the HSVtk gene coupled to the CEA promoter (AdCEAtk). However, MKN1 and HeLa cells infected with AdCEAtk remained resistant to GCV (IC50 > 300 microm of GCV). In addition, a bystander killing effect was demonstrated against MKN45 cells when only 20% of AdCEAtk-infected cells were mixed with uninfected cells. These data indicate the potential for targeted gene therapy using the cell type-specific promotor of the CEA gene against gastric cancers that produce CEA.
Adenoviridae↗
cDNA cloning of a murine homologue of Drosophila single-minded, its mRNA expression in mouse development, and chromosome localization.
A combination of the RT-PCR method and subsequent screening of the cDNA library of mouse skeletal muscle with the cDNA isolated by RT-PCR used as a probe led to isolation of cDNAs encoding a polypeptide (mSim) with bHLH and PAS domains which show high similarity to the corresponding regions of Drosophila Sim, a master regulator in neurogenesis. Experiments using a GST-fusion protein demonstrated that mSim heterodimerizes with Arnt (Ah receptor nuclear translocator), even more efficiently than AhR (Ah receptor) does with Arnt. RNA blot analysis using RNAs from various tissues of mice indicated that mSim transcript is expressed in several limited tissues such as muscle, kidney and lung of adult animals. Distribution of mSim mRNA was always accompanied with that of Arnt. All the results suggest a regulatory role of mSim in partnership with Arnt. Chromosomal location of the mSim gene was determined by fluorescent in situ hybridization to be localized on the C3.3-C4 band of mouse chromosome 16 which is syntenic with the human chromosome 21q22 carrying the Down syndrome critical region, where a gene highly homologous to the Drosophila sim was localized. Whole mount in situ hybridization using a unique part of mSim cDNA sequence showed that mSim mRNA was expressed in the ventral diencephalon, branchial arches and limbs. These findings will provide an approach to the cause of the Down syndrome as well as the elucidation of the functional roles of mSim in animal development.
Age Factors↗
In vivo antitumor effect of cytotoxic T lymphocytes engineered to produce interferon-gamma by adenovirus-mediated genetic transduction.
Immunotherapy with adoptive transfer of genetically-modified cytotoxic T lymphocytes (CTL) is a promising approach for cancer gene therapy. We developed an adoptive therapy model with murine tumor-specific CTL, to which very efficient (up to 100%) gene introduction was achieved by using recombinant adenoviral vectors. Through a comparative study on the antitumor effects of CTL genetically modified with cytokine genes, transduction with interferon-gamma gene resulted in a prominent increase in therapeutic efficacy of CTL in both metastatic and subcutaneous tumor models. Further additive effect was obtained by the adoptive cellular therapy in combination with vaccination of cytokine gene-modified tumor cells. Our findings provide a hopeful strategy of adoptive immunotherapy for human cancers.
Adenoviridae↗
Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene.
We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms.
Adenoviridae↗
GM-CSF-induced in vivo expansion of splenic dendritic cells and their strong costimulation activity.
Dendritic cells (DC), with their potent antigen-presenting and accessory activities, are involved in the stimulation of naive T cells. To examine the biological functions of DC, we developed a method for generating large numbers of murine splenic DC. First, DC were propagated in vivo by using a granulocyte-macrophage colony-stimulating factor-secreting tumor as a continuous in vivo source of the cytokine. The DC enriched in the spleen, especially in the white pulps, were purified after an overnight culture. We could reproducibly obtain 6 to 10 X 10(6) splenic DC per mouse. These DC were morphologically similar to interdigitating cells, expressed high levels of MHC class II and costimulatory molecules, and were highly allo-stimulatory in mixed lymphocyte reactions. Further analysis on T cell stimulation activity revealed that the DC had strong costimulatory activity on human T cells. Activated B cells, which express both B7-1 and B7-2, had little T cell costimulatory activity under the same assay conditions. A human histiocytic leukemia cell line, U937, that showed only weak costimulatory activity by itself, worked synergistically with DC and further intensified the T cell stimulation by DC. These findings suggest the presence of a T cell costimulation mechanism in DC, which is activated synergistically by monocytes or macrophages, and deserves further study.
Animals↗