Masanobu Yamada

An adoptive immunotherapy of patients with medulloblastoma by lymphokine-activated killer cells (LAK)

SummaryAn adoptive immunotherapy of 6 patients with medulloblastoma by lymphokine-activated killer (LAK) cells is described. They were from 2 to 9 years in age and had cerebrospinal fluid (CSF) dissemination of the tumours. All patients underwent the whole-neuraxis irradiation and chemotherapy. After the usual treatments, they were submitted to an adoptive transfer of one-haplotype identical LAK cells. The LAK cells were induced from peripheral blood lymphocytes (PBL) of their relatives with human recombinant interleukin-2 (rIL-2). 3–15×109 LAK cells were transferred intrathecally in 2–3 months. In 3 of 6 patients, neurological signs were improved and malignant cells had never been detected on CSF cytology after the adoptive immunotherapy. One among these 3 patients showed complete response in 20 months. Thus, this is an attractive approach for the treatment of medulloblastoma with CSF dissemination of the tumour which current therapeutic intervention can not cure.

Selective intra-arterial chemotherapy with a combination of etoposide and cisplatin for malignant gliomas: Preliminary report

We administered selective intra-arterial chemotherapy consisting of a combination of etoposide and cisplatin to 20 patients with malignant glioma (seven with recurrent and six with enlarged tumors after initial treatment, and seven newly diagnosed patients). Evaluation of efficacy was based on computed tomographic and magnetic resonance imaging findings. In the process of establishing a safe technique for superselective intra-arterial chemotherapy, we encountered cerebrovascular accidents in two patients (after etoposide in one and after etoposide plus cisplatin in the other). In these two cases, 100 mg/m2 of etoposide and 100 mg/m2 of cisplatin were delivered via the horizontal segment of the middle cerebral artery (M1) or the tip of the basilar artery, with the infusion time reduced to 20 minutes. Thereafter, the etoposide was diluted, and the doses of both drugs were reduced to 80 or 50 mg/m2, and finally to 60 mg/m2, and both were infused over 60 minutes. In addition, for prevention of local spasm, papaverine hydrochloride and nicardipine were given via the same catheter at 5-minute intervals during administration of etoposide and cisplatin. No complications developed in the later cases. Thereafter, selective intra-arterial infusion of etoposide and cisplatin into the anterior cerebral artery, middle cerebral artery, posterior cerebral artery, or the basilar artery for malignant gliomas in the basal ganglia, internal capsule, and brainstem--a procedure generally considered risky in terms of potential complications--was performed safely, with tolerable side effects. Computed tomography and magnetic resonance imaging indicated improvement in 13 patients, including four whose tumors completely disappeared. This method of intra-arterial chemotherapy may be useful as an adjuvant treatment for malignant glioma.

In vitro study on intrathecal use of 5-fluoro-2'-deoxyuridine (FdUrd) for meningeal dissemination of malignant brain tumors

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.

Myelin Basic Protein in the Cerebrospinal Fluid of Patients with Brain Tumors

We measured the level of myelin basic protein (MBP) in the cerebrospinal fluid (CSF) of patients with various kinds of tumors, including malignant tumors, using radioimmunoassay. The CSF had been obtained by lumbar puncture through an Ommaya reservoir or a shunt device placed in the lateral ventricle. The level of MBP was high (> 4 ng/ml) in the patients with meningeal dissemination of malignant tumors, but in those who showed a good response to chemotherapy and/or radiation, it decreased or returned to the normal level, with improvement on the computed tomography and magnetic resonance imaging, cytological, general CSF, and neurological findings. Of seven malignant gliomas without CSF dissemination, six showed an elevated level of MBP before selective intra-arterial chemotherapy with a combination of etoposide and cisplatin administered via a microcatheter placed at A1, M1, P1-P2, and the basilar top. All CSF specimens obtained during the period of the intra-arterial chemotherapy showed an abnormally high (> 4 ng/ml) level of MBP that exceeded the prechemotherapy level. The MBP level decreased or returned to normal in the patients with a good response to chemotherapy after intra-arterial chemotherapy. In some patients with multiple metastatic brain tumors, the MBP level was elevated before treatment and returned to normal after treatment (surgical removal, chemotherapy, and/or irradiation) in all except one. Thus, there was a clear correlation between the timing of treatment and changes in imaging studies and MBP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Retrovirus-mediated gene transfer targeted to malignant glioma cells in murine brain

A murine model for meningeal metastasis of malignant glioma was developed to study selective gene transfer into tumor cells and to establish a reliable means of determining the rate of tumor cell infection. A murine ecotropic retroviral vector was created in which the Escherichia coliβ‐galactosidase gene served as a marker for gene expression from the integrated retrovirus. This retrovirus exhibited a high rate of infectivity in RSV‐M mouse glioma cells in vitro. The recombinant retrovirus was injected directly into the cisterna magna of the mice. Staining of β‐galactosidase showed that the rate of gene integration was high in the disseminated glioma cells. These results suggest the possibility of retrovirus‐mediated gene therapy for meningeal dissemination of malignant glioma.

Clinical trial of intrathecal administration of 5-fluoro-2'-deoxyuridine for treatment of meningeal dissemination of malignant tumors.

Intrathecal administration of 5-fluoro-2′-deoxyuridine (FdUrd) was performed in patients with meningeal dissemination of malignant tumors during the period from January 1996 to September 1998, and they were followed up until February 1999. The study population consisted of 23 patients: 12 with lung cancer, 4 with breast cancer, 2 with colon cancer, 1 with malignant lymphoma, 2 with glioblastoma and 2 with metastatic brain tumors of unknown origin. FdUrd was administered intrathecally through an Ommaya reservoir placed in the lateral ventricle initially at a dose of 1u2009µg twice per week, and the dose was increased to 10u2009µg and administration schedule was also increased every day. Headache and nuchal pain were relieved in all patients regardless of responsiveness to intrathecal FdUrd therapy as determined from the findings in the cerebrospinal fluid (CSF). Patients showed no side effects during the course of intrathecal chemotherapy except for slight nausea in two patients and dull headache in one. Sixteen of the 23 patients showed decreased cell number in the cerebrospinal fluid (CSF). Positive cytological findings in CSF became negative in 6 of the 23 patients, and the levels of CSF tumor markers were decreased in 14. Responsiveness to intrathecal administration of FdUrd was defined as ‘response’ when both the cell number and tumor markers were decreased in both ventricular and spinal CSF or when the cell number was decreased in cases in which the tumor markers were not detected. Overall, 16 of the 23 patients (70%) showed complete or partial responses to intrathecal FdUrd therapy as determined from CSF findings.These results demonstrated the efficacy of intrathecal FdUrd chemotherapy without apparent neurotoxicity for treatment of meningeal dissemination of malignant tumors.

Fate of Jimpy‐Type Oligodendrocytes in Jimpy Heterozygote

Abstract: In the jimpy mutant mouse, as well as in many other animals with mutations in the myelin proteolipid protein (PLP) gene, Oligodendrocytes degenerate before their maturation. To analyze whether this degeneration is caused by the loss of function of PLP gene products related to oligodendrocyte maturation/survival acting extrinsically, expression of the PLP gene was investigated in the jimpy heterozygote, in which one‐half of the cells are jimpy type and the other half are wild type due to random Xchromosome inactivation. We first showed that jimpy PLP gene expression is normally regulated at the early stages of development in brains of jimpy hemizygotes and heterozygotes, at least to day 2 after birth. However, the great increase in the level of PLP gene transcripts observed in wild‐type mouse brain is suppressed in jimpy mouse brain. This increase was also suppressed in the jimpy heterozygote, and by 2 months after birth, very few jimpy‐type PLP gene transcripts were detected in heterozygotes. These results indicate that jimpy‐type Oligodendrocytes cannot survive or are still in the immature stage in the brain of jimpy heterozygotes. Thus, degeneration of jimpy Oligodendrocytes is not caused merely by the lack of trophic factors.

[Effects of phenytoin on cell-mediated immunity].

SummaryThe 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.

Anticonvulsant-induced Suppression of IFN-γ Production by Lymphocytes Obtained from Cervical Lymph Nodes in Glioma-bearing Mice

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.

Neural transplantation in mouse Parkinson's disease.

A complete recovery from the methamphetamine-induced rotational response was shown in C57BL/6 (H-2b) mice which had had unilateral 6-OHDA lesions in the nigrostriatal pathway about 60 days after transplantation of approximately 1 x 10(6) dopamine-rich cells from syngeneic or allogeneic (C3H/HeN, H-2k) mouse embryos (ED 15), without immunosuppressive agents. Morphological examination showed tyrosine-hydroxylase-immunoreactive cell clusters around the needle tract in the mice which were transplanted not only with syngeneic cells but also with allogeneic cells. This might indicate that so-called immunosuppressive agents are not necessary for grafted embryonic cells to survive in an allogeneic mouse brain.