Biomedical subjects
Publications and source records attributed to A Gee.
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Disseminated neoplasia is a leukemia-like disease that occurs in many species of bivalve molluscs worldwide, including the bay mussel (Mytilus trossulus). The etiology of the disease is undetermined, but an early report proposed that the anomalous bivalve cells were actually an invasive parasite rather than cancerous cells of host origin. Comparison of partial sequences of small subunit rRNA from normal and putative cancer cells was performed to resolve this issue. These studies showed a close phylogenetic relationship of the different forms of cancer cells to each other (similarity coefficient, 0.982), to the normal hemocytes (similarity coefficient, 0.990, 0.992), and to the oyster, Crassostrea virginica (similarity coefficient, 0.895-0.927). A large phylogenetic distance separates all 3 mussel hemocyte types from several representative protists (similarity coefficient, 0.702-0.761). These results indicate that the disseminated neoplastic cells in mussels are indeed proliferative host cells and not unicellular parasites.
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The Nalm-16 cell line was originally described as being of the null acute lymphoblastic leukaemia (ALL) phenotype. Using phenotypic and genotypic markers, we have demonstrated the line carries markers associated with cells of the B lineage. In addition, Nalm-16 binds a series of monoclonal antibodies characterized as predominantly recognising neuroectodermal tissues. Amongst these antibodies, UJ13A, 5.1.H11 and ERIC-1 have been shown to recognise the neural cell adhesion molecule (NCAM). Expression of the NCAM molecule is highly complex, with several isoforms of the protein resulting from the differential splicing of the NCAM mRNA transcript. Western-blot analysis of Nalm-16 cell extracts indicates that cells express the heavily polysialylated form of the molecule found on many embryonic tumours. Neuraminidase digestion indicates that the 140 kD isoform is predominantly expressed on Nalm-16, although the 120 kD isoform is present to a lesser degree. These findings have been confirmed using Northern-blot analysis.
We have developed a multiassay system consisting of fluorescence microscopy, immunocytology and tumor colony assay to monitor the removal of tumor cells from bone marrow. This system was tested in preclinical purging experiments in which neuroblastoma cells were seeded into bovine marrow and purged by treatment with monoclonal antibodies and immunomagnetic beads. Eight experiments were performed on two different neuroblastoma cell lines seeded at 2% and/or 5% contamination. We consistently demonstrated greater than a 3 log removal with one cycle of antibody/bead treatment and greater than a 1 log further reduction by addition of a second cycle. We also demonstrated removal of all detectable tumor stem cells by this purging method. We feel that this system will prove valuable for monitoring ex vivo tumor removal in future clinical studies and should be considered for use in other purging trials.
Two novel cytokines, stem cell factor (SCF) and PIXY321 (a fusion protein, granulocyte macrophage colony-stimulating factor+IL-3), have recently been demonstrated to enhance in vitro adult myelopoiesis. In this study, we compared the success of separating very early hematopoietic progenitor cells (CD34+) from both cord blood (CB) and adult bone marrow (ABM) and their differential response to SCF, PIXY321, and other later-acting colony-stimulating factors (CSF). Briefly, CD34+ cells were isolated from CB and ABM with an anti-CD34 MAb, HPCA-1, and incubated with various combinations of SCF, PIXY321, and other CSF. The percentage of CD34+ cells was decreased in CB compared to ABM before separation (0.54 versus 1.71%) (p = 0.05). Isolated CD34+ cells from CB and ABM were similar in lineage with respect to CD38, HLA-DR, CD33, and CD5, but decreased in CB with respect to B-lineage expression (CD19, CD10, and CD22) (p = 0.05). SCF increased colony forming unit-granulocyte-macrophage (CFU-GM) formation from CB CD34+ cells compared to unconditioned media and had a significant additive increase with IL-3 (p = 0.006) and granulocyte colony-stimulating factor (p = 0.03). SCF also had an additive increase in CB CFU-GM formation with PIXY321 (p = 0.007). PIXY321 had a similar increase in CFU-GM formation from both CB and ABM CD34+ cells compared to the combination granulocyte macrophage colony-stimulating factor + IL-3. When SCF was added to IL-3, PIXY321, or PIXY321 + IL-6, there was an increase in CFU-GM from CB versus ABM CD34+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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Bone marrow harvested from cancer patients for autologous bone marrow reinfusion (ABMR) after myeloablative treatment may be injured, in both its proliferating and stromal cell pools, by either previous treatment or manipulation at the time of harvest. We have examined the relative effects of seven covariates on hematologic recovery after ABMR in children with neuroblastoma (NBL) using univariate and step-up analysis. We measured recovery by times to achieve (1) white blood cell counts greater than 1,000/microL; (2) absolute neutrophil counts greater than 500/microL; and (3) platelet counts greater than 20,000/microL without transfusion. In univariate analysis, recovery was significantly associated with the amount of prior chemotherapy and the interval between last chemotherapy and marrow harvest. Patient sex, the number of granulocyte-macrophage colonies infused, harvest-to-freeze interval, and use of purging were marginally associated. After adjusting for potential confounders in a multivariate model, the amounts of chemotherapy and granulocyte-macrophage colonies infused were independently significant predictors of time to total white blood cell count recovery; chemotherapy courses and chemo-to-harvest interval were predictors of neutrophil count recovery; and sex, use of purging, and harvest-to-freeze interval were marginal predictors of platelet recovery. The speed of hematologic recovery after ABMR seems to depend mainly on pre-existing factors and marginally on manipulation of the marrow after harvest. These factors may affect both proliferating and stromal cell pools.
Cholestyramine was administered to hamsters at 6 doses in the diet for 1 week. Plasma cholesterol, LDL + VLDL cholesterol and HDL cholesterol were measured after this period. Bile acid excretion was measured in faeces collected over the final 24 h of the experiment. A dose-response curve for each parameter measured was constructed using data from individual hamsters. For the bile acid and the cholesterol measurements a maximum response was observed at the highest doses. A correlation between the bile acids excreted over 24 h and the LDL + VLDL cholesterol showed that the maximum effect of cholestyramine on lowering plasma and lipoprotein cholesterol occurred at a submaximal excretion level of bile acids. Comparison of the efficiency of cholestyramine in reducing plasma cholesterol in the hamster with limited data in the dog and in man suggest that a greater lowering of plasma cholesterol is achieved in the dog and in man for an equivalent increase in bile acid excretion caused by the sequestrant. As is already known, cholestyramine treatment caused an increase in hepatic cholesterol 7 alpha-hydroxylase and HMG-CoA reductase activity. Interestingly in this study the novel observation was made that the bile acid sequestrant reduced the activity of hepatic acyl-CoA: cholesterol acyltransferase.
1. The contribution of Li+ reabsorption in the loop of Henle to lithium clearance (CLi) and the possible mechanism(s) involved were assessed in healthy volunteers. Four mechanisms were considered: (a) passive reabsorption in the thin ascending limb, (b) solvent drag in the thin descending limb, (c) the Na+, K+, 2Cl- transporter in the thick ascending limb and (d) paracellular movement in the thick ascending limb. 2. Since alterations in the corticomedullary osmolal concentration gradient produced by fluid restriction (500 ml day-1) and subsequent water loading (15 ml kg-1) did not affect either CLi (28.5 +/- 2.1 vs. 28.2 +/- 1.9 ml min-1) or fractional lithium clearance (FELi; 23.5 +/- 2.0 vs. 23.0 +/- 1.9%), it is unlikely that substantial Li+ reabsorption occurs in the thin limbs by either passive movement or solvent drag. 3. Increasing plasma Li+ with unchanged plasma Na+ in salt-replete volunteers was associated with only small reductions in CLi (32.8 +/- 1.3 ml min-1, P less than 0.05) and FELi (27.3 +/- 1.8 vs. 25.3 +/- 2.0%, P less than 0.05). This suggests that substantial Li+ reabsorption on the Na+, K+, 2Cl- transporter does not occur. 4. Bumetanide increased FELi in salt-depleted (LS) and salt-replete (HS) volunteers and abolished the pre-diuretic difference in FELi between salt intakes (LS, 16.6 +/- 1.5 vs. 38.7 +/- 2.3%, P less than 0.001; HS, 30.1 +/- 1.5 vs. 40.5 +/- 2.0%, P less than 0.001). Changes in CPO4 and CHCO3 were not detected. Acetazolamide produced comparable increases in FELi (LS, 16.6 +/- 1.5 vs. 38.7 +/- 2.2%, P less than 0.001; HS, 30.1 +/- 1.5 vs. 43.1 +/- 2.4%, P less than 0.01); and CPO4 and CHCO3 were increased. When tubular flow to the loop of Henle was increased by acetazolamide, the bumetanide-induced increases in FELi were reduced (LS, 38.7 +/- 2.2 vs. 48.7 +/- 2.3%, P less than 0.001; HS, 43.1 +/- 2.4 vs. 48.1 +/- 2.6%, P less than 0.001). 5. These data are consistent with the view that (a) Li+ is reabsorbed by a bumetanide-sensitive mechanism in the loop of Henle, (b) approximately 20 and 10% of the filtered load, respectively, is reabsorbed in the loop in salt-depleted and salt-replete volunteers, (c) flow-dependent, voltage-driven paracellular movement in the thick ascending limb is the likely mechanism and (d) this mechanism could account for the difference in Li+ reabsorption between low and high salt intakes.
We conducted a pilot protocol at seven Pediatric Oncology Group (POG) institutions to examine the feasibility, toxicity, and efficacy of using a common regimen of high-dose chemoradiotherapy (HD CT/RT) supported by autologous or allogeneic marrow infusions in children with metastatic neuroblastoma (NBL) in first or second remission. During a 57-month period, we accrued 101 patients. We report here results for the 81 who completed treatment at least 2 years ago. The HD CT/RT regimen consisted of melphalan 60 mg/m2/d for three doses, and total body irradiation (TBI) either 1.5 Gy (n = 27) or 2.0 Gy (n = 54) twice daily for six doses. Twenty-three patients also received irradiation consisting of 1.2 Gy twice daily for 10 doses to persisting disease sites. Seventy-four were given autologous and seven allogeneic marrow, 64 autologous marrows being purged immunomagnetically. Fifty-four children were in first complete (CR) or partial (PR) remission and 27 in second CR or PR. As of October 1, 1990, follow-up was from 32 to 72 months. Forty-seven of these 81 children relapsed, 10 died of complications, one of unknown cause, and 23 continue in remission, including 21 of the 54 treated in first remission, and 16 who completed treatment more than 3 years ago. The 2-year actuarial event-free survival (EFS) probabilities are first CR (CR1) 32% (SE 10%), first PR (PR1) 43% (SE 9%), second CR (CR2) 33% (SE 27%), and second PR (PR2) 5% (SE 5%). Probability of EFS correlated with remission number (first better than second, P less than .001), with interval from diagnosis to HD CT/RT (greater than 9 months better than less than 9 months, P = .055), and with TBI dose (12 Gy better than 9 Gy, P = .031). These encouraging results may partly reflect selection for this treatment of patients with NBL who have a slower disease pace.
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Neutral cholesteryl ester hydrolase activity has been described in the cytosolic and microsomal fraction of rat liver, but the relationship of these activities to other parameters of hepatic cholesterol metabolism is not known. We have studied this in the hamster by manipulating the flux of cholesterol across the liver by dietary modifications. A bile acid sequestrant was used to stimulate LDL receptor activity and hence flux of cholesterol into the liver. A cholesterol-rich diet caused a hypercholesterolaemia and substantial uptake of cholesterol and deposition in the liver. Hypercholesterolaemia was also induced by a saturated fat-rich diet, but in contrast this reduced the flux of cholesterol into the liver. Animals were fed these diets for 1 week and then the livers removed and enzyme activities determined. These were 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol 7 alpha-hydroxylase, acyl-CoA: cholesterol acyltransferase, microsomal cholesteryl ester hydrolase and cytosolic cholesteryl ester hydrolase. Feeding the bile acid sequestrant increased the hydrolysis of cholesteryl ester in the liver over its synthesis. In contrast, both fat feeding and cholesterol feeding caused a reduction in the relative rate of hydrolysis of cholesteryl ester compared with synthesis. This was particularly marked with the cholesterol-rich diet. These results show that the hydrolysis of cholesteryl ester in hamster liver responds to dietary manipulation in a way that reflects the needs of the cell for cholesterol or the presence of an excess. It is suggested that a metabolically significant cholesteryl ester cycle may operate in the liver to a greater extent that had previously been thought.
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Many in vitro techniques have been developed for removing cancer cells from the marrow of patients who are to undergo autologous bone marrow transplantation (ABMT). These purging techniques can be classified as immunological or pharmacological. The immunomagnetic technique has been widely used in neuroblastoma patients. It depends on an interaction between target neuroblastoma cells in the marrow and a complex of specific monoclonal antibodies and magnetized microspheres, the target cells being selectively removed by passage through a magnetic field. Laboratory studies with neuroblastoma and acute lymphoblastic leukemia cells have shown the high efficiency of this technique in selectively removing cancer cells while retaining adequate numbers of normal hematopoietic cells for subsequent reinfusion into the patient. Clinical studies in several hundred neuroblastoma patients, as well as small numbers of acute lymphoblastic leukemia, breast cancer, and myeloma patients, suggest that this is a clinically safe and effective technique. However, no clinical trial has been conducted comparing ABMT with and without in vitro marrow purging. Until such time, we will regard immunomagnetic purging as "standard of care" for neuroblastoma patients receiving ABMT.