Loss of heterozygosity assay for molecular detection of cancer using energy-transfer primers and capillary array electrophoresis.
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Microsatellite DNA loci are useful markers for the detection of loss of heterozygosity (LOH) and microsatellite instability (MI) associated with primary cancers. To carry out large-scale studies of LOH and MI in cancer progression, high-throughput instrumentation and assays with high accuracy and sensitivity need to be validated. DNA was extracted from 26 renal tumor and paired lymphocyte samples and amplified with two-color energy-transfer (ET) fluorescent primers specific for loci associated with cancer-induced chromosomal changes. PCR amplicons were separated on the MegaBACE-1000 96 capillary array electrophoresis (CAE) instrument and analyzed with MegaBACE Genetic Profiler v.1.0 software. Ninety-six separations were achieved in parallel in 75 minutes. Loss of heterozygosity was easily detected in tumor samples as was the gain/loss of microsatellite core repeats. Allelic ratios were determined with a precision of +/- 10% or better. Prior analysis of these samples with slab gel electrophoresis and radioisotope labeling had not detected these changes with as much sensitivity or precision. This study establishes the validity of this assay and the MegaBACE instrument for large-scale, high-throughput studies of the molecular genetic changes associated with cancer. (+info)
The effects of internal radiation exposure on cancer mortality in nuclear workers at Rocketdyne/Atomics International.
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We examined the effects of chronic exposure to radionuclides, primarily uranium and mixed-fission products, on cancer mortality in a retrospective cohort study of workers enrolled in the radiation-monitoring program of a nuclear research and development facility. Between 1950 and 1994, 2,297 workers were monitored for internal radiation exposures, and 441 workers died, 134 (30.4%) of them from cancer as the underlying cause. We calculated internal lung-dose estimates based on urinalysis and whole-body and lung counts reported for individual workers. We examined cancer mortality of workers exposed at different cumulative lung-dose levels using complete risk-set analysis for cohort data, adjusting for age, pay type, time since first radiation monitored, and external radiation. In addition, we examined the potential for confounding due to chemical exposures and smoking, explored whether external radiation exposure modifies the effects of internal exposure, and estimated effects after excluding exposures likely to have been unrelated to disease onset. Dose-response relations were observed for death from hemato- and lymphopoietic cancers and from upper aerodigestive tract cancers, adjusting for age, time since first monitored, pay type, and external (gamma) radiation dose. No association was found for other cancers, including cancers of the lung. Despite the small number of exposed deaths from specific cancer types and possible bias due to measurement error and confounding, the positive findings and strong dose-response gradients observed suggest carcinogenic effects of internal radiation to the upper aerodigestive tract and the blood and lymph system in this occupational cohort. However, causal inferences require replication of our results in other populations or confirmation with an extended follow-up of this cohort. (+info)
chlD gene function in molybdate activation of nitrate reductase.
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chlD mutants of Escherichia coli lack active nitrate reductase but form normal levels of this enzyme when the medium is supplemented with 10-3 M molybdate. When chlD mutants were grown in unsupplemented medium and then incubated with molybdate in the presence of chloramphenicol, they formed about 5% the normal level of nitrate reductase. Some chlD mutants or the wild type grown in medium supplemented with tungstate accumulated an inactive protein which was electrophoretically identical to active nitrate reductase. Addition of molybdate to those cells in the presence of chloramphenicol resulted in the formation of fully induced levels of nitrate reductase. Two chlD mutants, including a deletion mutant, failed to accumulate the inactive protein and to form active enzyme under the same conditions. Insertion of 99-Mo into the enzyme protein paralleled activation; 185-W could not be demonstrated to be associated with the accumulated inactive protein. The rates of activation of nitrate reductase at varying molybdate concentrations indicated that the chlD gene product facilitates the activation of nitrate reductase at concentrations of molybdate found in normal growth media. At high concentrations, molybdate circumvented this function in chlD mutants and appeared to activate nitrate reductase by a mass action process. We conclude that the chlD gene plays two distinguishable roles in the formation of nitrate reductase in E. coli. It is involved in the accumulation of fully induced levels of the nitrate reductase protein in the cell membrane and it facilitates the insertion of molybdenum to form the active enzyme. (+info)
Microvascular integrity and the time course of myocardial sodium accumulation after acute infarction.
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Loss of membrane permeability caused by ischemia leads to cellular sodium accumulation and myocardial edema. This phenomenon has important implications to left ventricular structure and function in the first hours after myocardial infarction. We hypothesized that during this period of time, after prolonged coronary occlusion and complete reflow, the rate of myocardial sodium accumulation is governed by microvascular integrity. We used 3-dimensional (23)Na MRI to monitor myocardial sodium content changes over time in an in vivo closed-chest canine model (n=13) of myocardial infarction and reperfusion. Infarcts with microvascular obstruction (MO) defined by both radioactive microspheres and contrast-enhanced (1)H MRI showed a slower rate of sodium accumulation as well as lower blood flow at 20 minutes and 6 hours after reperfusion. Conversely, the absence of MO was associated with faster rates of sodium accumulation and greater blood flow restoration. In addition, infarct size by (23)Na MRI correlated best with infarct size by triphenyltetrazolium chloride and contrast-enhanced (1)H MRI at 9 hours after reperfusion. We conclude that in reperfused myocardial infarction, sodium accumulation is dependent on microvascular integrity and is slower in regions of MO compared with those with patent microvasculature. Finally, (23)Na MRI can be a useful tool for monitoring in vivo myocardial sodium content in acute myocardial infarction. (+info)
Evaluation of toxicity and efficacy of 186Re-hydroxyethylidene diphosphonate in patients with painful bone metastases of prostate or breast cancer.
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Twenty-eight patients (12 men with prostate cancer, 16 women with breast cancer) were included in a phase II trial to evaluate the efficacy of 186Re-hydroxyethylidene diphosphonate (HEDP) on pain from bone metastasis and the toxicity of this agent. METHODS: After intravenous administration of 1295 MBq 186Re-HEDP, the efficacy was evaluated by means of a daily log. RESULTS: We observed an objective response in 67% of prostate cancer patients and in 36% of breast cancer patients. The mean duration of response was 45 d for prostate cancer patients and 24 d for breast cancer patients. No major adverse effects were observed. Marrow toxicity did not exceed grade 2 for white blood cells and grade 3 for platelets using National Cancer Institute criteria. CONCLUSION: 186Re-HEDP provides safe symptomatic relief of pain in prostate cancer patients. The benefit of this treatment is less clear in breast cancer patients. Further studies should be conducted to evaluate treatment by 186Re-HEDP at an earlier stage of the disease. (+info)
Demonstration of highly specific toxicity of the alpha-emitting radioimmunoconjugate(211)At-rituximab against non-Hodgkin's lymphoma cells.
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The ability of an alpha-emitter conjugated to a chimaeric anti-CD20 monoclonal antibody to kill selectively human B-lymphoma cells in vitro is reported. Two B-lymphoma cell lines RAEL and K422, and normal haematopoietic progenitor cells from human bone marrow aspirates were incubated with(211)At-rituximab (Rituxan(R) or MabTheratrade mark) and plated in clonogenic assays for survival analyses. Following 1 h incubation with(211)At-rituximab, in concentrations which gave an initial activity of 50 kBq ml(-1), a high tumour cell to normal bone marrow cell toxicity ratio was obtained; 4.1 to 1.0 log cell kill. Biodistribution studies of(211)At-rituximab in Balb/c mice showed similar stability as that of the iodinated analogue. The data indicate that testing of(211)At-rituximab in human patients is warranted. (+info)
A phase I radioimmunotherapy trial evaluating 90yttrium-labeled anti-carcinoembryonic antigen (CEA) chimeric T84.66 in patients with metastatic CEA-producing malignancies.
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Chimeric T84.66 (cT84.66) is a genetically engineered human/murine chimeric IgG, with high affinity and specificity to carcinoembryonic antigen (CEA). The purpose of this Phase I dose escalation therapy trial was to evaluate the toxicities, biodistribution, pharmacokinetics, tumor targeting, immunogenicity, and organ and tumor absorbed dose estimates of cT84.66 labeled with 90Y. Patients with metastatic CEA-producing malignancies were first administered 5 mCi 111In-labeled DTPA-cT84.66 (5 mg), followed by administration of the therapy dose of 90Y-labeled DTPA-cT84.66 1 week later. The therapy infusion was immediately followed by a 72-h administration of DTPA at 250 mg/m2/24 h. Dose levels of administered activity ranged from 5 to 22 mCi/m2 with three to six patients per level. Serial nuclear scans, blood samples, and 24-h urine collections were performed out to 5 days after infusion. Human antichimeric antibody response was assayed out to 6 months. Patients were administered up to 3 cycles of therapy every 6 weeks. Radiation absorbed doses to organs were estimated using a five compartment model and MIRDOSE3. Twenty-two patients received at least one cycle of therapy, with one individual receiving two cycles and two receiving three cycles of therapy. All were heavily pretreated and had progressive disease prior to entry in this trial. Reversible leukopenia and thrombocytopenia were the primary dose-limiting toxicities observed. Maximum tolerated dose was reached at 22 mCi/ m2. In general, patients with liver metastases demonstrated more rapid blood clearance of the antibody. Thirteen patients developed an immune response to the antibody. Average radiation doses to marrow, liver, and whole body were 2.6, 29, and 1.9 cGy/mCi 90Y, respectively. Dose estimates to tumor ranged from 66 to 1670 cGy (8.7 to 52.2 cGy/mCi 90Y) for each cycle of therapy delivered. Although no major responses were observed, three patients demonstrated stable disease of 12-28 weeks duration and two demonstrated a mixed response. In addition, a 41-100% reduction in tumor size was observed with five tumor lesions. 90Y-labeled cT84.66 was well tolerated, with reversible thrombocytopenia and leukopenia being dose limiting. Patients with extensive hepatic involvement by tumor demonstrated unfavorable biodistribution for therapy with rapid blood clearance and poor tumor targeting. Average tumor doses when compared with red marrow doses indicated a favorable therapeutic ratio. Stable disease and mixed responses were observed in this heavily pretreated population with progressive disease. This trial represents an important step toward further improving the therapeutic potential of this agent through refinements in the characteristics of the antibody and the treatment strategies used. Future trials will focus on the use of peripheral stem cell support to allow for higher administered activities and the use of combined modality strategies with radiation-enhancing chemotherapy drugs. Further efforts to reduce immunogenicity through humanization of the antibody are also planned. Finally, novel engineered, lower molecular weight, faster clearing constructs derived from cT84.66 continue to be evaluated in preclinical models as potential agents for radioimmunotherapy. (+info)
Medical use of byproduct material; policy statement, revision. Nuclear Regulatory Commission. Final policy statement; revision.
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The Nuclear Regulatory Commission (NRC) is revising its 1979 policy statement on the medical use of byproduct material. These revisions are one component of the Commission's overall program for revising its regulatory framework for medical use, including its regulations that govern the medical use of byproduct material. The overall goals of this program are to focus NRC regulation of medical use on those medical procedures that pose the highest risk and to structure its regulations to be risk-informed and more performance-based, consistent with NRC's "Strategic Plan for Fiscal Year 1997-Fiscal Year 2002." The policy informs NRC licensees, other Federal and State agencies, and the public of the Commission's general intentions in regulating the medical use of byproduct material. (+info)