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A case of psammoma body rich gliofibroma is reported. Computed tomography (CT) showed a high-density mass without contrast enhancement in the right cerebellar hemisphere. Magnetic resonance images (MRI) demonstrated a mass with a mixture of high- and iso-intensity regions without meningeal attachment on both T1- and T2-weighted images. Pathological examinations revealed a biphasic pattern consisting of tumor cells in sparce cellularity and dense fibrous connective tissue. Numerous psammoma bodies of uniform size were found in the stroma. The tumor cells expressed glial fibrillary acidic protein and S-100, but not p53, vimentin and EMA. This is the first report describing a case of psammomatous gliofibroma.

Testican family proteins are putative extracellular heparan/chondroitin sulfate proteoglycans of unknown function. We identified recently N-Tes, which is a product of testican 3 splicing variant gene, as an inhibitor of membrane-type matrix metalloproteinases (MT-MMPs). The inhibitory function is common among testican family members except for testican 2, which was shown to uniquely abolish inhibition of MT1-MMP- or MT3-MMP-mediated pro-MMP-2 activation by other testican family members. Testican 2 inactivates N-Tes by binding to the COOH-terminal extracellular calcium-binding domain of N-Tes through its NH(2)-terminal unique domain as demonstrated by coimmunoprecipitation analysis, and, thus, testican 2 was unable to inactivate a N-Tes deletion mutant lacking the extracellular calcium-binding domain (N-Tes-Delta 122). Migration of U251 cells on collagen, which was dependent on MT1-MMP activity under serum-free condition, was inhibited by N-Tes or N-Tes-Delta 122 deposited on collagen. Testican 2 was not incorporated into collagen by itself, and was deposited only in the presence of N-Tes, suggesting that testican 2 binds to N-Tes deposited on collagen. Binding of testican 2 to N-Tes deposited on collagen allowed migration of cells expressing MT1-MMP. Unlike wild-type N-Tes, N-Tes-Delta 122 did not bind to testican 2, and, thus, expression of testican 2 did not recover cell migration blocked by N-Tes-Delta 122. In situ hybridization showed that neurons are a major source of all of the testican family members in the normal brain. The quantitative reverse transcription-PCR analysis demonstrated that all of the testican family members are expressed prominently in normal brain, and their expression levels decrease as tumor grade increases. The expression level of testican 2 was the highest among testican family members regardless of histological grade of astrocytic tumors. These results suggest that abundant distribution of testican 2 may contribute to glioma invasion by inactivating other testican family members including N-Tes, which all inhibit MT-MMPs. We propose that N-Tes-Delta 122, which is resistant to testican 2, may have therapeutic potential as a barrier against glioma invasion.

The matrix metalloproteinase (MMP) family plays an important role in the degradation of extracellular matrix (ECM) in various physiological and pathological conditions. Accumulated evidence has suggested that MMPs contribute to cancer cell invasion of the surrounding normal tissues and metastasis through the cell-surface ECM degradation. Strong correlations have been reported between elevated MMP levels and tumor cell invasiveness in human gliomas. Among them, attention has been focused on gelatinases (MMP-2 and MMP-9) and membrane type MMPs (MT-MMPs). We discuss here the biological significance of these MMPs in the glioblastoma invasion processes. A better understanding of cell-ECM interactions will help in developing therapeutic strategies to decrease the invasion of gliomas.

Ets transcription factors are associated with tumor malignancy. We reported previously that the stable transfection of the dominant-negative form of Ets-1 (Ets-DN) in the glioma cell line U251 induced down-regulation of urokinase-type plasminogen activator mRNA expression and invasiveness (M. Nakada et al., J. Neuropathol. Exp. Neurol., 58: 329-334, 1999). Here we analyzed effects of Ets-DN expression on cell adhesion, migration, and phosphorylation of focal adhesion kinase. U251 cells expressing Ets-DN (U251-DN) showed reduced cell adhesion, spreading, and extension of actin stress fibers on dishes coated with fibronectin but not on dishes coated with collagen. Migration of U251-DN cells was found to be significantly inhibited compared with that of parental cells when examined by wound-induced migration assay on fibronectin-coated dishes. Phosphorylation levels of focal adhesion kinase in U251-DN cells were also attenuated on dishes coated with fibronectin. Reduced expression level of integrin alpha5 subunit in U251-DN cells was demonstrated by semiquantitative reverse transcription-PCR analysis. Semiquantitative reverse transcription-PCR of surgical samples of brain tumors revealed that the expression level of Ets-1 mRNA correlated with that of integrin alpha5 mRNA in glioma. The experimental metastatic ability of U251-DN cells examined in chick embryo was considerably lower than that of parental cells. These results suggest that Ets-1 contributes to glioma malignancy by up- regulating expression of the integrin alpha5 subunit, which composes integrin alpha5beta1 and mediates intracellular signaling and the subsequent acceleration of the invasive process, including cell adhesion and migration.

Drug resistance is a major clinical problem in the chemotherapy of human gliomas. The multidrug resistance-associated protein (MRP), a membrane transporter related to non-P-glycoprotein multidrug resistance, is overexpressed in some drug-selected cancer cell lines. To investigate whether MRP is involved in the intrinsic drug resistance of human gliomas, surgical specimens of 20 gliomas (11 glioblastomas, 6 anaplastic astrocytomas, and 3 astrocytomas), 3 normal brain specimens, and 4 glioma cell lines (U87MG, U251MG, U373MG, and T98G) were analyzed. The expression of MRP was studied by RT-PCR and immunohistochemistry in the surgical specimens. The MRP expression levels in the cell lines were assessed by the quantitative RT-PCR and Western blot analyses. Sensitivity to adriamycin (ADM), etoposide (VP-16), cisplatin (CDDP), and 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), were determined by MTT assay, and antisense treatment was evaluated in the cell lines. The expression of MRP was detected in 9 of 11 glioblastomas and 3 of 6 anaplastic astrocytomas. The quantitative analyses of the cell lines revealed that the MRP mRNA and protein levels were increased 4.5-fold in the T98G cells as compared to U87MG. T98G cells showed the highest resistance to all drugs. Western blot analysis revealed that treatment with the antisense oligonucleotide reduced the level of MRP expression to 25% of the sense oligonucleotide treatment in T98G cells. The sensitivity to ADM, VP-16 and CDDP was significantly increased in the antisense-treated cells as compared with the sense-treated cells. These results suggest that the MRP expression may be related to the intrinsic multidrug resistance in human gliomas.

Recent physiological and anatomical studies have demonstrated that a major fraction of brain interstitial and cerebrospinal fluid drains into cervical lymph nodes (CLN) in a number of experimental animals. To investigate the role of CLN in brain tumour immunity, temporal profiles of MHC class II molecule expression and T lymphocyte subsets in brain tumours, CLN and other lymphoid tissues were analysed by immunocytochemistry. A total of 64 Wistar rats weighing 250 g were used. Two weeks after the transplantation of C6 glioma cells (10(6) cells/1 microl) into a rat brain, expression of MHC class II molecules was induced in the brain and all systemic lymphoid tissues examined. However, the subsequent appearance of CD4 or CD8 positive cells was strictly confined to CLN, and coincided with the infiltration of such cells into the brain tumour 2 weeks after transplantation. In the group of animals in which cervical lymphadenectomy was followed by intracerebral transplantation of C6 glioma cells, infiltration of CD4 or CD8 positive cells into the brain tumour was delayed until 3 weeks after the transplantation, and the production of such cells was by the spleen. These results suggest that CLN act as regional lymph nodes in brain tumour immunity.

We performed intraoperative radiotherapy (IORT) in 19 patients with various brain tumors. IORT was given for primary tumors in 2 patients with malignant glioma, but was used for treating recurrent tumors in the other 17 patients. The former 2 patients respectively received 33 Gy by IORT alone and 30 Gy by IORT in combination with 50 Gy of external beam radiotherapy (EBRT), and survived for 12 and 9 months. The latter 17 patients had received EBRT at 4 to 112 months before IORT. In this group, single doses of 23-40 Gy were delivered by IORT after removing as much tumor as possible. The median survival time after IORT was 12 months for 9 patients with glioblastoma or anaplastic astrocytoma, while it was 51 months for 8 patients with less infiltrative tumors (ependymoma, anaplastic ependymoma, and anaplastic oligodendroglioma). One patient with ependymoma and another with anaplastic ependymoma are currently alive with no evidence of disease at 7 and 11 years after IORT, respectively. Symptomatic brain necrosis occurred in 3 patients following IORT, but the symptoms were relieved in 2 of them by the removal of necrotic brain tissue. It is concluded that IORT combined with extensive tumor removal has an acceptable toxicity in previously irradiated patients and can be effective for selected recurrent malignant brain tumors.

Mixed purine-pyrimidine oligodeoxynucleotides were designed to form collinear DNA triplexes with pyrimidine-rich elements in the EGFR gene promoter. Their effects as mediators of human epidermal growth factor receptor (EGFR) gene transcription and subsequent gene expression were evaluated using human squamous cell carcinoma (A431) and human glioma cell line (U251MG and U87MG). Gel shift analysis indicated that the oligonucleotide forms a collinear triplex within the duplex Sp-1 binding site. An in vitro assay system revealed a correlation between triplex formation and the repression of EGFR transcription. We postulate that guanine residues are not always optimum in apposition to G-C pairs to form triple helices in the target. Site-specific oligodeoxynucleotides binding to a DNA duplex may serve as the basis for an alternative program of gene control in vitro.

We report the MR findings of a biopsy-proven gliosarcoma of the posterior cranial fossa. Multiple homogeneously enhancing lesions had shaggy margins and broad-based dural attachments, which may reflect the gliomatous and sarcomatous element of this tumour.

Two weeks after the inoculation of 1.5 x 10(5) 9L glioma cells into the rat brain, the uptake of radiolabelled drugs into the brain and the experimental 9L glioma during the first cerebral circulation was measured with a liquid scintillation counter and analyzed by the method of Oldendorf (1970). The expression of P-glycoprotein, which is known to be associated with the efflux of drugs, was also studied, using anti-P-glycoprotein monoclonal antibody, C-219. Furthermore, the ultrastructure of brain capillaries, tumor vessels, and glioma cells was studied by conventional and immunoelectron microscopy. Sucrose (control), the transport of which through the blood-brain barrier is known to be negligible, accumulated to fivefold higher levels in the tumor than in normal brain. Ranimustine (MCNU), 5-fluorouracil (5-FU), and doxorubicin showed little accumulation in the normal brain, whereas nimustine (ACNU) showed an increased accumulation. MCNU and doxorubicin showed negligible accumulation in the glioma cells despite diffusion into the tumor interstitial space. In contrast, ACNU and 5-FU showed an increased accumulation in tumor cells. The accumulation of 5-FU in the cultured 9L glioma cells was decreased by ATP inhibitors or by low temperature. Although both brain capillary endothelial cells and glioma cell membrane were immunohistochemically positive for P-glycoprotein, the tumor vasculature showed low expression of P-glycoprotein. The endothelial cells of tumor vessels ultrastructurally showed increased fenestrations, swelling, and disrupted junctions. Accordingly, it is suggested that hydrophobic drugs such as doxorubicin, being pumped out by P-glycoprotein, do not accumulate in 9L glioma cells as do other lipophilic drugs such as ACNU, or drugs such as 5-FU, which accumulate by a carrier-mediated mechanism.

Recently a 17p deletion and p53 gene mutations were reported in human gliomas, but the relationship of the timing of p53 gene mutation and oncogenesis of glioma is still obscure. We examined eight pairs of primary and recurrent gliomas. Four of eight had a histological malignant transformation. In the group with malignant transformation, three out of four pairs had a mutation in the p53 gene only in recurrence. None of the mutations in either primary or recurrent glioma was detected in the group with no histological change. All point mutations occurred within the evolutionarily conserved regions. This suggests that the p53 mutations occurred during the progression and were important in the malignant transformation in the some kinds of gliomas.

To predict the efficacy of anticancer drugs such as ACNU [1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl-3-nitrosoure a hydrochloride] and MCNU [1-(2-chloroethyl)-3-(methyl-alpha-D-glucopyranos-6-yl)-1-nitrosou rea] in the treatment of malignant gliomas, the usefulness of the chick embryo assay as a chemosensitivity test was studied. Fifty-seven surgical specimens including benign tumors were examined by this method. All tumor specimens tested could be grafted on the chorioallantoic membrane of chick embryo; the evaluable ratio was 100%. Twenty-one patients with previously untreated malignant glioma could be evaluated to test the predictability of the clinical effects, judged by computed tomography. There were 7 (78%) instances in which the assay response corresponded to a clinical partial response (true-positive). There were 2 (22%) false-positives for the assay, 0 (0%) false-negative and 12 (100%) true-negatives. The over-all predictive accuracy was 90% (19/21). Thus, a high-degree of positive association exists between the chick embryo assay and the clinical outcome. This in vivo assay system for malignant glioma is advantageous for chemosensitivity tests because of its convenience, rapidity, and inexpensiveness.

The neurotoxicity of local administration of nitrosoureas in malignant gliomas was investigated clinicopathologically. Twenty patients were entered into this study: 13 were treated with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) and 7 with methyl 6-[3-(2-chloroethyl-3-nitrosoureido]-6-deoxy-alpha-D-glucopyranoside (MCNU). On the average, a single dose of 20 mg of ACNU was administered 15 times, for a total dose of 295 mg in each case, while a single dose of 11 mg of MCNU was given 2 times, for a total dose of 24 mg. These nitrosoureas provoked greater toxicity when the administration dose was larger or the indwelling multiperforated Silastic basket was in direct continuity with the ventricle or the basal cistern. Usually ACNU was well tolerated, whereas MCNU induced marked brain edema. Side effects consisted of headache, nuchal stiffness, vomiting, motor weakness, and cranial nerve palsy for ACNU, and headache, vomiting, abnormal respiration, and arrhythmia for MCNU. Pathological changes were represented by capsule formation, spongy degeneration and reactive gliosis of adjacent white matter, occlusion of neighboring arteries, and demyelination of cranial nerves in the patients treated with ACNU, while they were represented by focal brain necrosis in two patients treated with MCNU. The differences in neurotoxity of ACNU and MCNU conceivably derive from the different blood-brain delivery of these drugs.

The clonal analysis in the ultrastructure of tumor-lymphocyte interaction was carried out in order to investigate the precise mechanism responsible for CTL-mediated cytolysis of tumor cells. A glioma-derived cell line (GI-1) and autologous tumor-specific cytotoxic T lymphocyte (CTL) clones were established. The CTL lines were composed of the morphologically homogeneous lymphocytes with intracytoplasmic electron-dense secretory granules. After the stimulation by GI-1, the size of the CTLs increased, and the intracytoplasmic organellas were developed. It was noted that the intracytoplasmic secretory granules markedly increased in number and size, and many of them exhibited an "immature" appearance. On the other hand, the tumor cells underwent a progressive degeneration. In contrast, the stimulation by other antigens caused only small morphological changes in the CTLs. It is suggested, therefore, that the secretory function of tumor-specific CTLs is activated by the stimulation of the specific antigen, and that soluble factors in the secretory granules in the CTLs may be closely associated with the mechanism of target cell lysis.

We have recently shown that exogenous expression of the mouse interferon-gamma (IFN-gamma) gene augmented the cell-killing potential of a line of cytotoxic T lymphocytes (CTLs) specific against a murine glioma line (203-glioma). In the present work, we further investigated the in vivo antitumor effects of the E gamma-6 and E gamma-9 sublines of this CTL line transfected with the IFN-gamma gene. Using the Winn assay to test the neutralization of subcutaneous gliomas, we determined that these CTL sublines were more effective than the E-4 parent CTL line and that suppression of the tumor growth was dependent on the number of effector cells (CTLs). Moreover, intravenous injection of E gamma-9 cells was more effective in suppressing the tumor growth than intravenous injection of E-4 cells. These results suggest that transfection of antitumor effector cells with the IFN-gamma gene could improve the efficacy of adoptive immunotherapy against cancer.

The anti-tumour effect of mouse interferon (IFN) on an intracranially transplanted 203-glioma in C 57BL mice and the natural killer (NK) activity of spleen cells were studied. As a clinical trial, five patients with glioblastomas were treated with human fibroblast IFN and the anti-tumour effect of IFN and the NK activity of peripheral blood lymphocytes were also studied. The NK activity increased after the beginning of IFN therapy but there was no remarkable anti-tumour effect of IFN in both mouse and human studies. There was no marked correlation between the increased NK activity and the anti-tumour effect of IFN in this study.

The effects of prostaglandin D2 (PGD2) on the growth of mouse malignant glioma cells were studied in vitro and in vivo. The in vitro studies consisted of various concentrations of prostaglandins (PG's) being added to cultures of mouse glioma cells. At concentrations above 2.5 micrograms/ml, PGD2 strongly inhibited the proliferation of glioma cells, whereas PGE2 had no effect at the same value. Exposure to 5.0 micrograms/ml PGD2 for more than 2 hours resulted in inhibition of glioma cell proliferation. This growth-inhibitory effect of PGD2 was related to the inhibition of DNA synthesis of the cells. The in vivo studies were performed with a subcutaneously transplanted mouse glioma model. Injection of 0.5 mg/kg PGD2 into the tumor was more effective than the same concentration given by intraperitoneal injection. In mice with intracranially transplanted glioma, daily intraperitoneal injection of 0.5 mg/kg PGD2 had no significant effect on survival time.

Changes of natural killer activity (NK activity) by local 60Co irradiation in intracranial tumor-bearing mice were studied by the method of 51Cr release assay. Local irradiation was administered 10 days after intracranial transplantation of 203-Glioma which had been originally induced by 20-methylcholanthrene in C 57 BL mice. Irradiation suppressed the growth of tumor and prolonged the mean survival time. The 50% survival time of untreated mice was about 2.5 weeks but that of mice treated by a single dose of 1000 rad and 1500 rad of irradiation was about 4.5 weeks and 6.5 weeks respectively. NK activity of spleen cells in these mice was serially examined. NK activity was gradually increased in mice treated by local irradiation, while it was gradually decreased in mice without treatment. On the other hand, NK activity remained unchanged in non-tumor-bearing control mice. Mice treated with 1000 rad and 1500 rad of irradiation showed 44.0% and 47.6% of % specific 51Cr release respectively 11 days after irradiation while normal mice showed 18.0%. The increased NK activity after local irradiation suggested that local irradiation might have enhanced the immunological defence mechanism against the tumor in the tumor-bearing hosts. Some characteristics of effector cells in this assay system were examined. The cytotoxicity of spleen cells was removed by the treatment of anti-BAT serum and complement but was not removed by the treatment of anti-Thy-1. 2 serum and complement. Since NK activity reflects the immunological resistance to tumors to some extent, it is felt important to clarify the significance of changes of NK activity in patients with brain tumors in relation to various treatments including surgery, radiotherapy, chemotherapy and immunotherapy in the next step.

During a 6.5-year period from March 1978, 136 patients with brain tumors, which were diagnosed as gliomas or metastatic brain tumors during surgical operation, were treated by the application of sustained release anticancer pellets in seven institutes. Histological diagnosis disclosed that the majority of the tumors were malignant gliomas (61 cases), metastatic brain tumors (35 cases), and benign astrocytoma (17 cases). The anticancer agents (250 mg of 5FU, 1.5 mg of MMC, 250 mg of BUdR and 6,000 iu of urokinase) were sealed in a Silastic silicone tube of 6 mm in diameter, 0.25 mm in wall thickness, and 12 mm in length. The daily delivery of the drugs was (2-3)/10,000 of the original volume. This treatment was combined with surgical and irradiation therapy with or without general chemotherapy. The median survival time of patients with malignant gliomas was 545 days (18 months). The 1-year survival rate was 65.63%, and the 3-year survival rate 16.2%. On the other hand, in patients with benign astrocytomas, the median survival time was 1,500 days (4.1 years), 1-year survival rate 83.75%, and 3-year survival rate 42.84%. In those with metastatic brain tumors, the median survival time was 280 days, 1-year survival rate 43.87%, and 3-year survival rate 24.84%. The present findings revealed that the tube-type anticancer pellets cannot inhibit the growth of the brain tumors located 5 cm from the pellets.

Suppressor T cells in syngeneic tumor-bearing mice that inhibited in vitro generation of tumor antigen-specific cytotoxic T lymphocytes were characterized with respect to the kinetics, the nature and the target specificity, using murine malignant glioma (a methylcholanthrene-induced malignant ependymoblastoma, 203-glioma). Suppressor cell activity was assessed by the inhibition of tumor cell killing activity of cytotoxic T lymphocytes, which were prepared from splenic T enriched lymphocytes of mice immunized with 1 X 10(6) mitomycin C (50 micrograms/ml, 45 minutes)-treated 203-glioma cells twice at an interval of 7 days. It was confirmed that suppressor T cells were generated in 203-glioma-bearing mice, and they were tumor antigen-specific as evidenced by the fact that sensitized splenic T lymphocytes from mice bearing other syngeneic EL4 thymoma or allogeneic P 815 mastocytoma or YAC-1 T cell lymphoma did not exhibit the inhibition of the cytotoxic T lymphocyte activity against 203-glioma cells. Significant suppressor cell activity was detected in spleen cells 1 to 5 days after the subcutaneous inoculation of 203-glioma cells with the peak activity on day 3 and it disappeared as early as on day 7, suggesting strongly that the turn-over of suppressor T cells is very quick. Surface markers of suppressor T cells in 203-glioma-bearing mice were checked on day 3 with the results that the suppressor cell activity was eliminated by the treatment with anti-Lyt-2 monoclonal antibody and complement, indicating that the phenotype of suppressor T cells is Lyt-1-.2.3+.(ABSTRACT TRUNCATED AT 250 WORDS)

Four patients with glioblastoma and one patient with astrocytoma (grade III) were treated with recombinant IFN ( rIFN -alpha A, Ro 22-8181) and the effect of IFN on clinical symptoms, CT findings and side effects of IFN were studied. Neurological symptoms were improved in one patient, stable in one patient and worsened in three patients. In all cases, there was no remarkable change of CT findings but in one case a slight decrease in tumor size was recognized. With regards to IFN side effects general malaise, anorexia, fever, nausea and vomiting were observed clinically, decrease of leukocytes, platelets, erythrocytes, hematocrit, hemoglobin and increase of GOT, GPT, LDH, AL-P were noted in the laboratory findings. These symptoms and change in laboratory findings were not serious, and they recovered spontaneously during or after IFN therapy. In one patient, an increase in IFN neutralizing antibody titer was detected. Since the biological activity of IFN may be diminished and anti-tumor effect cannot be expected in such a patient, the appearance of IFN neutralizing antibody may indicate an important problem in IFN therapy.

The effects of IFN (interferon) on the activation and differentiation of syngeneic murine malignant glioma-specific killer T cell were investigated in C57BL/6 adult mice in order to clarify the potential usefulness for anti-tumor local immunotherapy. It was confirmed that the specific killer T cell against 20-methylcholanthrene-induced 203-glioma was generated in mice after intracranial and subcutaneous inoculation of the tumor cells. The cytotoxic activities of splenic cells obtained from intracranial and subcutaneous tumor-bearing mice were assessed on MLTC (mixed lymphocyte-tumor cell culture) for 18 hours by microcytotoxicity assay modified Takasugi and Klein's method. The addition of IFN to MLTC resulted in a similar 1.5- to 2.0-fold increase of generated cytotoxic activities. Prior treatment of sensitized splenic cells with IFN resulted in an enhancement of the specific cytotoxic activity for the target tumor cells. IFN enhanced cytotoxic activities in MLTC only in the first 3 hours. These cytotoxic activities were eliminated by the treatment of sensitized lymphocytes with anti-Thy-1 antibody and complement before adding IFN. Therefore, it was found that IFN was able to enhance killer T cell activity of sensitized lymphocytes. Normal lymphocytes did not exhibit any cytotoxic activity even after the treatment with IFN. On the other hand, IFN had a cytostatic effect on the growth of 203-glioma cells. This effect of IFN on 203-glioma cells was not observed when IFN was removed from the suspension (by washing 203-glioma cells).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of adult thymectomy in C 57 BL/6 mice on in vivo and in vitro responses to syngeneic methylcholanthrene-induced glioma (203-glioma) were investigated in order to analyse the role of T cell subpopulation in relation to the antitumor immunity. The tumor growth in adult mice thymectomized 3 weeks before subcutaneous inoculation of tumor cells was significantly suppressed. On the other hand, in mice thymectomized 7 or 10 weeks before tumor cell inoculation, the tumor growth was enhanced resulting in shorter mean survival time. The cytotoxic activity of the regional lymph node T cells in the former mice was increased from the beginning after tumor cell inoculation with peak observed on day 14 and maintained for about 4 weeks, while it was extremely decreased in the latter mice. The marked enhancement of cytotoxic activity in the former mice is probably due to a reduced proportion of short-lived T lymphocyte population after adult thymectomy. In contrast, the low level of cytotoxic activity in the latter mice may be due to a gradual reduction of long-lived T lymphocyte population in addition to short-lived T lymphocyte population after adult thymectomy. The cytotoxic activity was specific for 203-glioma cells and almost completely eliminated with anti-Thy-1 monoclonal antibody and complement. The surface markers of these killer T cells were checked with the results that in normal mice Lyt-1-.2.3+ and Lyt-1+.2.3+ cells participate in cytotoxic reaction. In mice thymectomized 3-10 weeks before tumor cell inoculation, however, Lyt-1+.2.3+ killer T cells were not detected suggesting strongly that the progenitors of Lyt-1+.2.3+ killer T cells are short-lived cells in contrast to those of Lyt-1-.2.3+ killer T cells which survive more than 10 weeks after adult thymectomy. The tumor growth was also significantly suppressed by the intravenous adoptive transfer of sensitized lymphocytes obtained from mice thymectomized 3 weeks before tumor cell inoculation. This effect of tumor suppression was disappeared by the pretreatment of infused lymphocytes with anti-Thy-1 monoclonal antibody and complement. These evidences may suggest that in tumor bearing mice short-lived suppressor T cells or their precursors exist and regulate the growth and differentiation of killer T cells and that adult thymectomy affects immunoregulation, possibly by altering the generation of suppressor T cells.

Eleven lymphocyte clones were established from the peripheral blood lymphocytes of a patient with gliosarcoma by means of autologous tumor stimulation and the limiting-dilution technique with recombinant interleukin-2. Ten of the 11 clones were cytotoxic against the autologous tumor cell line GI-1. Seven of the 10 clones were also cytotoxic against allogeneic brain-tumor lines and HeLa cells, one clone was cytotoxic against several target cells, and two clones were specifically cytotoxic against GI-1 and allogeneic brain-tumor cells. One of the 11 clones was not cytotoxic against any target cells tested. Lymphokine-activated killer cells induced by recombinant interleukin-2 alone exhibited cytotoxic activity against all target tumor cells tested. Surface phenotypic analysis revealed that all lymphocyte clones expressed CD3 antigen, some expressed CD4 antigen, and others expressed CD8 antigen. These clones seemed to be antigen-specific cytotoxic T lymphocyte clones. Analysis with these antigen-specific cytotoxic T lymphocyte clones may be useful in the elucidation of tumor-specific or tumor-associated antigens on autologous tumor cells.

The effects of interleukin 2 (IL2) and interferon (IFN) on the generation and lytic activation of syngeneic murine malignant glioma (a methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin, 203-glioma)-specific cytotoxic T-lymphocyte (G-CTL) were investigated. The surface marker analysis showed that G-CTLs from both intracranial and s.c. tumor-bearing mice were composed of thymectomy-resistant (mature) Lyt-1-.2.3+ and thymectomy-sensitive (immature) Lyt-1+.2.3+ CTLs, which markedly decreased concurrently with increased intracranial pressure. G-CTLs were confirmed to be activated with target specificity by both factors in a different way. The CTL activation by IL2 (20 units/ml) remained for a longer time, although a lag time of 5 days after initial culture was required. IL2 influenced Lyt-1+.2.3+ CTLs to proliferate and develop the lytic potential. In contrast, even a 3-h incubation with IFN (1000 units/ml) could enhance the cytotoxicity, but the augmenting effects were observed no longer than 5 days later. IFN activated Lyt-1-.2.3+ CTLs and increased their proportion of the total cell population with a simultaneous decrease of Lyt-1+.2.3+ CTLs. Therefore, it was suggested that IL2 may provide a growth of CTL populations and that IFN can accelerate recruitment of new effectors, causing activation of the lytic process.

The concentration of serum immunoglobulins (IgG, IgM, IgA) in 63 patients of primary brain tumors were determined by the method of single radial immunodiffusion. 1) The mean values and standard deviations of three major serum immunoglobulins in 18 cases of meningioma were as follows; IgG, 1297.0 +/- 387.9 mg/dl, IgM, 165.1 +/- 153.4 mg/dl, IgA, 219.5 +/- 76.7 mg/dl. These values were not significantly different with those in normals. 2) 45 cases of gliomas showed; IgG, 1190.5 +/- 378.3 mg/dl, IgM, 160.0 +/- 75.9 mg/dl, IgA, 189.8 +/- 91.5 mg/dl. These values were in normal levels as compared with normal serum immunoglobulins. 3) Values of serum immunoglobulins in 19 cases of glioblastoma multiforme were compared with those in 18 cases of astrocytoma to know the relationship between histological malignancy and levels of serum immunoglubulins. But no significant difference was observed between these values. The levels of three major serum immunoglobulins either in meningiomas or in gliomas were not significantly different with those in normals. Therefore, the levels of serum immunoglobulins could not be used as a diagnotic procedure in cases of primary brain tumors.

The purposes of the current study were: (1) to investigate the immunoregulatory effects of T-cell growth factor (TCGF) on the activation and differentiation of syngeneic cytotoxic T lymphocyte (CTL) populations generated against a 20-methylcholanthrene-induced ependymoblastoma, 203-glioma, in C57BL/6 mice; and (2) to determine whether the glioma-specific CTL clone (G-CTLL) could be established by TCGF, and whether the in vivo efficacy of the cloned cells could be rendered more effective in adoptive therapy. It was found that TCGF largely allows the CTL populations to proliferate and thus can activate the depressed cytotoxic activity in tumour-bearing mice. Two lines of G-CTLL were successfully obtained by the limiting dilution technique. The G-CTLL retained a TCGF-dependent proliferative growth and a marked cytotoxic activity with target specificity for over 18 months, characterized by a surface phenotype of Lyt-1-.2.3+, Lyt-2 antibody blocking of cytotoxicity and the production of immune interferon in response to mitogen and tumour antigen. In the Winn assay and the adoptive transfer assay, the therapeutic effects were detected in intracranially inoculated tumours in mice. The in vivo efficacy was dependent on the dose of G-CTLL and on the time of the intravenous administration, although the transfer was inversely ineffective in conditions of increased intracranial pressure. The mechanism responsible for the in vivo effect was probably due to the adoptive immunity and/or the tumour-specific interferon production of G-CTLL.

The efficacy of glioma-specific cytotoxic T-lymphocyte for a syngeneic murine malignant glioma (a 20-methylcholanthrene-induced ependymoblastoma, 203-glioma) was investigated. The cytotoxic clone (G-CTLL 1), established and expanded exponentially by T-cell growth factor, has retained target specificity for more than 6 months. In adoptive therapy and Winn assay, the in vivo antitumor activity of G-CTLL 1 was demonstrated against mice inoculated intracranially with 203-glioma cells. The therapeutic effects in adoptive immunotherapy were largely dependent on dose and time of i.v. administration, although the therapy was rather ineffective in condition of increased intracranial pressure due to the tumor growth. The mechanisms responsible for the in vivo protection were probably related to the killing activity of G-CTLL 1 or the tumor-specific production of immune interferon by G-CTLL 1.

The study analyses 85 cases of brainstem glioma in the past 35 years, 69 of which include patients under 16 years of age. The incidence of brainstem glioma was 2.4% of all intracranial tumours, and 9.4% of intracranial tumours in children. There were two peaks in age distribution, in the first and in the fourth decades. In children, the tumours were located mainly in the pons, so VIth and VIIth cranial nerve palsies, and pyramidal and cerebellar signs were frequently seen. In adult cases, the tumours ranged in location from the midbrain to the medulla, so neurological symptoms caused by lesions of the whole brainstem axis were seen. The left side was dominant in both age groups. The choice of treatment was steroid administration and radiation. Chemotherapy was not effective. Even after these treatments, the median survival period from onset was no longer than 10.5 months. We conclude that the treatment of brainstem gliomas in children should be distinguished from adult cases, which in the latter may be considered to be merely one of the gliomas which may occur at any other sites. Since brainstem gliomas in children may be congenital, we must redirect our treatment of these lesions to treatment of congenital tumours.

Two human glioma-specific cytotoxic T-lymphocyte (G-S-CTL) lines were established by autologous tumor stimulation (ATS) with the aid of lectin free interleukin 2 (IL 2). Coculture of patient's peripheral blood lymphocytes and autologous irradiated glioma cells and subsequent addition of partially purified IL 2 enhanced the tumoricidal activity of the lymphocytes. These CTL lines possessed cross-cytotoxic activity against autologous allogeneic glioma cells and exhibited low cytotoxic activity against non-glial tumor cells. They did not lyse autologous lymphoblasts. This phenomenon suggested the existence of a common glioma-specific antigen recognized by the CTL lines. T-cell subset depletion test revealed that the major surface phenotype of G-S-CTL line, responsible for cytotoxic activity was OKT 3 positive, OKT 4 negative and OKT 8 positive. G-S-CTL lines were composed of a low proportion of OKT 8 positive subpopulation after primary ATS and successive propagation with IL 2. The proportion of OKT 8 positive subpopulation was increased by secondary ATS, which enhanced the cytotoxic activity to glioma cells more effectively.

The immunoregulatory effects of TCGF (T-cell growth factor) on the generation and growth of syngeneic murine malignant glioma (20-methylcholanthrene-induced 203-glioma)-specific killer T-cell were investigated in C57BL/6 adult mice in order to clarify the immunopotential usefulness for anti-tumor local adoptive immunotherapy against malignant brain tumor. TCGF was prepared and assayed. Briefly, 5 x 10(6) ml mouse spleen cells were cultured with 2 microgram/ml concanavalin A in RPMI-1640 medium supplemented with 2% fetal calf serum for 24 hours. Culture supernatants were concentrated by ammonium sulphate precipitation (55 to 80% saturation) and purified by gel filtration (Sephadex G-100, a molecular weight from 30 to 36,000 daltons) and ion exchange chromatography (DEAE-cellulose, elution with 0.15 M in NaCl at ph 7.4). The purified TCGF had no IFN activity. Assays for TCGF was performed for quantitative analysis using 203-glioma-specific killer T cell clone (G-CTLL), which was obtained by limiting dilution method (0.3 cells/well in 96 well microtiter plate) and maintained for over 6 months in the presence of TCGF. Titer (U/ml) of TCGF was defined as the quantity of TCGF required to obtain one-half of the maximal stimulation of G-CTLL proliferation assay. It was confirmed that the specific killer T-cell against 203-glioma was generated in mice after intracranial as well as subcutaneous inoculation of the tumor cells. The killer T-cell activity of spleen cells, however, began to be severely impaired 2 weeks after intracranial inoculation concurrently with the increased intracranial pressure due to developing the tumor growth. Sensitized lymphocytes obtained from intracranial and subcutaneous tumor-bearing mice were assessed for CTL (cytotoxic T-lymphocyte) activity in MLTC (mixed lymphocyte-tumor cell culture) for 18 hours by microcytotoxicity assay. The specific cytotoxicity against 203-glioma cells was enhanced when sensitized lymphocytes from intracranial and subcutaneous tumor-bearing mice were pre-cultured with optimal TCGF (20 U/ml) for over 5 days. After the treatment of sensitized lymphocytes with anti-Thy-1 monoclonal antibody and complement, however, the specific cytotoxicity of sensitized lymphocytes was eliminated almost completely. Therefore, it was thought that TCGF possesses immunoregulatory effects of enhancement of killer T-cell activity. On the contrary, TCGF had no influence on normal T lymphocytes and the growth of 203-glioma cells in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Thirteen patients with malignant brain tumours were treated by continuous intra-carotid infusion of ACNU in combination with systemic administration of phenobarbital. This aimed to maintain an ACNU concentration around the tumour cells higher for a relatively longer period and to make an ACNU concentration in the systemic circulation lower. Improvements on clinical states and CT findings were observed in 10 patients. The protective effect of phenobarbital against bone marrow suppression by ACNU was also suggested in this study. No complications were experienced in this treatment. This results strongly suggested that this technique is useful to treat patients with malignant brain tumours.

The authors examined the growth rate of mouse 203 glioma cells in vitro and found it to be markedly inhibited after exposure to ACNU for 5 minutes at a drug concentration of 100 micrograms/ml. Rats that had undergone intracranial implantation of T1 neurogenic tumor were treated by 5 mg/kg of ACNU administered either intravenously or intra-arterially. The median survival times for the control animals and the animals undergoing intravenous or intracarotid administration of ACNU were 23, 29, and 46 days, respectively. The difference in survival time between the intravenous and intracarotid administration groups was statistically significant (p less than 0.01) when examined by the Cox-Mantel test. In a clinical trial, 17 patients with glioblastoma were treated by ACNU, eight intravenously and nine by the intra-arterial route. The drug was given in doses of 2 to 3 mg/kg at least twice before and twice after a course of postoperative radiotherapy. Intra-arterial administration was performed over a period of 5 minutes under local anesthesia. The median postoperative survival time for the patients in the intra-arterial group was 12.5 months, compared with 9.0 months for those in the intravenous group. The survival rate for the intra-arterial group was slightly higher, although statistically not significant, probably because the number of cases was small. The degree of thrombocytopenia due to ACNU tended to be less marked in the intra-arterially treated patients. The theoretical advantages of the intra-arterial administration of ACNU are discussed.

Results of radiotherapy for 164 supratentorial malignant gliomas were analyzed based on recent histological subclassification into glioblastoma multiforme (GBM) and anaplastic astrocytoma (AA). Patients with AA had a better prognosis than those with GBM. The 5-year survival rate was 1% for GBM and 16% for AA. The size of radiation field did not influence survival significantly, and whole brain radiation appeared of no benefit. Survival time prolonged with an increase of radiation dose between 45 Gy and 72 Gy. The optimal radiotherapy for the disease is discussed.

In an attempt to facilitate the long-term proliferative growth and subsequent cloning of cytotoxic T lymphocytes (CTL's) against syngeneic murine 203-glioma (20-methylcholanthrene-induced ependymoblastoma of C57BL/6 mouse origin), sensitized T lymphocytes from tumor-bearing mice were cultured in the presence of T cell growth factor (TCGF). Of five clones established by a limiting dilution technique, two clones (G-CTLL 1 and 2) exhibited tumor-specific cytotoxicity. G-CTLL 1 cells, which possessed much higher cytotoxic activity than G-CTLL 2 cells, were further analyzed. G-CTLL 1 cells were maintained in a TCGF-dependent exponential proliferative culture for over 18 months and continued to mediate an extremely high cytotoxic activity with the target specificity (50- to 100-fold increases over the peak cytotoxic activity of sensitized T lymphocytes in tumor-bearing mice). Their phenotypes of surface antigens were Thy-1+ (weak positive), Lyt-1.-2.+3+, and asialo-GM1-, and their cytotoxicity was blocked by adding only anti-Lyt-2 monoclonal antibodies. These results indicated that the cloned cells originated from CTL's. The cloned cells were characterized by the production of immune interferon with the glioma antigen-stimulation, suggesting that the immune interferon could enhance the cytotoxic activity of the CTL clone at the site of a clone-target cell recognition event.