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For in-beam positron emission tomography (PET), which is an in situ monitoring method for charged particle therapy, we developed the world's first open-type PET system “OpenPET.” Following our previous studies for small prototypes, in this paper, we finally developed a whole-body dual ring OpenPET (WBDROP). This scanner has two separated rings. Each ring has two detector rings of 40 detector blocks, each of which has the capability of 4-layer depth-of-interaction identification. The ring diameter is 66 cm, and the adjustable open gap is fixed as 9 cm. We found that the system sensitivity was 4.4%. The average spatial resolution, which was measured through filtered back projection, was about 3.5 mm. For in-beam PET, imaging performance was confirmed through phantom experiments. Phantom study results with 11C beam and 10C beam irradiations of about 3 Gy showed that the beam stopping position in the target could be measured with a precision of better than 2 mm. The developed WBDROP promises high performance for in-beam PET imaging.

Algae are eaten as healthy foods in Asian countries. We summarize our recent results on the immunoregulatory role of polysaccharide fractions from edible algae on immediate hypersensitivity, delayed-type hypersensitivity, and antitumor immune responses. They are divided into two types. One is to manipulate immune response through toll-like receptor 4 (TLR 4). The other uses different receptors to modify the immune response. Petalonia binghamiae polysaccharide fraction and Spirulina pacifica complex polysaccharide attenuate the delayed-type hypersensitivity and tumor growth by reducing the production of inflammatory cytokine, IL-17 through TLR4. This is suggested from the results that these polysaccharide fractions could suppress the delayed-type hypersensitivity and tumor growth in C3H/HeN but not in tolllike receptor 4 mutant, C3H/HeJ mice. Alginic acid, the polysaccharide from brown algae suppressed tumor growth in both C3H/HeN and C3H/HeJ mice and did not suppress delayed-type hypersensitivity response significantly, when administered intraperitoneally. We propose the potential usefulness of edible algae as the fine tuning reagents of the immune response. We also summarize the recent advancement in the area of regulation of immune responses in relation to these polysaccharides.

It is well known that phenytoin can cause impairment of cellular immunity. The authors investigated the potential role of other anticonvulsant drugs in the development of antitumor immunity in murine malignant glioma models. The survival rate was determined in murine glioma models using syngeneic 203 glioma cells following treatment with four anticonvuisants, which are most commonly administered to glioma patients, i.e., phenytoin, phenobarbital, valproate and zonisamide. In a second set of experiments, we further examined the effect of these drugs on interferon-γ (IFN-γ) secretion by lymphocytes prepared from cervical lymph nodes (CLN) in the same models. The IFN-γ production of CLN lymphocytes as measured by ELISA method was markedly impaired in the early stage of tumor-bearing mice treated with phenytoin or zonisamide, and the median survival time (MST) of controls and of mice treated with either phenytoin or zonisamide was 13, 10 and 11 days, respectively, which was not a statistically significant difference. Phenobarbital and valproate did not affect either IFN-γ production or their survival rate. In addition, immunohistochemistry showed a reduction in tumor-infiltrating lymphocytes containing CD4 and CD8 antigens in the mice treated with phenytoin and zonisamide. Two anticonvulsants, phenytoin and zonisamide, showed a significant inhibitory effect on IFN-γ production by CLN lymphocytes in murine glioma models, although there was no statistically significant difference in MST between controls and the anticonvulsant-treated mice. These drugs might have some detrimental influence on the prognosis of brain tumor patients when combined with the latent immune dysfunction accompanying the tumor-bearing state.

1. Human medulloblastoma (ONS-76), a central nervous system (CNS)-derived undifferentiated cell line, was found to possess glial characteristics as defined by responses in the interferon (IFN) system; ONS-76 cells produced as much IFN-β as human fibroblast and glioma cells by viral infection and poly(I):poly(C) induction. 2. Major histocompatibility complex (MHC) class I antigens were also induced under IFN-β stimulation. ONS-76 cells expressed neurofilament protein, as shown by Northern blot analysis, and morphological differentiation was induced by dibutyryl cyclic AMP (dcAMP). 3. Expression of IFN-β and MHC class I antigens was suppressed in ONS-76 cells during the dcAMP-induced differentiation. 4. These results showed that ONS-76 cells possessed a glial properly in IFN system responses and a neuronal property in cytoskeleton protein, suggesting that the precursors of medulloblastoma may be characterized as bipotent neuronal and glial progenitors in CNS.

We have searched for suitable promoters to regulate the expression of suicide genes for use in gene therapy. We have shown that the 1.3‐kb fragment of the mouse myelin basic protein (MBP) promoter region initiates transcription in mouse glioma cells more efficiently than glial flbrillary acidic protein (GFAP) or myelin proteolipid protein (PLP) promoter. Among three different lengths of the MBP promoter, the shortest (256‐bp) core promoter region initiates transcription as efficiently as 650‐bp or 1.3‐kh MBP promoter lengths in RSV‐M glioma cells. To assess the suitability of the MBP promoter for use in clinical trials of malignant glioma gene therapy, we also had to show that it (the 1.3‐kb length in this case) Is effective in human glioma cells, as well as in murine glioma cells. The activity of the MBP promoter is much higher than that of GFAP or PLP promoter in most human glioma cells, suggesting that the MBP promoter would be best for directing toxic gene expression in gene therapy for patients with malignant glioma. Human glioma cells in which the MBP promoter was strongly active were sensitive to ganciclovir when they were transduced with MBP promoter/herpes simplex virus thymidine kinase gene‐bearing retroviruses. In conclusion, retrovirus‐targeted gene therapy for malignant glioma using this MBP promoter is a promising candidate for clinical trials.

The effects of phenytoin on cellular immunity were examined in murine models. Fresh splenocytes were obtained from mice which had received 1 mg/day of phenytoin i.p. for 28 days. The serum concentration of phenytoin in these animals was 10–10 µg/ml. The proliferative response of splenocytes to mitogens was assessed by 3H-thymidine incorporation. The cytotoxic activities of cells such as natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and lymphokine-activated killer (LAK) cells were estimated by a 4-h 51Cr release assay. The 3H-thymidine incorporation of splenocytes was reduced significantly (P<0.01) in phenytoin-treated mice. The NK and CTL activities of splenocytes from phenytoin-treated mice were significantly suppressed. However, the LAK activity of phenytoin-treated mice was equal to that of control mice.

In order to study the mechanism of the resistance to chemotherapeutic agents, especially ACNU [1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride], two variant cell lines (C6/ACNU and 9L/ACNU) resistant to ACNU were selected in vivo from rat C6 and 9L glioma, respectively. Uptake and efflux of ACNU in these resistant cells were studied with Ethylene[14C]ACNU. The result indicated that the resistance exhibited by both sublines were due to both the reduced uptake of the drug and the increased efflux. The study of the effects of oxidative phosphorylation inhibitor, DNP (2,4-dinitrophenol), on the uptake and retention of ACNU suggested that there is an active outward transport mechanism for ACNU in both glioma sublines and that enhanced activity of this efflux mechanism renders cells highly resistant to the cytotoxic action of ACNU. In an attempt to clarify the more detailed biochemical mechanisms of this active efflux system, we surveyed various membrane-modifying agents which potentiate the sensitivity of these resistant cells to ACNU. Among a number of membrane-modifying agents, reserpine was found to retain ACNU in the resistant cells and to enhance the action of ACNU on these resistant cell lines. It may be concluded that drugs such as reserpine may overcome a mechanism of ACNU resistance.

ABSTRACTSerum-induced inactivation of retroviruses is the most critical limitation for in vivogene transfer therapy. To solve this problem, we searched for reagents that protect retroviruses from inactivation. The effects of the protease inhibitors FOY-007 and FOY-305 and of an inhibitor of the complement pathway FUT-175, all of which have been used clinically, were investigated. All of these agents protected against the inactivation of retroviruses by human serum, with 1 MFUT-175 providing the most effective protection. Thus, the co-administration of FUT-175 with retroviruses may make retrovirus-mediated in vivogene transfer feasible for the treatment of patients. FUT-175 dose-dependently inhibited the classical pathway of complement in a hemolysis protection assay of sensitized sheep erythrocytes with guinea pig serum or by cell-lysis assay of mouse fibroblasts with human serum. However, increasing the FUT-175 concentration by 10-fold (10 M)did not produce further protection against retroviral inactivation in most human sera. There was also no correlation between the serum-induced inactivation of retroviruses and either the amount of anti--galactosyl (anti--Gal) antibody or the complement activity in human serum. These results suggest that retroviruses are not inactivated by utilizing the same pathway leading to cell lysis by the classical complement system.

ABSTRACTThe bystander effect describes the killing of nearby unmodified cells and herpes simplex thymidine kinase (HTK)-transduced cells by ganciclovir (GCV) treatment. This effect is thought to be produced by contact between these cells. In this study, we showed that injected glioma cells migrated rapidly to a place distant from the injection point whereas injected virus-producing fibroblast cells did not migrate in a murine brain model. Moreover, the initially injected glioma cells and glioma cells injected at a later time mix very well, even at a place distant from the injection point. This suggested that glioma cells migrating after injection could be targeted by HTK-modified glioma cells introduced in a second injection and be killed together by GCV treatment. Therefore, we injected HTK-modified glioma cells 3 days after injection of wild glioma cells to investigate whether wild-type glioma cells that migrated to a place distant from the injection point could also be killed by GCV treatment. Tumor growth was suppressed after the GCV treatment. Suppression of tumor growth of wild glioma cells is not solely mediated by the immune response, which may be triggered by the killing of HTK-modified glioma cells with GCV, because inoculation of HTK-modified glioma to the contralateral side followed by GCV treatment did not cure the initial wild glioma. Moreover, the migration of the second inoculum of glioma cells is necessary for effective killing, because early administration of GCV resulted in insufficient killing.

We investigated 5-fluoro-2′-deoxyuridine (FdUrd) as a potential agent for intrathecal treatment of malignant brain tumors with meningeal dissemination. We examined the neurotoxicity of FdUrd in vitro using primary cultures of neurons from C57BL/6 mice (ED14). Tumoricidal activity was also studied in four glioma cell lines and one medulloblastoma cell line. In addition, thymidine phosphorylase (TPase) and thymidine kinase (TK), which are key enzymes for FdUrd metabolism, were measured in the cerebrospinal fluid (CSF) of 36 patients with brain tumors. The antitumor activity of FdUrd for murine glioma cells was approximately 20- to 200-fold higher than that of 5-fluorouracil (5-FU). Against human cell lines, it was 3- to 500-fold higher than that of 5-FU. The neurotoxic effect of FdUrd on cultured neurons was far less than that of 5-FU or 5-fluorouridine (FUrd). Cerebrospinal fluid contained no detectable thymidine phosphorylase in most patients with brain tumors. Several studies have indicated that FdUrd is rapidly converted to 5-FU in the presence of thymidine phosphorylase, so that a high dose of FdUrd must be administered to obtain good efficacy. However, a high dose FdUrd frequently cause severe toxicity. In contrast, the data obtained here suggest that no enzymatic conversion of FdUrd to 5-FU should occur in the CSF. In addition, FdUrd has an excellent antitumor activity and minimal neurotoxicity. We therefore conclude that intrathecal FdUrd is a potential therapy for CSF dissemination of malignant brain tumors.

The prevalence of artificially administered nutrition and hydration (AANH) in different age groups among patients with advanced cancer remains unknown. The present study investigated the current utilization of AANH according to age groups in palliative care units.

We demonstrate a new method of imaging crystal lattice strain with spatial resolution on the nanometer scale. The method, Fourier transform mapping (FTM), is based on the mapping of spot positions in local Fourier-transformed images from a transmission electron microscope image. Lattice deformation caused by island formation due to strained epitaxial growth is imaged and the relief of the strain in the lateral direction, as well as that in the normal direction, is clearly visible in the lattice-deformation image.

We present a new technique for imaging p-njunctions in semiconductors based on Auger electron spectroscopy. By measuring fine Auger electron spectra of the semiconductor material (e.g., silicon LVV Auger), the position of the Fermi level in the band gap of the semiconductor is estimated from the energy shift of the Auger spectra. The position of the Fermi level depends on the type of dopant. By applying a correlation calculation to measure the Auger energy shift, which is smaller than the band gap of the semiconductor, we can successfully image two-dimensional p-njunctions in LSI devices.