In vivo gene transfer into rat hearts with Epstein-Barr virus-based episomal vectors using a gene gun.
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Animals↗
Biomedical subjects
E Satoh
Publications and source records attributed to E Satoh.
Highly efficient suicide gene expression in hepatocellular carcinoma cells by epstein-barr virus-based plasmid vectors combined with polyamidoamine dendrimer.
The present study was aimed at devising an efficient nonviral strategy for suicide gene therapy of hepatocellular carcinoma (HCC). To improve the efficiency of DNA delivery and expression, we applied Epstein-Barr virus (EBV)-based plasmid vectors instead of conventional plasmid vectors and combined them with cationic liposome (EBV/lipoplex) or polyamidoamine dendrimer (PAAD) (EBV/polyplex). When the beta-galactosidase gene was transferred to HuH7, PLC/PRF/5, or HLE cells, < or =50-fold higher beta-galactosidase activities were demonstrated in the cells transfected with EBV vector compared with those transfected with conventional plasmid vectors. PAAD-mediated transfection of HCC with pSES.Tk (an EBV-based vector carrying the herpes simplex virus-1 thymidine kinase gene) resulted in a marked reduction in viable cell number by the addition of ganciclovir (GCV). The HCC cells transfected with pSES.Tk/PAAD showed 100- to 1000-fold higher susceptibilities to GCV than those transfected with pS.Tk (a conventional plasmid vector carrying herpes simplex virus-1 thymidine kinase gene)/PAAD. The pSES.Tk-transfected HCC cells were effectively killed by day 9 in culture with a clinically feasible concentration of GCV (25 microM), whereas the pS.Tk-transfected cells survived the culture. These results demonstrate highly efficient suicide gene transfer into various HCC cells by EBV-based plasmid vectors in vitro, suggesting the possible application of this nonviral vector system to gene therapy of HCC.
Antineoplastic Agents↗
Targeted killing of carcinoembryonic antigen (CEA)-producing cholangiocarcinoma cells by polyamidoamine dendrimer-mediated transfer of an Epstein-Barr virus (EBV)-based plasmid vector carrying the CEA promoter.
The present study reports a novel nonviral method to efficiently and specifically target carcinoembryonic antigen (CEA)-producing cholangiocarcinoma (CC) cells in vitro. Epstein-Barr virus (EBV)-based and conventional plasmid vectors were constructed that possess the beta-galactosidase (beta-gal) or herpes simplex virus-1 (HSV-1) thymidine kinase (Tk) genes as well as tandem repeats of the human genomic sequence -82 to -42 bp from the transcriptional start site of the CEA gene. The plasmids were transfected by means of polyamidoamine dendrimer into CEA-positive (HuCC-T1) or -negative cell lines. Transfection of the conventional plasmid vector with the CEA promoter and beta-gal gene resulted in a very low or undetectable level of marker gene expression even in the CEA-positive cell line. Transferring the HSV-1 Tk gene by conventional plasmid did not affect the susceptibility of HuCC-T1 cells to ganciclovir. In marked contrast, strong beta-gal expression was specifically obtained in HuCC-T1 cells by transfecting the EBV-based plasmid in which the CEA promoter and a ubiquitous promoter (SRalpha) are employed to drive the EBV-encoded nuclear antigen 1 (EBNA1) and beta-gal genes, respectively (pTES.beta). Furthermore, CEA-positive but not -negative tumor cells were rendered highly susceptible to ganciclovir when transfected with the EBV-based vector that carries the CEA promoter-EBNA1 and SRalpha-HSV-1 Tk genes (pTES.Tk). These results strongly suggest that the EBV-based plasmid vector/cationic polymer system (EBV/polyplex) equipped with the CEA promoter provides an efficient nonviral method for the targeted gene therapy of CEA-producing malignancies.
Carcinoembryonic Antigen↗
Effective suicide gene therapy in vivo by EBV-based plasmid vector coupled with polyamidoamine dendrimer.
This study demonstrates in vivo effectiveness of a nonviral vector system, Epstein-Barr virus (EBV)-based plasmid vector coupled with polyamidoamine (PAMAM) dendrimer (EBV/polyplex), in suicide gene therapy of cancer. The EBV-based vector is a plasmid vector containing EBV nuclear antigen 1 (EBNA1) gene and oriP from EBV genome. HSV-1 tk gene was transferred into Ewing's sarcoma cell lines, A4573 and KP-EWS-YI, by using an EBV-based plasmid vector, pSES.Tk, or a conventional plasmid vector, pS.Tk. Cells transfected with pSES.Tk/dendrimer showed approximately 100 times lower ID50 to ganciclovir (GCV) compared with those transfected with pS. Tk/dendrimer. Intratumoral injection of pSES.Tk/dendrimer but not pS. Tk/dendrimer drastically suppressed the growth of tumors which had generated from A4573 or Huh7 hepatocellular carcinoma (HCC) cells inoculated into severe combined immunodeficiency (SCID) mice. The treatment with pSES.Tk/dendrimer also resulted in significant prolongation of survival of the mice implanted with A4573. These results suggest that the EBV/polyplex system could be useful for in vivo suicide gene therapy of cancer. Gene Therapy (2000) 7, 53-60.
Animals↗
Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters.
In chronic heart failure, down-regulation of beta-adrenergic receptor (beta-AR) occurs in cardiomyocytes, resulting in low catecholamine response and impaired cardiac function. To correct the irregularity in the beta-AR system, beta-AR gene was transduced in vivo into failing cardiomyocytes. The Epstein-Barr virus (EBV)-based plasmid vector carrying human beta2-AR gene was injected into the left ventricular muscle of Bio14.6 cardiomyopathic hamsters whose beta-AR is down-regulated in the cardiomyocytes. The echocardiographic examinations revealed that stroke volume (SV) and cardiac output (CO) were significantly elevated at 2 to 4 days after the beta2-AR gene transfer. Systemic loading of isoproterenol increased the cardiac parameters more significantly on day 2 to day 7, indicating that the adrenergic response was augmented by the genetic transduction. The same procedure did not affect the cardiac function of normal hamsters. Immunohistochemical examinations demonstrated human beta2-AR expression in failing cardiomyocytes transduced with the gene. RT-PCR analysis detected mRNA for the transgene in the heart but not in the liver, spleen, or kidney. The procedures may provide a feasible strategy for gene therapy of severe heart failure. Gene Therapy (2000) 7, 2087-2093.
Animals↗
Pyemotes dermatitis caused by indirect contact with husk rice.
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Animals↗
Keratoacanthoma developing on a pigmented patch in incontinentia pigmenti.
Cutaneous manifestations of incontinentia pigmenti (IP) have classically been described as three sequential stages: an initial vesicobullous stage, a verrucous stage and a stage of swirled pigmentation. Verrucous lesions tend to last longer than vesicobullous eruptions, often persisting until 1 year of age. However, adult patients with verrucous lesions are rare. We report a case of keratoacanthoma with marked dyskeratosis on a pigmented patch in a 20-year-old woman. This tumor, like subungual keratotic tumors of IP, might have been developed as one of the late manifestations of the disease.
Adult↗
[Renal inflammatory pseudotumor after embolization for arteriovenous malformation: a case report].
A 61-year-old man was admitted to our hospital with the chief complaint of asymptomatic macrohematuria. Because he was diagnosed with arteriovenous malformation of the right kidney by angiography, embolization was performed. However, 2 years and 3 months later, bleeding from the right kidney was detected. Computed tomography (CT) revealed a low density area (1.8 cm) in the right kidney protruding to the renal pelvic adjacent to the old embolized lesion. Since we could not make a conclusive diagnosis as malignant disease by ureteroscopy and angiography, we have decided to follow the case carefully. Nine months later, macroscopic hematuria worsened, and the low density area by CT in the right kidney expanded. Therefore, we finally decided to perform right total nephroureterectomy. The tumor was histologically diagnosed as renal inflammatory pseudotumor. This is the 14th case reported in Japan. We here report the renal inflammatory pseudotumor with a review of the Japanese literature.
Arteriovenous Malformations↗
[A case of urinary stone in ureterocele extracted transurethrally after ESWL].
A 66-year-old man was referred to our hospital with chief complaints of difficulty in urination and terminal micturition pain. Ureteroceles were identified bilaterally, and a ureteral stone (19 x 12 mm) existed in the right ureterocele. After crushing the stone by extra corporeal shock wave lithotripsy (ESWL), we removed the stone transurethrally with a small incision in the right ureterocele. The vesicoureteral reflux (VUR) was not detected postoperatively. Now, we recommend the combination of ESWL and a small transurethral incision of the urelerocele for the treatment of ureteral stones in a ureterocele in order to prevent postoperative VUR.
Aged↗
Effects of sevoflurane and enflurane on lower esophageal sphincter pressure and gastroesophageal pressure gradient in children.
PURPOSE: The effects of sevoflurane and enflurane on the intraluminal pressure of the lower esophagus (LE), lower esophageal sphincter (LES), and stomach were investigated in paralyzed and mechanically ventilated children under general anesthesia. METHODS: A total of 14 children, ASA physical status class I without risk factors for regurgitation, scheduled for orthopedic surgery were studied. After induction of anesthesia, we inserted a gastrointestinal pressure sensor nasally and monitored the intraluminal pressure of the LE, LES, and stomach under various concentrations of sevoflurane or enflurane with 66% nitrous oxide in oxygen prior to surgical incision. The barrier pressure (BrP), which is the difference between LES pressure and intragastric pressure, was calculated. RESULTS: Sevoflurane at 2.0 and 2.5 minimum alveolar concentration (MAC) decreased LES pressure, and enflurane at 2.0 and 2.5 MAC decreased both LES pressure and BrP in anesthetized children. The intraluminal pressure of the LE and stomach were not altered in either group. CONCLUSION: Sevoflurane and enflurane have an inhibitory effect on LES smooth muscle in anesthetized children. However, since the reduction was relatively low, even at high concentrations, these inhalation anesthetics are unlikely to influence gastroesophageal reflux during anesthesia.
Journal Article↗
Immunohistochemical and functional characterization of pH-dependent intestinal absorption of weak organic acids by the monocarboxylic acid transporter MCT1.
The participation of the monocarboxylic acid transporter MCT1 in the intestinal absorption of weak organic acids has been clarified by functional characterization, by use of stably transfected cells, and by immunohistochemical location of the transporter in intestinal tissues. Immunohistochemical analysis by use of the anti-MCT1 antibody showed that MCT1 is distributed throughout the upper and lower intestines, especially in the basolateral membrane and, to a lesser extent, in the brush-border membrane. When the transporter gene rat MCT1 was transfected into MDA-MB231 cells, transport of benzoic acid, a model weak organic acid that has been generally believed to be transported across the cell membranes by passive diffusion, and lactic acid in rat MCT1-transfected cells was significantly increased compared with transport in cells transfected with the expression vector pRc-CMV alone (mock cells). The observed transport was pH-dependent and activity increased between pH 7.5 and pH 5.5, whereas pH-dependence in mock cells was moderate. Rat MCT1-mediated benzoic acid uptake was saturable, with an apparent Km value of 3.05 mM. In addition, MCT1 increased the efflux of [14C]benzoic acid from the cells. Several weak organic acids were also transported by rat MCT1. These results show that pH-dependent intestinal absorption of weak organic acids, previously explained in terms of passive diffusion according to the pH-partition hypothesis, is at least partially accounted for by MCT1-mediated transport energized at acidic pH by utilization of the proton gradient as a driving force.
Amino Acid Sequence↗
[A clinical study on renal pelvic and ureteral tumor associated with bladder tumor with special reference to risk factors of subsequently recurrent bladder tumor].
PURPOSE: The purpose of this report is to analyze the clinical feature of renal pelvic and/or ureteral tumor (RUT) associated with bladder tumor (BT) with special reference to risk factors of subsequently recurrent BT. METHODS: Of the 49 patients with RUT who underwent surgery and were diagnosed pathologically as transitional cell carcinoma at the Department of Urology, Osaka National Hospital from April 1986 to October 1996, 20 patients (40.8%) had associated BTs. These patients were categorized to the following 4 groups, Group 1: 5 patients with BT preceding RUT, Group 2: 5 patients with concomitant BT, Group 3: 10 patients with subsequent BT following RUT operation and Group 4: 29 patients without any associated BT. The clinical course of these 4 groups were studied and compared with each other retrospectively. RESULTS: In group 1, the first BTs preceded RUTs by 19 to 81 months (mean 54.6 months). And during this relatively long period, the preceding BTs were treated by TUR for each recurrence, 1 to 9 times (mean 5.2 times). Two of 5 were bilateral RUT cases, which were observed only in this group. In group 2, the prognosis were relatively poor (5-year survival rate: 0%), because all RUTs of this group were high stage. And also the concomitant BTs were showing invasive feature during the observation period, despite they were superficial at first. Thus 3 of 5 underwent radical cystectomy. On the other hand, in group 3, the subsequent BTs, which developed at 2 to 26 month (mean 13.4 month) after RUT operation, were all superficial and resectable by TUR. The 5-year disease specific survival rate was 50% in group 1, 0% in group 2, 63.5% in group 3, 64.9% in group 4. Group 2 had the most poor prognosis. However there was no significant difference in prognosis among the 4 groups. Incidence of preoperative urine positive cytology was significantly higher in group 3, than in group 4 (87.5% vs. 44.8%). CONCLUSIONS: These results indicated that the RUTs with associated BTs have distinct clinical features depending on the sequence of association with the BTs. Especially the RUTs with concomitant BTs should be watched carefully as a high risk group with poor prognosis and possible development of invasive BTs. Positive urine cytology prior to RUT operation may reflect biological activity of tumor cell for dissemination in the lower urinary tract and we suggested preoperative urine cytology was possible predictor of subsequently recurrent BTs after RUT operation in this study.
Adult↗
[Intravesical bacillus Calmette-Guerin instillation therapy for carcinoma in situ of the urinary bladder and prediction of effects by urinary cytologic examination].
PURPOSE: We investigated the effects of intravesical BCG instillations for carcinoma in situ (CIS) of the urinary bladder. And we have retrospectively analyzed the prediction of effects by fresh urinary cytologic examinations before instillation. MATERIAL AND METHODS: 33 patients were treated for bladder CIS (1991-1997) with a median follow-up of 30 months (range from 9 to 90 months). The patients (27 males and 6 females) ranged in age from 46 to 91 (average 71 years) and received 6 to 12 weekly BCG Tokyo 172 strain 80 mg instillations. They were divided into 3 groups based on tumor history: primary (9), secondary (15), concurrent (9). The prediction of effects were analyzed by scoring fresh urinary cytologic examinations before instillation. RESULTS: 22 cases (67%) were responded and they have remained free of disease for follow-up period. The statistic evaluation proved to show the significance between the effects of treatment and the sum of scoring (cellular appearance and existence of large nuclear cells). CONCLUSION: We confirmed the effects of this treatment. The prediction of effect of this treatment seemed to be indicative by fresh urinary cytologic examinations before instillation, especially cellular appearance and existence of large nuclear cells.
Adjuvants, Immunologic↗
[Long-term immunological study on postoperative adjuvant treatment with interferon-gamma in patients with renal cell carcinoma who underwent nephrectomy].
Previously, we reported the short-term immunological effects of postoperative adjuvant interferon-gamma (IFN-gamma) administration to renal cell carcinoma patients as determined by three-color flow cytometry. We now report the results of a long-term study on a larger number of subjects. Thirty-three patients with renal cell carcinoma received a prophylactic intramuscular injection of IFN-gamma (300 x 10(4) units per week) after nephrectomy. We evaluated immunological changes by measuring peripheral blood lymphocyte subsets including activated cytotoxic T lymphocytes (ACTL), cytotoxic T lymphocytes (CTL), activated suppressor T lymphocytes (ASTL), helper T lymphocytes (HTL), activated suppressor-inducer T lymphocytes (AITL), and suppressor-inducer T lymphocytes (SITL). We also estimated the natural killer (NK) activity by a cytolytic test. All 33 patients were examined for at least 12 months after the start of IFN-gamma injection, and 18 patients were examined for 30 months including the 6-month period following discontinuation of IFN-gamma injection. We found significant enhancement of the ACTL subset from the second week to the sixth month after the start of IFN-gamma injection. On the other hand, we found a significant decrease in the percentage of the HTL and SITL subsets for a long time after the start of injection. NK activity significantly increased throughout the period of administration, and it continued to increase for six months after discontinuation of IFN-gamma injection.
Adult↗
Gene transfer in vitro and in vivo with Epstein-Barr virus-based episomal vector results in markedly high transient expression in rodent cells.
The EBV-based plasmid vectors, which carry oriP and EBNA1 gene from EBV genome, can be retained in the nucleus and replicate in human cells. Rodent cells are not permissive for the EBV plasmids, in terms of the plasmid replication. However, the EBV vector facilitates not only the long term maintenance of the plasmid but also high level gene expression at a transient phase after transfection. It has not been elucidated if rodent cells show this high level transient expression. We demonstrate that rodent cells transfected with an EBV vector expressed a marker gene more intensively than those with a conventional plasmid vector did. The high marker gene expression was also seen in rat myocardium injected in vivo with the EBV plasmid. The present data indicate that the EBNA1-oriP system functions in "non-permissive" rodent cells at transient phase, and may require different cellular factor(s) for the transient expression and plasmid replication.
Animals↗
Successful transfer of ADA gene in vitro into human peripheral blood CD34+ cells by transfecting EBV-based episomal vectors.
We report a novel non-viral system for transfecting human immature hematopoietic cells in vitro. Epstein-Barr virus (EBV)-based episomal vectors carrying human adenosine deaminase (ADA) gene cDNA were transfected by electroporation into human peripheral blood (PB) CD34+ cells. The transgene-specific mRNA were detected from 37 to 100% of CFU-c (colony forming unit in culture) colonies derived from the transfected cells. A two-fold increase in enzyme activity was also found. These results indicate the successful transfer and expression of genes in human immature hematopoietic cells using the EBV-based episomal vector system.
Adenosine Deaminase↗
Intracellular nucleotide-mediated gating of SUR/Kir6.0 complex potassium channels expressed in a mammalian cell line and its modification by pinacidil.
1. We have examined the properties of intracellular nucleotide-mediated gating of K+ channel constructs composed of the sulphonylurea receptor 2B and the inwardly rectifying K+ channel subunits Kir6.1 and Kir6.2 (SUR2B/Kir6.1 and SUR2B/Kir6.2 complex K+ channels) heterologously expressed in human embryonic kidney (HEK) 293T cells. In the cell-attached form, both types of K+ channel were activated by pinacidil. 2. In inside-out (IO) patches, the SUR2B/Kir6.2 channels opened spontaneously and were inhibited by intracellular ATP (ATPi). Pinacidil attenuated the ATPi-mediated channel inhibition in a concentration-dependent manner. In contrast, the SUR2B/Kir6.1 channels required intracellular nucleoside di- or tri-, but not mono-, phosphates for opening. The potency of adenine, guanine or uracil nucleotides to activate SUR2B/Kir6.1 channels was enhanced by pinacidil. 3. In the presence of pinacidil, adenine and guanine, but not uracil, nucleotides exhibited bell-shaped concentration-dependent activating effects on SUR2B/Kir6.1 channels. This was due to channel inhibition caused by adenine and guanine nucleotides, which was unaffected by pinacidil. 4. From power density spectrum analysis of SUR2B/Kir6.1 currents, channel activation could be described by the product of two gates, a nucleotide-independent fast channel gate and a nucleotide-dependent slow gate, which controlled the number of functional channels. Pinacidil specifically increased the potency of nucleotide action on the slow gate. 5. We conclude that Kir6.0 subunits play a crucial role in the nucleotide-mediated gating of SUR/Kir6.0 complex K+ channels and may determine the molecular mode of pinacidil action.
Adenosine Diphosphate↗
A marked stimulation of Fe2+-initiated lipid peroxidation in phospholipid liposomes by a lipophilic aluminum complex, aluminum acetylacetonate.
In the present study, the efficacy of a lipophilic Al complex, aluminum acetylacetonate, as a stimulator of Fe2+-initiated lipid peroxidation in phospholipid liposomes was examined, and results were compared with those from the liposomes treated with AlCl3. The extent of lipid peroxidation was assessed by the formation of thiobarbituric acid-reactive substances (TBARS). The results indicated that the stimulatory effect of Al complex on Fe2+-initiated lipid peroxidation in phosphatidylcholine liposomes was more effective than that of AlCl3 under the same conditions. The concentration dependence of Al complex on TBARS production showed that the concentration of the complex required to induce half-maximal stimulation of TBARS production was 43 microM. In contrast, the stimulatory effect of AlCl3 was not observed until the AlCl3 concentration is increased above 300 microM. In addition, it was found that there is a linear relationship between the TBARS values and the residual amounts of Fe2+ at an earlier stage (within 2 min after the addition of Fe2+) of the lipid peroxidation in PC liposomes with different concentrations of Al complex, suggesting that Fe2+ oxidation process is closely related to the stimulatory effect of Al complex. The stimulatory effect of Al complex upon the lipid peroxidation completely disappeared by treatment of Al complex-treated liposomes with Triton X-100. The results of fluorescence anisotropy measurements using 12-(9-anthroyloxy)stearic acid-labeled liposomes suggested that treatment of the liposomes with Al complex caused a decrease in their lipid fluidity. Furthermore, it was found that there is a correlation between the extents of the fluorescence anisotropy and the Fe2+ oxidation parameters in the liposomes with different concentrations of Al complex. From these results, it is suggested that the Al effect on Fe2+-initiated lipid peroxidation in the phospholipid liposomes is markedly enhanced by incorporation of Al complex into the liposomal membranes and that an acceleration of Fe2+ oxidation due to a strengthened packing between the acyl chains in the lipid layer may be one possible mechanism for the occurrence of a marked stimulatory effect of Al complex on Fe2+ initiated lipid peroxidation.
Aluminum↗