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Darell D. Bigner

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Stephen C. Saris, Sandra H. Bigner and Darell D. Bigner

✓ A model was developed for in vivo study of the human glioma-derived D-54 MG cell line in the brains of immunosuppressed Fischer 344 rats. The rats were injected with horse anti-rat thymocyte serum before and after intracerebral inoculation with 5 or 10 µl of a D-54 MG tumor cell suspension. Reproducible mortality distributions were obtained, with deaths occurring 18 to 34 days after intracerebral inoculation. Tumors grew as well circumscribed intracerebral masses with sheets of anaplastic cells, areas of necrosis bordered by concentrated nuclei, and minimal lymphocytic infiltration. Cytogenetic analysis revealed the same general chromosome distribution and markers in the heterotransplanted glioma cells as in the cultured line. Blood-brain barrier disruption was demonstrated by intracerebral tumor staining after intravenous injection of Evans blue dye. The in vivo growth of D-54 MG in immunosuppressed rats provides a reliable experimental model for the study of chemotherapy, immunodiagnosis, and immunotherapy of a human glioma-derived tumor in an animal sufficiently large to evaluate intracarotid or intratumoral injection of therapeutic agents.

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Dennis E. Bullard, Mario Bourdon and Darell D. Bigner

✓ Different methods were evaluated for delivering iodine-125 monoclonal antibodies (Mab's) to the central nervous system in 40- to 99-gm Fischer rats. By evaluating interhemispheric, interregional, and brain:blood ratios of Mab's, the efficacy of intracarotid (IC) or intravenous (IV) administration of Mab's with and without prior IC perfusion with 0.9% NaCl (normal saline, NS), 1.4 M mannitol, or 1.6 M arabinose, or of femoral artery perfusion with 1.4 M mannitol was evaluated. No difference was seen between IC and IV administration of Mab's with or without prior perfusion. Intracarotid perfusion with hyperosmolar agents was required to disrupt the blood-brain barrier (BBB) and to significantly elevate brain levels of Mab's. The brain and blood levels of Mab's were elevated in all regions of the brain following hyperosmolar BBB disruption. However, the levels were significantly higher in the ipsilateral hemisphere, with cross-over occurring primarily in the vascular distribution of the contralateral anterior cerebral artery. Intracarotid hyperosmolar perfusion produced 450% to 500% increases in ipsilateral and 240% to 280% increases in contralateral hemispheric brain:blood Mab ratio levels compared to those achieved with NS perfusion. For IC perfusion of mannitol or arabinose, flow rates ranging from 0.017 to 0.052 ml/sec were equally effective in disrupting the BBB. Insignificant morbidity and mortality rates were noted up to 2 weeks following BBB disruption. Additional ligation of major extracranial branches of the external and internal carotid arteries prior to IC perfusion did not result in a selective increase in hemispheric Mab levels. Temporally, following hyperosmolar BBB disruption, brain:blood Mab ratios remained elevated bilaterally at 7 days after Mab delivery, with the ipsilateral hemispheric levels remaining significantly elevated compared with the contralateral hemispheric levels until Day 5, when the ratio returned to the nonperfused range. Catheterization was required in the small animals and was performed under magnification in 10 to 20 minutes, with less than an 8% overall morbidity and mortality. The methodology developed should prove helpful in delivery of Mab's or other agents in rat tumor models and experimental models for other disease entities.

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Dennis E. Bullard and Darell D. Bigner

✓ The development of monoclonal antibodies has resulted in marked expansion in understanding the central nervous system (CNS). This has been especially true in the study of human neuroectodermal tumors where monoclonal antibodies have been used as physiological probes to define and characterize human neuroectodermal tumor-associated antigens. Utilizing monoclonal antibodies, neuroectodermal tumor-associated antigens have been described in four broad categories; biochemically defined markers, shared nervous systemlymphoid cell markers, shared neuroectodermal-oncofetal markers, and putative restricted tumor markers. Preliminary data have demonstrated the ability to localize animal and human tumors in vitro, ex vivo, and in vivo. Early application of monoclonal antibody technology to neuroimmunology and neuro-oncology has resulted in a new awareness of the complex relationships that exist within the CNS. Their specificity and reproducibility may provide the means to qualitatively and quantitatively define the phenotypic heterogeneity of human neuroectodermal tumors. Potentially, monoclonal antibodies, alone or as carriers of radionuclides, drugs, or toxins, may allow successful diagnosis and treatment of human neuroectodermal tumors.

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Dennis E. Bullard and Darell D. Bigner

✓ Methods for transiently disrupting the blood-brain barrier (BBB) which are consistent with survival are described for immature Fischer 344 rats weighing 40 to 99 gm. A catheter was retrogradely inserted into the external carotid artery to the level of the bifurcation. Perfusion of 1.4 M mannitol or 1.6 M arabinose, at a rate of 0.01 to 0.1 ml/sec for 30 seconds, resulted in transient BBB disruption as measured by Evans blue dye (EBD) staining. Higher flow rates or perfusion with 10% to 30% dimethyl sulfoxide were associated with a mortality rate ranging from 0% to 44%. Perfusion with 0.9% sodium chloride or intrafemoral artery perfusion with 1.4 M mannitol did not disrupt the BBB. Optimum BBB disruption as measured by EBD staining was achieved with 1.6 M arabinose at 0.026 ml/sec for 30 seconds, at which time all of the 42 experimental animals had BBB disruption; all of the animals so treated survived 2 weeks following perfusion. This technique will allow the efficacy of delivering chemotherapeutic agents following BBB disruption to be tested in several of the more commonly used small-animal models for brain-tumor research.

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Darell D. Bigner and Charles B. Wilson

✓ The authors provide a summary of a workshop on “Cancer of the Brain.” This conference reviewed the current knowledge about the etiology and pathogenesis of human brain tumors and the experimental induction of comparable animal brain tumors, and considered new lines of research.

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W. Jerry Oakes, Henry S. Friedman, Sandra H. Bigner, Nancy H. Bullock and Darell D. Bigner

✓ The development of the Cavitron ultrasonic surgical aspirator (CUSA) has facilitated neurosurgical intervention for removal of central or peripheral nervous system tumors adjacent to or within vital structures. However, laboratory studies defining the phenotypic and genotypic properties of these tumors, both in cell culture and as xenografts in immunoincompetent animals, require viable tumor fragments free of microbial or red blood cell contamination. This report describes the use of a readily available sterile trap with the CUSA which, in conjunction with centrifugation and ammonium chloride lysis of the bloody aspirate, allowed collection of concentrated viable human medulloblastoma tumor cells. These cells were successfully established in cell culture and as transplantable xenografts in athymic mice.

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Immunobiology of primary intracranial tumors

Part 2: The evaluation of chemotherapy and immunotherapy protocols using the avian sarcoma virus glioma model

M. Stephen Mahaley Jr., Robert E. Gentry and Darell D. Bigner

✓ The avian sarcoma virus-induced glioma model in rats was used to study the effectiveness of immunotherapy, namely, Bacillus Calmette-Guérin (BCG) with or without sarcoma cells and/or chemotherapy with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), upon survival times. The most consistent prolongation of survival time was produced by triple therapy: BCG + intraperitoneally administered sarcoma cells + intravenously administered BCNU.

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Darell D. Bigner, Olin M. Pitts and Carol J. Wikstrand

✓ The introduction of active specific immunotherapy as an adjunct to conventional therapy of the brain-tumor patient creates the risk of the concomitant induction of experimental allergic encephalomyelitis (EAE). The lack of resolution concerning the total group of central nervous system (CNS) antigens which may be encephalitogenic, and the lack of definition of the necessary conditions for the induction of an anti-CNS myelin response complicate the design of an immunotherapeutic regimen for brain-tumor patients. We report here the ready induction of EAE in four of four guinea pigs and both of two nonhuman primates (Macaca fascicularis) with human glioblastoma multiforme (GBM) tissue injected with either complete or incomplete Freund's adjuvant (CFA, IFA). Immunization protocols utilizing encephalitogenic GBM tissue and adjuvant which did not result in EAE induction were established in both of two macaques, and the production of significant levels of antibodies specifically reactive with immunizing GBM-derived cultured cell lines in all of 12 macaques without EAE induction was demonstrated. As the lower detection limit of the sodium dodecyl sulfate-polyacrylimide gel electrophoresis (SDS-PAGE) assay for human myelin basic protein (HBP) was 0.6 µg HBP/gel, and an extract prepared from WR-GBM tumor tissue contained less than 0.6 µg of detectable HBP/25 µg of pH 3 extractable protein, and as 100 to 1000 µg of purified human basic protein (HBP) failed to induce EAE in three of three macaques, it was hypothesized that 1) GBM tissue may act as an adjuvant and markedly lower myelin basic protein (MBP) threshold doses for EAE induction, that 2) MBP encephalitogenic fragments capable of EAE induction may be present in GBM tissue but difficult to quantitate in precipitates by in vitro methods, or that 3) secondary encephalitogenic antigens unrelated to MBP may be present in GBM tissue. The threat of EAE induction and the potential difficulty of its detection in the deteriorating brain-tumor patient receiving active specific immunotherapy warrants a biological screen in immunizing CNS material in experimental animals prior to administration to patients in immunotherapy protocols.

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Thomas L. Roszman, William H. Brooks, William R. Markesbery, Gary J. Aziz and Darell D. Bigner

✓ The mitogenic responsiveness of peripheral blood lymphocytes obtained from Fischer 344 rats inoculated with avian sarcoma virus was studied. In addition, quantitative alterations in lymphocyte subpopulations were determined in these animals. Only peripheral blood lymphocytes from rats bearing astrocytomas had significantly diminished responses to concanavalin A when compared to control responses. The percentage of lymphocyte subpopulations detected in either the peripheral blood or spleen of tumor-bearing rats did not differ from values obtained with control rats. However, rats bearing astrocytomas had a marked decrease in the absolute number of the various lymphocyte subpopulations as a result of lymphopenia. Neither the sarcoma-bearing rats nor the virus-inoculated rats that did not develop tumors exhibited this lymphopenia. In addition, sera from rats bearing astrocytomas diminished the concanavalin A reactivity of spleen cells obtained from normal rats. The results of this study establish the avian sarcoma virus-induced rat astrocytoma model as a useful immunological parallel for the human disease.