Chimeric honeybees (Apis mellifera) produced by transplantation of embryonic cells into pre-gastrula stage embryos and detection of chimerism by use of microsatellite markers.

The production of chimeras, by use of cell transplantation, has proved to be highly valuable in studies of development by providing insights into cell fate, differentiation, and developmental potential. So far, chimeric honeybees have been created by nuclear transfer technologies. We have developed protocols to produce chimeric honeybees by use of cell transplantation. Embryonic cells were transplanted between pre-gastrula stage embryos (32-34 hr after oviposition) and hatched larvae were reared in vitro for 4 days. Chimeric individuals were detected by use of microsatellite analysis and a conservative estimation approach. 4.8% of embryos, posteriorly injected with embryonic cells, developed into chimeric honeybee larvae. By injection of cells pre-stained with fluorescent cell tracer dye, we studied the integration of transplanted cells in the developing embryos. Number of injected cells varied from 0 to 50 and cells remained and multiplied mainly in the area of injection.

The Cooperative Breast Cancer Tissue Resource: archival tissue for the investigation of tumor markers.

Investigators continue to search for reliable markers of prognosis of breast cancer. For many analyses, laboratory techniques permit the use of archival paraffin-embedded tissue collected years previously and readily linked to clinical and follow-up information. Laboratory investigators have often expressed the need for such a tissue resource. We have developed a publicly available resource of archival breast cancer specimens. The pathological material has been collected and reviewed by investigators at four institutions and currently includes breast cancer specimens from more than 9300 cases. Institutional pathologists reviewed slides and blocks using a common protocol and coding scheme. Clinical information and details of follow-up came from data routinely collected by the institutions' cancer registries. Coded data are maintained centrally in a single database. A subset of the data may be searched on the World Wide Web to determine the availability of cases with specified characteristics. The material collected by this Cooperative Breast Cancer Tissue Resource is generally representative of breast cancer diagnosed in community hospital settings in the United States. Seventy-two percent of the living cases have been followed for at least 5 years, and follow-up status is updated regularly. Interested laboratory investigators may apply to the Resource for the use of these tissues. This Resource is proving valuable to laboratory investigators who require large numbers of specimens for validation studies of prognostic markers of breast cancer.

Reproducibility of p53 immunohistochemistry in bladder tumors. National Cancer Institute, Bladder Tumor Marker Network.

The National Cancer Institute Bladder Tumor Marker Network conducted a study to evaluate the reproducibility of immunohistochemistry for measuring p53 expression in bladder tumors. Fifty paraffin blocks (10 from each of the five network institutions) were chosen at random from among high-grade invasive primary bladder tumors. Two sections from each block were sent to each laboratory for staining and scoring, and then all sections were randomly redistributed among the laboratories for a second scoring. Intra- and interlaboratory reproducibility was assessed with regard to both staining and scoring. For overall assessments of p53 positivity, the results demonstrated that intralaboratory reproducibility was quite good. Concordance across the five participating laboratories was high for specimens exhibiting no or minimal nuclear immunostaining of tumor cells or high percentages of tumor cells with nuclear immunoreactivities. However, there was a reduced level of concordance on specimens with percentages of stained tumor cells in an intermediate range. The discordancies were due mainly to staining differences in one of the five laboratories and scoring differences in another laboratory. These results indicate that some caution must be used in comparing results across studies from different groups. Standardization of staining protocols and selection of a uniform threshold for binary interpretation of results may improve assay reproducibility between laboratories.

Providing human tissues for research: how to establish a program.

The Cooperative Human Tissue Network is a group composed of cooperating academic institutions that supply human tissues to researchers studying a wide range of neoplastic and other diseases. The experience of the Cooperative Human Tissue Network in establishing methods of prospective tissue collection and in developing tumor banks is discussed to aid institutions in establishing tissue resources for their local investigators, who may wish to use human tissues in current or future research projects. The advantages to pathology departments and to associated medical institutions of establishing an organized tissue resource include ensuring proper institutional review board approval of research projects using human tissues, protecting diagnostic specimens, creating new opportunities for extramural research, increasing the speed of diagnostic specimen transport to surgical pathology, and providing educational and research opportunities for pathologists and pathology residents. Methods of tissue collection, processing, storage, data collection, and supply are outlined. Also, resources necessary to begin organized tissue collection, including personnel, space, equipment, and supplies, are discussed.

Zinc metabolism in patients with exocrine pancreatic insufficiency.

OBJECTIVE: Metabolism of zinc was studied by administering radioactive zinc (Zn65), and by performing metabolic balance studies in eight patients with exocrine pancreatic insufficiency and eighteen control subjects. METHODS: Retention of radioactive zinc was measured by total body counter, and its urinary and fecal excretion by gamma scintillation counter. Metabolic balance studies were carried out by measuring dietary zinc intake as well as fecal and urinary excretion of zinc by atomic absorption spectrophotometry in this group of patients. RESULTS: These studies revealed a 50% reduction in intestinal absorption of Zn65 in patients with exocrine pancreatic insufficiency as compared to alcoholic and non-alcoholic control subjects. In addition, there was a 2 to 4 fold increase (p<0.05) in urinary excretion of zinc in subjects with pancreatic disease. In pancreatic insufficiency, reduced zinc absorption and increased urinary zinc excretion were balanced by lower (p<0.05) endogenous excretion of zinc as evidenced by reduced excretion of Zn65 in feces during the second 4-day period. The mean biological half-life of Zn65 tended to be lower in patients with pancreatic insufficiency as compared to alcoholic control subjects, however the difference did not reach statistical significance. CONCLUSION: These observations indicate marked alterations in zinc metabolism in patients with advanced chronic pancreatic disease and provide greater insight into development of zinc deficiency in this group of patients.

The Cooperative Human Tissue Network. An update.

BACKGROUND: During the past decade, the National Cancer Institute became aware of a lack of availability of human tissues for research, especially in the fields of molecular biology, genetics, and immunology. METHODS: In 1987, the National Cancer Institute (NCI) established the Cooperative Human Tissue Network (CHTN) by funding three institutions that had extensive experience in the procurement and distribution of tissues for research. RESULTS: Since its inception, the CHTN has been expanded to five member institutions, with the addition of the Columbus Children's Hospital (the Pediatric Division for the procurement of pediatric tissues) and Case Western Reserve University. Each of the five divisions have established regional networks of institutions that supply tissues to the major divisions. From 1987 through 1991, the CHTN has distributed more than 29,000 human tissue samples and supplied approximately 500 researchers throughout the country. CONCLUSIONS: This report describes the development and current status of the CHTN, a valuable resource to the research community.

External gamma-ray counting of selected tissues from a Thorotrast patient.

Results of gamma-ray measurements of selected tissues from a patient who was injected with Thorotrast almost 36 y ago are reported. The purposes of this study were: 1) to determine the relative tissue distribution and activities of specific radionuclides in the 232Th decay chain, specifically 228Ra (as measured by 228Ac), 212Pb, and 224Ra (measured directly and as measured by 212Pb), and 2) to evaluate the level of radioactive disequilibrium among the daughter products. The spleen and liver had the highest concentrations of radioactivity. Bone also appears to be a long-term sink for 232Th daughter products based on estimates from a small portion of one rib. Larynx and esophagus contained measurable activity, which may have been due to their proximity to the "Thorotrastoma." Radioactivity in the remaining measured tissues were low, as expected. Secular equilibrium could be demonstrated in bone, pancreas, larynx, esophagus, and breast. Significant disequilibrium was observed for spleen, liver, kidney, and red blood cells. Radioactivity measurements reported here will be useful in estimating radiation doses to selected tissues. Such dose estimates are valuable in refining current risk estimates (e.g., liver) and in identifying tissues at risk for further epidemiologic studies. These results, while consistent with other published studies, should be interpreted with caution since measurements were made on only one patient.

Kinetic analysis of zinc metabolism in humans after simultaneous administration of 65Zn and 70Zn.

Zinc kinetics were studied and compared after oral simultaneous administration of two tracers, radioactive (65Zn) and stable (70Zn) isotope, to four normal human volunteers. Both tracers and zinc concentration were measured in plasma, red blood cells (RBC), urine, and feces for up to 78 days. Radioactive zinc was also measured by external counting over whole body, liver, and thigh. Data from each individual were analyzed using a compartmental model for zinc metabolism. Values calculated for absorption, fractional zinc excretion in urine, exchange with RBC, and secretion into gut using 70Zn data did not differ from values calculated using 65Zn data. Results show that human zinc metabolism can be investigated using stable isotopes as tracers to determine parameters of whole body zinc metabolism, including zinc absorption, excretion, and secretion.

Effect of long-term freezer storage, thawing, and refreezing on selected constituents of serum.

We determined the effect of long-term freezer storage and repeated thawing and freezing of serum on concentrations of electrolytes (sodium, potassium, calcium, and phosphate), enzymes (aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, and creatine kinase), total protein, tumor markers (carcinoembryonic antigen and alpha-fetoprotein), and other substances. Vials (1 ml) of frozen serum from a single blood drawing from 40 women with no breast disease and 70 with benign breast disease were analyzed annually from 1983 to 1987. Blood had been obtained from 40 subjects in 1978, 40 in 1980, and 30 in 1983. Thawing and refreezing studies were done in two ways: (1) serum samples from 30 subjects with benign breast disease were thawed at weekly intervals for 6 weeks and (2) serum samples from 30 patients with stage IV breast cancer were analyzed for alpha-fetoprotein and carcinoembryonic antigen, and serum specimens from 23 patients with benign breast disease and 7 control subjects were analyzed for lactate dehydrogenase and creatine kinase after thawing and keeping the samples at room temperature for up to 4 hours and then refreezing them. For measuring laboratory variability, duplicate samples were processed. Long-term storage (up to 10 years) and repeated thawing and refreezing did not affect the results of any tested constituents of serum. Although most measurements showed statistically significant variability over test cycles, these differences were thought to be due to laboratory variability.

Special communication. The Cooperative Human Tissue Network.

This report is to make the general scientific community aware of availability of human tissues through this network. During the short period of its existence, this network has developed useful procedures for distributing human tumor tissue in a rapid, scientifically useful, cost-effective manner to investigators to whom this tissue would not otherwise be available. The growth in the number of specimens being distributed matches the previous experience of each of the member institutions in internal distribution activities. It is expected that the availability of tissue will lead to new research initiatives and grant applications. This Network has been established as a model which can in the future be expanded to meet the needs of the scientific community at large. The National Cancer Institute encourages investigators engaging in cancer research who have need of human tissues to submit their requests to the Cooperative Human Tissue Network.

Contrasting effects of verapamil and nifedipine on pH of ischemic myocardium in the dog.

It remains unknown whether the actions of verapamil to depress and nifedipine to enhance contractile function of ischemic myocardium influence the degree of myocardial ischemic injury. Thus, we measured intramyocardial pH using fiberoptic pH probes in 43 anesthetized open-chest dogs pretreated for 30 min with verapamil, or nifedipine in doses that decreased aortic pressure 10 to 15 mm Hg before ligation of the left anterior descending coronary artery for 15 min. Drugs were continued during the 15-min ischemic period until the animals were euthanized without reperfusion: verapamil, 10-20 micrograms/kg/min and nifedipine, 2 to 4 micrograms/kg/min i.v. Verapamil-treated dogs showed higher pH of ischemic subendocardium after 15 min ischemia (6.75 +/- 0.07) than did the nifedipine (6.48 +/- 0.04) or placebo (6.43 +/- 0.05) groups, even if the animals were paced (6.71 +/- 0.11) to prevent the negative chronotropic effect of verapamil (P less than 0.01). Neither verapamil nor nifedipine changed collateral myocardial blood flow from 0.10 +/- 0.02 in the subendocardium and 0.17 +/- 0.03 ml/min/g in the subepicardium. Left ventricular function estimated by left ventricular dp/dt was depressed 15% by verapamil and enhanced 26% by nifedipine. Thus, verapamil, but not nifedipine, relieves acidosis of ischemic myocardium after acute coronary occlusion in doses that sustain a 10 to 15 mm Hg decrease in aortic pressure. Nifedipine, in doses that produced the same 10 to 15 mm Hg decrease in mean aortic pressure, did not increase intramyocardial pH, as it enhanced contractile function, estimated by left ventricular dp/dt.(ABSTRACT TRUNCATED AT 250 WORDS)

Body cell mass in cancer-bearing and anorexic patients.

We used a sensitive whole body counter which measures potassium-40 (40K) to determine total body potassium and to estimate body cell mass (BCM) in 104 previously untreated patients with upper gastrointestinal malignancies, 233 normal volunteers, and 18 patients with anorexia nervosa. BCM was greater in normal males than in females. In both normal males and females, the BCM tended to decrease with age, both as an absolute measure and as a percentage of body weight. Anorexia nervosa patients experienced marked weight loss (30.5%), and had significant depletion of absolute BCM, but exhibited relative sparing of BCM as indicated by a rise in BCM as a percentage of body weight. This may reflect a normal adaptation and predominant fat utilization in chronic malnutrition. The cancer patients, on the other hand, had significant weight loss (12.7% for females, 13.9% for males) and demonstrated a proportional decline in BCM, with no change in BCM as a percentage of body weight. These findings support the contention that, in the cancer-bearing patient, weight loss consists of a significant depletion of both fat and BCM. The challenge to the clinicians caring for cancer patients is repletion of this supremely functional body compartment.

Kinetic analysis of zinc metabolism and its regulation in normal humans.

Zinc metabolism was studied in 32 normal volunteers after oral (n = 25) or intravenous (n = 7) administration of 65Zn. Data were collected from the blood, urine, feces, whole body, and over the liver and thigh regions for 9 mo while the subjects consumed their regular diets (containing 10 mg Zn ion/day) and for an additional 9 mo while the subjects received an exogenous oral supplement of 100 mg Zn ion/day. Data from each subject were fitted by a compartmental model for zinc metabolism that was developed previously for patients with taste and smell dysfunction. These data from normal subjects were used to determine the absorption, distribution, and excretion of zinc and the mass of zinc in erythrocytes, liver, thigh, and whole body. By use of additional data obtained from the present study, the model was refined further such that a large compartment, which was previously determined to contain 90% of the body zinc, was subdivided into two compartments to represent zinc in muscle and bone. When oral zinc intake was increased 11-fold three new sites of regulation of zinc metabolism were identified in addition to the two sites previously defined in patients with taste and smell dysfunction (absorption of zinc from gut and excretion of zinc in urine). The three new sites are exchange of zinc with erythrocytes, release of zinc by muscle, and secretion of zinc into gut. Regulation at these five sites appears to maintain some tissue concentrations of zinc when dietary zinc increases.

Zinc metabolism in adrenal cortical insufficiency: effects of carbohydrate-active steroids.

Detailed studies of zinc kinetics were performed in two patients with adrenal cortical insufficiency to investigate the effects of carbohydrate-active steroids (CAS) on zinc metabolism. Zinc- 69m was administered intravenously to each patient under two conditions: (1) treated with CAS replacement therapy and (2) untreated, ie, without hormone treatment for five to six days. Radioactivity was measured in blood plasma, red blood cells, urine, and stool and by means of external probes placed over liver and thigh. Data were analyzed using a previously developed multicompartmental model, which describes the early phase of zinc metabolism. The results of these studies suggest that CAS promotes the internalization of zinc into red blood cells and liver cells. These results are consistent with previous in vitro and in vivo studies in which CAS was shown to induce the synthesis of metallothionein in liver cells.

Comparative effects of verapamil, diltiazem, and nifedipine on hemodynamics and left ventricular function during acute myocardial ischemia in dogs.

The calcium channel-blocking drugs verapamil, diltiazem, and nifedipine are being used with increasing frequency in patients with angina pectoris due to coronary artery disease. Although each of these agents possesses negative inotropic potential, their relative effects on myocardial function in relation to their vasodilator potencies are unknown. We understood to study this in 20 conscious dogs that had partial occlusions of their circumflex coronary arteries during therapy with placebo, verapamil, nifedipine, or diltiazem. Myocardial blood flow was measured by use of microspheres, and left ventricular function was measured by radionuclide angiography. Drug effects were compared at doses causing equal decreases in mean arterial pressure and coronary vascular resistance of nonischemic myocardium. Global ejection fraction and ejection fraction of the ischemic region were significantly decreased by verapamil (p less than .01) and increased by nifedipine (p less than .001); diltiazem caused no significant changes. Verapamil significantly increased peak diastolic filling rate (p less than .001); nifedipine also increased diastolic filling rate, but only at doses that markedly decreased mean arterial pressure and coronary vascular resistance. The effect of diltiazem on diastolic filling rate was not significantly different than placebo. For doses causing an equal decrease in mean arterial pressure, verapamil decreased heart rate (p less than .001), and diltiazem and nifedipine increased heart rate (p less than .001). We conclude that the relative potencies of these three calcium channel blocking agents on left ventricular systolic and diastolic function during myocardial ischemia are different when compared with their relative vasodilator potencies. These differences may have important clinical implications.

Phase I study of the adoptive immunotherapy of human cancer with lectin activated autologous mononuclear cells.

In previous in vitro studies, the authors showed that phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes (PBL) from cancer patients to generate cells that were lytic for fresh autologous tumor but not for lymphocytes or lymphoblasts. Thus, after IRB approval, a phase I clinical protocol was instituted in cancer patients who had failed all other therapy to determine the toxicity and effects, in vivo, of the infusion of large numbers of such PHA activated autologous PBL. Ten patients were treated on the protocol, six with sarcoma, one with melanoma, and three with colorectal cancer. Up to a total of 1.7 X 10(11) PBL were obtained from 7 to 15 successive leukaphereses, the cells from each leukapheresis being incubated in vitro in medium containing PHA and human AB serum for 2 days and then reinfused following the next leukapheresis 2 days later. Toxicity encountered included fever and chills in 10/10 patients, headaches in 5/10, nausea and vomiting in 3/10, and requirement for erythrocyte transfusion in 8/10. No evidence for autoimmune disease, abnormal serum chemical or coagulation studies, or pulmonary emboli was found. 111Indium trafficing studies showed distribution of infused cells mainly to the spleen and liver, with some accumulation in the lungs and tumor especially after repeated infusions. In 9/10 patients, activated PBL were detected in the peripheral circulation by the sixth leukapheresis. Evidence for this was found by assaying the incorporation of tritiated thymidine (3H-Tdr) into, and lysis of fresh tumor cells by, unstimulated PBL from successive leukaphereses. No tumor regression was seen in these patients with bulk disease. These studies demonstrated that large numbers of PHA-activated PBL can be safely obtained and infused into humans, achieving an increase in the number of circulating activated cells with evidence of migration of cells to tumor, lungs, liver and spleen. Further studies of the use of activated lymphocyte infusion in conjunction with chemotherapy in humans are in progress.

Metabolism of human apolipoproteins A-I and A-II: compartmental models.

The metabolism of radioiodinated apolipoproteins (apo) A-I and A-II have been examined using the techniques of compartmental modeling. The model for apoA-I contains two plasma compartments decaying at different rates. One component of apoA-I has a residence time of 3.8 days and the second has a residence time of 6.1 days. In contrast, the apoA-II model has only one plasma component, with a residence time of 5.5 days, which decays through two distinct pathways. Twenty-seven percent of apoA-II decays through a pathway that takes 1.1 days longer to reach the urine than the remaining 73% which decays through the more direct path. These differences in the metabolism exist in both male and female populations. Comparison of fasting and nonfasting concentrations of apoA-I revealed that apoA-I concentration was elevated 0.5 standard deviations in the nonfasting samples while there was no significant difference in the apoA-II concentrations. The fasting apoA-I concentrations were found to be less stable over the study period when compared to fasting apoA-II concentrations. These findings are interpreted as indicating that apoA-I and apoA-II each have a separate metabolism which overlaps when they are present on the same lipoprotein particle. Furthermore, these findings are consistent with the concept that apoA-I metabolism is influenced more by perturbations such as dietary modulation.