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Electroporation facilitates transfer of chemicals or plasmid DNA from extracellular milieu into cells by increasing the permeability of the cell membrane. Delivery of electric pulses to established tumors thereby can improve the susceptibility of tumors to an anti-cancer agent administered. We examined whether electroporation-mediated transfer of cytokine genes into solid tumors could produce anti-tumor effects in the tumor-bearing mice. Plasmid DNA containing cytokine genes were injected into human esophageal T.Tn tumors developed in nude mice and electric pulses were then delivered. Administration of murine GM-CSF or human IL-2 gene followed by electroporation significantly suppressed the subsequent growth of T.Tn tumors and prolonged the survival of the inoculated mice. In contrast, electroporation-mediated introduction of a control gene, human GM-CSF gene, whose products do not bind to murine GM-CSF receptors, did not achieve any anti-tumor effects. In vivo transfection of cytokine genes with electroporation could be a possible therapeutic strategy for established solid tumors.

The present study was addressed on the effect of 3,3',4,4',5-pentachlorobiphenyl (PenCB) to the expression of glucose regulated protein (GRP) 78 and GRP94 in liver endoplasmic reticulum of rat by treatment with the schedule after acute or subacute exposure. In the acute exposure, male Wistar rats received PenCB in corn oil at once a dose of 25 mg/kg i.p., then at 5 days after treatment the microsomes were prepared. Free- and pair-fed control groups were given the vehicle. The microsomal proteins were separated on SDS-PAGE, transferred to membrane and blotted using anti-sera to the GRPs. The reduction of GRP78 and GRP94 was associated significantly with the acute exposure. In subacute exposure, the rats received PenCB in corn oil at once a dose of 0.1 or 1.0 mg/kg i.p. At 4 weeks after treatment, liver microsomes were obtained. The expression level of GRP78 and GRP94 are also decreased at 1.0 mg PenCB/kg treatment as similar as the acute exposure. But the reduction was not notable at 0.1 mg PenCB/kg treatment. GRP78 and GRP94 are a member of GRPs and the expression is regulated by glucose in cells as stress proteins. GRP78 and GRP94 have also the function for chaperone protein. Chaperone proteins have important physiological functions against synthesized and/or denatured proteins, which include assembling, folding of proteins. Our results suggested that a part of the toxicity of PenCB is associated to significant decrease of the chaperone proteins in the endoplasmic reticulum.

We report here that a highly toxic coplanar polychlorinated biphenyl (PCB), 3,3',4,4',5-pentachlorobiphenyl (PenCB) induces molecular chaperones, HSP70 and HSP90 in liver cytosol of rats. Male Wistar rats received PenCB in corn oil once at a dose of 25 mg/kg i.p. Pair-fed control groups were treated with the vehicle and given the amount of chow matched with that taken by the PenCB-treated animals, and free-fed controls were given the vehicle. The liver cytosolic HSP70 level in rats treated with PenCB was 5-fold higher than those in free-fed controls, though that for pair-fed controls was approximately 2-fold higher than that in free-fed controls. The liver cytosolic HSP90 alpha and HSP90 beta levels were also higher in PenCB-treated rats than in both control groups, but the induction extent was lesser than that for HSP70. Inductive effect on the chaperones was examined with a single different dose of PenCB 0, 0.5, 1.0, 5.0, 10 and 25 mg/kg. Marked induction of the HSP70 level was observed with a minimum dose of PenCB 0.5 mg/kg. The HSP90 alpha level was induced with PenCB-dose dependent manner although the HSP90 beta induction was greatest with a dose of PenCB 5.0 mg/kg. HSP70 and HSP90 are essential for cells under normal conditions and act as molecular chaperones. HSP90 is well known to modulate the function of sex steroid hormone or aromatic hydrocarbon receptors while HSP70 is required for receptor-HSP90 heterocomplex assembly. The role of molecular chaperones may be involved in the endocrine disrupting properties of coplanar PCB and dioxins.

We have examined the antitumor effect of the retrovirally expressed interleukin-2 (IL-2) gene in murine colon carcinoma cells (Colon 26) on a disseminated model within the peritoneal cavity. Intraperitoneal injection of IL-2-producing Colon 26, but not wild-type cells, into syngeneic mice did not cause animal death and conferred T-cell-dependent, tumor-specific protective immunity on the inoculated mice. Direct administration of the retrovirus harboring IL-2 gene into the peritoneal cavity of the mice that had intraperitoneal tumor of wild-type cells significantly prolonged the survival of the mice compared with that of the mice that received control retrovirus. In the surviving mice treated with the retrovirus we also observed the induction of protective immunity with tumor specificity. Integrated retrovirus DNA was not detected in any organs examined. Thus, in vivo IL-2 gene transfer by retrovirus enabled tumor-bearing mice to generate antitumor immunity without unnecessary retroviral insertion into host genome.

The inoculation of cytokine-producing tumor cells into syngeneic animals can produce antitumor effects. We previously reported that injection of a mixed population of murine colon carcinoma (Colon 26) cells transduced with interleukin-4 (IL-4) gene with granulocyte macrophage-colony stimulating factor (GM-CSF) gene developed subcutaneous tumors in syngeneic mice, but these tumors thereafter regressed spontaneously (Oncology 1997;54:69-73). In this study, we found the generation of tumor-specific acquired immunity in the mice which had rejected the tumors of a mixed population of IL-4 and GM-CSF producer cells, and that the immunity was mediated by mature T cells. Moreover, local secretion of both IL-4 and GM-CSF in the vicinity of tumors was found to be crucial for this rejection. These data suggest that enhanced host defense brought about by the combined expression of IL-4 and GM-CSF genes in tumor cells is a potential therapeutic strategy.

We examined the antitumor effect of low and high interleukin-2 (IL-2) producers of murine colon carcinoma cells (Colon 26) which were generated by transduction with IL-2 gene in an experimental lung metastasis model using syngeneic mice. Intravenous injection of the low IL-2 producer cells formed multiple lung metastatic foci and the survival of the mice was not different from that of the mice injected with wild-type cells. However, the mice administrated with the high producer cells survived significantly longer. Subcutaneous inoculation of the low producers, although it caused the development of local tumors at the inoculation sites in some of the mice tested, inhibited lung metastasis of wild-type cells subsequently inoculated and prolonged the survival of the mice rechallenged with Meth A cells, syngeneic fibrosarcoma cells. In contrast, inoculation of the high producers did not cause the development of subcutaneous tumors and inhibited the experimental metastasis of parental but not Meth A cells inoculated thereafter. Thus, the amount of secreted IL-2 from tumor cells differentially influences antitumor effects by inducing tumor specific and nonspecific immunity.

We have examined the antitumor effect of murine neuroblastoma cells (C1300) engineered to produce cytokines. Retrovirally transduced cells with human interleukin-2 (IL-2) or murine GM-CSF gene, but not murine IL-4 gene, abolished their tumorigenicity in syngeneic mice, although their in vitro growth rate and expression of class I antigens of the major histocompatibility complex were unchanged. Inoculation of wild-type cells into the mice, which had rejected IL-2 or GM-CSF producers, did not develop tumors, indicating that protective immunity was induced. In an experimental hematogenous metastasis model, we found that the numbers of metastatic foci in the liver caused by intravenous administration of IL-2 or GM-CSF producers were significantly reduced compared with those by the injection of wild-type or vector virus-transduced cells. No significant differences in their adhesiveness to extracellular matrices and ability to differentiate were observed among parent and transduced cells. Thus, these results indicate that IL-2 or GM-CSF secretion, in the vicinity of neuroblastoma cells, produced antitumor effect and reduced metastatic ability.

We have examined antitumor effect of human pancreatic carcinoma cells (AsPC-1) retrovirally transduced with interleukin-12 (IL-12), IL-15 or IL-18 gene in nude mice. The tumor growth of IL-12-expressing AsPC-1 cells was significantly retarded and that of IL-15-expressing cells was also impeded compared with that of wild-type cells, although their in vitro cell growth remained unchanged. However, the expression of IL-18 in AsPC-1 cells did not generate any antitumor effect since the tumor growth of the transduced cells was the same as that of wild-type cells. Thus, the differential actions of these cytokines on non-T cells can generate a variety of antitumor effect in nude mice, although their actions on T cells lineage favor the stimulation of Th1-type helper T cells.

We examined the correlation between the amount of exogenous protein secreted and that of its viral transcript from PA317 packaging cells transduced with a cytokine gene. Amphotropic packaging cells for the retrovirus expressing murine interleukin-15 (IL-15) gene were cloned, and we examined 22 clones for both the amount of IL-15 secreted with enzyme-linked immunosorbent assay and the IL-15 transcript released as infectious virions by slot blot-hybridization analysis. The study revealed that there was no statistical correlation between them, and suggests that the integrated transcript is used discordantly. Thus, the examination of secreted protein from packaging cells cannot be substituted for laborious assays to determine the amount of virion transcripts when isolating high titer packaging cells.

We have reported that a 54 kDa protein in rat liver cytosol is highly inducible by treatment with 3,3',4,4',5-pentachlorobiphenyl (PenCB) or 3-methylcholanthrene (MC) using SDS-polyacrylamide gel electrophoresis. Internal amino acid sequences of this protein in the rat liver were highly homologous to those of selenium binding protein (SeBP) or acetaminophen binding protein (APBP) in mouse liver cytosol. In this paper, the purification and characterization of this protein were demonstrated. MC was given at a dose of 20 mg/kg for 3 consecutive days. The liver cytosolic 54 kDa protein was purified twice from the MC-treated male Wistar rats by Rotofore Cell procedure to apparent single on SDS-polyacrylamide gel electrophoresis, and the rabbit antiserum against this protein was obtained. Male Wistar rats were given PenCB in corn oil at a single dose of 25 mg/kg i.p. The liver cytosol was prepared on the 5th day after the treatment and subjected to immunoblot analysis. The 54 kDa protein was markedly induced in the liver cytosol of PenCB-treated rats. Immunoblot analysis after two-dimensional gel electrophoresis suggested that there could be isoforms of 54 kDa protein. The induction of the 54 kDa protein with PenCB was assumed to be mediated through Ah-receptor. The physiological role of the 54 kDa protein was discussed together with SeBP and APBP, the role of which has not yet been elucidated.

Two human Burkitt's lymphoma cell lines (HBL-4 and HBL-5) were established individually from two patients with small noncleaved cell lymphoma (Burkitt's type). The HBL-4 cell line is Epstein-Barr virus (EBV)-negative, and the HBL-5 cell line is EBV-positive. Cytogenetically, both cell lines had the same chromosomal translocation, t(8;14)(q24;q32) as those observed in the primary malignant cells from individual patients. Morphologic, immunophenotypic, cytogenetic, and molecular studies confirmed that both cell lines were derived from the primary lymphoma cells in vivo. HBL-4 cells lacked CD23(H107), CD11a(LFA-1), and latent membrane protein (LMP) but expressed CD54(ICAM-1) at low levels, whereas HBL-5 cells showed the high level of expression of CD54 and faint expression of LMP but lacked CD11a. In addition, the EBV-positive lymphoblastoid cell line (LCL) expressed CD11a, CD23, CD54, and LMP at high levels. Therefore, an HBL-5 phenotype with expression of CD54 and LMP tends toward an LCL phenotype, and the augmentation of CD54 on the HBL-5 cells in comparison with primary lymphoma cells is likely to be upregulated by LMP, probably resulting from the EBV infection. There was little difference in the BrdUrd uptake in vivo and in vitro, doubling time, tumorigenicity, and dynamics of tumor growth in athymic nude mice between both cell lines. These findings indicate that the potentiality of cell growth and tumorigenicity of these two cell lines are unlikely to be related with EBV.

A new recording system for fluorescein video-angiography using a highly sensitive color charge coupled device (CCD) camera was developed. An image processing unit installed in a personal computer made it possible to process video signals from the camera and/or a video tape recorder. Functions of the unit are on-line and/or off-line operations on image data such as smoothing, digital subtraction, differentiation etc. Application of the unit improved the quality of images observed on a monitor screen. This system enabled observation of the ocular fundus with illumination of low intensity, which reduced physical and psychological suffering of patients. In addition continuous and whole sequences of fundus fluorographs were recorded, without using strong stroboscopic lights. Extraction of a clear image from ambiguous records at any moment was possible through off-line processing on the stored data. Furthermore quantification of the blood flow in any blood vessels was possible by subtracting two successive images. With this system, quantitative expression of a slight difference in the color of the fundus could be made through calculation of the ratios among R-, G-, and B-components of signals at any place. Finally, comparative study of images of the fundus with natural color could be made with those of fluorography. These features may provide further information on the physiology and pathology of the fundus.

To decrease both the intensity of the exciting light and the amount of sodium fluorescein, we attached a compact image intensifier incorporating a microchannel plate with a fundus photoscope and tried to record video-fluorescein angiography with low steady light. We preliminarily examined the relationships of exciting and emitted light intensity with various concentrations of sodium fluorescein. The strongest fluorescence was obtained with a concentration of 0.01 mg/ml solution of sodium fluorescein. When the compact image intensifier was attached to the equipment, the exciting light intensity could be decreased to 1/300 with an exciting filter of lambda max of 486 nm. When sodium fluorescein of 0.01 mg/ml solution was injected directly into the heart of Rana catesbeiana, complete video-fluorescein angiography with sufficient brightness and resolution was possible. With the same machine, we recorded video-fluorescein angiography in a normal human subject with injection of 5 ml of 10% sodium fluorescein. The ordinary observing light of the machine was strong enough for video-recording. This exciting light intensity caused no trouble and elicited no complaint from the examinee. These results showed that the use of a compact image intensifier incorporated microchannel plate with a fundus photoscope made it possible to record video-fluorescein angiography and to decrease exciting light intensity and the amount of fluorescein dye. This means that light damage to photoreceptors by exciting light and problems induced by allergic reaction with fluorescein dye might be decreased. These aspects might be very important to obtain information about both retinal circulation and disruption of the blood-retinal barrier and also to perform safe fluorescein angiography examination.

Single units were recorded through glass microelectrodes placed on the optic disk or on the retina of the opened eye of the frog (Rana catesbiana). Units were classified as A-, B-, and C-fibers according to conduction velocities. By the method of collision between naturally elicited and electrically elicited impulses, many of the B-fibers and some A- and C-fibers, which showed unusual behavior to photic stimulation, were found to be efferent fibers. Retinal effects of the efferent nerves were studied by repetitive stimulation and cooling of the optic nerve. The effects were found to be both inhibitory and excitatory.

H3-thymidine labeled lymphocytes from thymus and lymph nodes of donor rats were washed and injected in to the intestine of recipient rats on the 11th and 19th day of gestation; subsequent labeling of maternal and embryonal cells was studied autoradiographically 24 hours after injection. In 12-day embryos, numerous stem cells or hemocytoblasts were labeled frequently intensely. In 20-day embryos, stem cells or hemocytoblasts scattered throughout the liver were often labeled. In other fetal tissues at this stage, cells in thymus, spleen, mesenteric lymph node and intestine were labeled but scarcely and weakly. In mothers, labeling in lymphoid tissues was scarce but definite, in thymus, mesenteric lymph node and spleen. These results suggest that nuclear materials from lymphocytes emigrated into the intestinal canal of the mother could be reutilized by maternal and embryonal cells.