Radiation safety education for laboratory animal science.

Students enrolled in the laboratory animal science graduate program at MCP Hahnemann University seek to gain entrance to veterinary school or to manage an animal facility within an academic institution, pharmaceutical or biotechnology company conducting biomedical research. Ongoing interaction between faculty in the radiation oncology, radiation safety, and lab animal science disciplines revealed an acute need for radiation safety education for laboratory animal science students who will likely interact with researchers either designing and writing protocols for animal studies using radiation or radioactive materials, or veterinary staff who will use sources of radiation to diagnose and/or treat possible animal injuries and diseases. A core course in the Radiation Sciences graduate program was modified to address the needs of these students, instructing them in radiation safety, detection and counting instrumentation, and radiation biology. These fundamental areas were integrated to help the students gain a sound, basic knowledge of radiation and radioactive materials used in biomedical research.

External beam radiation enhances antibody mediated radiocytotoxicity in human glioma cells in vitro.

Enhanced accumulation of monoclonal antibodies in tumor tissue has been observed as a result of external beam irradiation (EBR). This effect was mainly attributed to increased vascular leakage due to unspecific radiation damage of vascular endothelial cells. The aim of this study was to investigate the effects of EBR on expression and antibody-binding of epidermal growth-factor receptor (EGF-R) in human glioma cells in-vitro. High-grade glioma cells were irradiated with conventional x-rays (0-3600 Rad) and surface binding, internalization and radiocytotoxicity of 125I-labeled monoclonal antibody (MAb) 425, specific for human EGF-R, was tested. EBR showed a short-term dose and time dependent increase of specific MAb 425 binding and internalization in receptor positive cell lines U87-MG and A1207. This effect was probably due to a mitotic block and an increase in cellular volume. Combination of EBR and 125I-425 showed additive effects on cell vitality/survival and was more pronounced in contact inhibited cells as compared to cells growing in a log-phase. We assume that cells exposed to 125I-labeled MAb 425 are only able to accumulate a critical number of DNA double-strand breaks when the doubling-time is prolonged e.g. under contact-inhibition or radiation induced mitotic blockade. We conclude that EBR has no negative effects on EGF-R expression, MAb-binding and internalization. The combination of EBR and 125I-MAb 425 enhances cytotoxic efficacy and thus supports adjuvant use in the clinical management of high-grade glioma.

Identification of fatty acid methyl esters (FAMEs) in postmortem tissue. A new marker of alcohol abuse?

Based on a method that combines thin layer chromatography and gas chromatography, it proved possible to detect postmortem fatty acid methyl esters (FAMEs) in human tissues (pancreas, liver, heart and adipose tissue). The highest concentrations were found in the pancreas, medium concentrations in the liver and adipose tissue and the lowest concentration in heart tissue. All tissues contained higher concentrations of unsaturated FAMEs than saturated FAMEs. In order to evaluate the influence of alcohol consumption on the formation of FAMEs, the decreased were divided into three groups: control group, chronic alcoholics (who were alcohol free at the time of death), and acutely alcohol-intoxicated subjects (so intoxicated at the time of death with no evidence of chronic alcohol abuse). In comparison to the control group, the organs of the chronic alcoholics showed only slight values, the tissues of the acutely intoxicated subjects were obviously higher. Based on the varying concentration distribution in the three groups, it seemed that postmortem differentiation of the alcohol consumption which existed before the time of death would be possible. After further investigation with a greater number of samples, FAMEs could become a useful supplement to existing alcohol markers in the future.

Biodistribution of 125I-MAb 425 in a human glioma xenograft model: effect of chloroquine.

Chloroquine has been shown to increase the cellular retention and nuclear incorporation of 125I-labeled monoclonal antibody (MAb) 425, a murine anti-epidermal growth factor receptor monoclonal antibody, in human high-grade glioma cells in vitro. The objective of this study was to examine the effect of chloroquine on the biodistribution of 125I-MAb 425 in an intracerebral xenogeneic transplant of glioma cells. Nude rats were stereotaxically implanted in the right hemisphere with A1207 human high-grade glioma cells. After 14 days, animals were injected i.v. with chloroquine (40 mg/kg) followed 2 h later by an 125I-MAb 425 (9 MBq) infusion. Tissue distributions were performed up to 168 h post 125I-MAb 425 injection. From 24 to 168 h, tumor-to-contralateral left brain ratios increased from 9 to 15 for 125I-MAb 425 alone, and 7 to 13 for the 125I-MAb 425/chloroquine combination, respectively. A single administration of chloroquine did not result in any significant difference in radiolabeled MAb accumulation in either the tumor site or other tissues. We conclude that chloroquine did not increase the amount of 125I-MAb 425 into the tumor; however, it is safe to administer i.v. at the 40 mg/kg dose. Under these experimental conditions, the increased radioactive accumulation observed for in vitro data did not translate into similar in vivo results.

In vitro evaluation of iodine-125-labeled monoclonal antibody (MAb 425) in human high-grade glioma cells.

Human high-grade glioma cell lines (A1207, U-87MG, U-373MG, and F39) with high levels of epidermal growth factor receptor (EGF-R) expression were incubated for 2-48 h with 1 microCi/ml of the EGF-R-specific 125I-MAb 425 and measured for surface-bound, cytoplasmic, and nuclear radioactivity. The A1207 and U-373MG cell lines showed the highest surface-bound radioactivity with 215.9 +/- 8.7 nCi (30 h) and 287.8 +/- 23.2 nCi (24 h)/10(6) cells, respectively, whereas the U-87MG and the F39 cell lines bound significantly less antibody (48.8 +/- 5.4 nCi [48 h] and 31.1 +/- 0.7 nCi [24 h]). Surface-bound antibody was efficiently internalized into the cytoplasm. The U-373MG, U-87MG, and A1207 cell lines achieved 19.8% +/- 2.1 internalization of the surface-bound antibody in contrast to > 40% for the F39 cell line. Only the A1207 cell line showed significant nuclear radioactivity. There was no correlation between the reported EGF-R number and amount of antibody bound or internalized. We conclude that binding and uptake of the 125I-MAb 425 is specific for human glioma cells and shows saturation kinetics independent of receptor density.

Histone H1 suppresses tumor growth of leukemia cells in vitro, ex vivo and in an animal model suggesting extracellular functions of histones.

Purified histone H1 exerts growth inhibition of leukemia cells independent of lineage, stage, and maturation. At 200 micrograms/ml, H1 proved cytotoxic in 19 of 21 of the tested leukemia-derived cell lines and for 11 of 16 of the fresh tumor samples from leukemia patients. In all cases, normal peripheral blood mononuclear cells and bone marrow cells remained unaffected. Multicellular spheroids from the Burkitt's lymphoma cell line IM-9 were growth arrested at 500 micrograms H1/ml. The clonogenic growth of the Burkitt's lymphoma cell line Daudi was arrested at 160 micrograms H1/ml. Synthetic H1-peptides as well as peptides and proteins with biochemical properties similar to H1 had no inhibitory growth effect at equimolar concentrations. Furthermore, 250 micrograms H1 injected into a Burkitt's lymphoma (Daudi), xenotransplanted into nude mice, arrested tumor growth. As shown by electron microscopy and flow cytometry, incubation of leukemia cells with H1 resulted in severe plasma membrane damage and ultimately cytolysis. This report characterizes a 33-kd protein that binds H1 and is responsible for the cell death via destruction of the cell membrane integrity. New extranuclear functions of histones are presented.

Epidermal growth factor receptor 425 monoclonal antibodies radiolabeled with iodine-125 in the adjuvant treatment of high-grade astrocytomas.

Fifty-nine patients with primary presentation of high-grade gliomas of the brain, 13 with astrocytomas with anaplastic foci and 46 with glioblastoma multiforme, were treated with surgical intervention and definitive postoperative radiation therapy followed by multiple intravenous administration of iodine-125-labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The total cumulative labeled antibody doses ranged from 40 to 296 mCi. The administration of the radiolabeled antibody was performed in most instances within 3 months following completion of the primary surgery and radiation therapy. No significant life-threatening toxicities were observed during the trial. At one year, 34 (58%) of the 59 patients in the trial were alive. The median overall survival for both groups was 13.5 months.

Enhancement of monoclonal antibody efficacy: the effect of external beam radiation.

External beam irradiation has been shown to enhance accumulation of monoclonal antibodies (MAb) in tumors in vivo. This effect is mainly attributed to an unspecific damage of vascular endothelial cells resulting in an increased vascular leakage. The aim of our studies was to determine the effects of external beam radiation on the expression and function of the epidermal growth factor receptor (EGF-R) in vivo. Expression and internalization of EGF-R was tested in vivo, employing 125I-MAb 425 that binds specifically to the human EGF-R. Irradiation of human high-grade glioma cell lines U87-MG and A1207 with increasing doses (0-3600 Rad) of 240 kVp X-rays, markedly enhanced the binding of 125I-MAb 425 to the cell surface. This effect could only be observed for a few days following irradiation. No correlation of the radiation dose and overexpression of EGF-R were found. At the same time, irradiation stimulated significant and dose-dependent internalization of 125I-MAb. Internalization and intranuclear accumulation of 125I-MAb are necessary to explain the radiocytotoxic effects of 125I. The combination of external beam irradiation and labeled MAb 425 showed at least additive effects on tumor cell survival, when the interval between irradiation and MAb treatment was short. Our data support the clinical observations in the adjuvant treatment of high grade gliomas with 125I-MAb 425 following surgery and external beam radiation.

Report of congenital anaplastic astrocytoma discovered in a newborn.

Reports of congenital anaplastic astrocytoma have been rare, representing less than 2% of all brain tumors. We present a case of a congenital anaplastic astrocytoma discovered during the first 24 hours after delivery. A recent literature review found 12 additional cases since 1972. The presenting signs, most common findings on neuroimaging, treatment, and outcome are examined.

Brain uptake of thallium-201 from the cerebrospinal fluid compartment.

The present report elucidates the movement of 201Tl through the cerebrospinal fluid compartment and its subsequent uptake by normal brain. Autoradiographic studies of rat brain, after stereotaxic 201Tl injection into either the lateral or fourth ventricle, reveal that 201Tl moves freely through the cerebrospinal and extracellular fluid compartments. Subsequent to lateral ventricular injection, central thalamic and specific hypothalamic nuclei are heavily labeled. Densely labeled mesencephalic nuclei include the periaqueductal grey and oculomotor nuclear complex. Labeling of giant cells within the vestibular complex is suggestive of neuronal uptake. Major fiber tracts are devoid of label confirming that 201Tl uptake does not occur in white matter. Most labeling following fourth ventricle injection occurs within the caudal medulla and cervical spinal grey. Our findings suggest that 201Tl uptake by normal brain from the cerebrospinal fluid occurs as a function of thallium concentration and neuronal activity.

Assessing short-term recognition memory with forced-choice psychophysical methods.

The rationale and methodology for using computer-controlled forced-choice psychophysical methods to assess short-term recognition memory in human subjects are presented. Here, we use non-verbal computer-synthesized auditory and visual stimuli with an adaptive psychophysical procedure. Sequence-length thresholds (SLTs, span lengths) for randomly generated binary auditory and visual-sequential patterns and simultaneous visual-spatial patterns are determined to assess short-term memory capacity. The SLTs can also be used to equate for initial retention level for delayed matching-to-sample (DMS) or delayed matching-to-non-sample (DMNS) tasks which assess memory decay. The DMS/DMNS tasks have also been modified for use with the forced-choice paradigm. In contrast to many verbal paradigms requiring immediate ordered recall, non-verbal stimuli in a forced-choice paradigm provide a more direct measure of sensory memory because long-term memory, complex encoding/decoding processes, and motor-sequencing factors are minimized or avoided. Furthermore, the forced-choice recognition memory tasks are applicable over a broad age range, are less sensitive to socio-economic factors and educational level, and avoid complex instructions. Taken together, these factors enhance the applicability of these tasks in children and adults with CNS lesions, particularly where cognitive status may be compromised.

Malignant astrocytomas treated with iodine-125 labeled monoclonal antibody 425 against epidermal growth factor receptor: a phase II trial.

Twenty-five patients with primary presentation of malignant astrocytoma, astrocytoma with anaplastic foci, and glioblastoma multiforme were treated with surgical resection and definitive radiation therapy followed by intravenous or intra-arterial administration of Iodine-125 labeled monoclonal antibody-425, which binds specifically to human epidermal growth factor receptor. The patients presented with primary untreated disease, positive contrast enhanced computed tomography scans of the brain, and compatible clinical symptoms. In this Phase II clinical trial, the patients had surgical debulking or biopsy followed by definitively administered external beam radiation therapy and one or multiple doses (35 to 90 mCi per infusion) of radiolabeled antibody. The total cumulative doses ranged from 40 to 224 mCi. The administrations of the radiolabeled antibody were performed in most cases 4-6 weeks following completion of the primary surgery and radiation therapy. Ten patients had astrocytoma with anaplastic foci and 15 had glioblastoma multiforme. No significant life-threatening toxicities were observed during this trial. At 1 year 60% of the patients with astrocytoma with anaplastic foci or glioblastoma multiforme are alive. The median survival for both groups was 15.6 months.

A novel heterobifunctional linker for formyl to thiol coupling.

A maleimide hydrazide has been synthesized as a heterobifunctional cross-linking agent for thiol to formyl coupling. This linker has been applied to the coupling of the monoclonal antibody 17-1A, or an Fab' derived therefrom, to polyaldehyde dextran onto which the antineoplastic agent ellipticine has been attached. High binding avidities for the unshed antigen on the SW1116 colorectal tumor cell are retained in these drug-dextran-linker-antibody conjugates.

Characterization of astrocytomas, meningiomas, and pituitary adenomas by phosphorus magnetic resonance spectroscopy.

Phosphorus magnetic resonance (MR) spectroscopy allows noninvasive measurement of phosphate-containing compounds and pH within brain cells. The authors obtained localized phosphorus MR spectra from 10 normal brains, four low-grade astrocytomas, six glioblastomas, four meningiomas, and three pituitary adenomas and found differences in the spectra of each tumor type. Compared to normal brain, the spectra from low-grade astrocytomas showed a significant reduction of the phosphodiester (PDE) peak. Glioblastomas were characterized by a significant reduction of the PDE peak, elevation of the phosphomonoester (PME) peak, and a relatively alkaline intracellular pH. The spectra from meningiomas and pituitary adenomas were markedly different from the glial tumors. Meningiomas showed significant reductions in phosphocreatine, PDE, and inorganic phosphate, as well as a relatively alkaline pH. Pituitary adenomas resembled meningiomas, but had a much higher PME peak. Although the number of tumors studied was small, there appears to be a characteristic spectrum associated with these different tumor types. The present findings can be useful in the preoperative identification of these tumors and in furthering understanding of their growth and metabolism in vivo.

Misonidazole conjugates of the colorectal tumor associated monoclonal antibody 17-1A.

The radiation sensitizer misonidazole has been linked to the monoclonal antibody 17-1A which recognizes a nonshed antigen of a human gastrointestinal tumor. Linkage was accomplished through a hemisuccinate of misonidazole attached by a mixed anhydride coupling and gave a conjugate whose plasma half-life (for drug cleavage) was ca. 70 h. The degree of substitution on the antibody could be precisely regulated by varying the reactant ratios. The binding avidities of the resulting conjugates to the SW1116 colorectal tumor cells decrease logarithmically with increasing drug load. Four to six misonidazoles per antibody represented the optimum drug loading on this system. Enzymatic cleavage of the conjugate-drug union took place at both the ester and the amide linkages with the former scission predominating.

Early metabolic changes following chemotherapy of human gliomas in vivo demonstrated by phosphorus magnetic resonance spectroscopy.

The authors have used phosphorus magnetic resonance spectroscopy to monitor pH changes in malignant gliomas following treatment with intravenous and intra-arterial 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). Image-guided, localized phosphorus spectra of human gliomas in situ were obtained using a 1.5-T whole body combined imaging and spectroscopy system. Initial intravenous BCNU treatment was followed by a transient decrease of tumor intracellular pH by 0.15 +/- 0.03 pH units (mean +/- SD). Superselective intra-arterial administration of the same drug was followed by an increase of tumor intracellular pH by 0.15 +/- 0.6 pH units (mean +/- SD). These changes occurred prior to any changes on x-ray, computed tomography (CT), or magnetic resonance imaging (MRI). In addition to enhancing our understanding of the metabolic effects of BCNU, such changes may correlate with drug efficacy or toxicity and may be useful in guiding therapy in the future.