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Biomedical subjects

W M Miller

Publications and source records attributed to W M Miller.

At least 55 records · Page 3Linked to original sources
PubMed1993

Expansion of primitive human hematopoietic progenitors in a perfusion bioreactor system with IL-3, IL-6, and stem cell factor.

Present methods for long-term hematopoietic culture (LTHC) employ a static culture environment which is not well-characterized. Primitive long-term culture-initiating cell (LTC-IC) numbers have been shown to decline in conventional static human LTHC, even with exogenous cytokine combinations. We have expanded human hematopoietic cells from umbilical cord blood on a preformed marrow stroma with synergistic cytokine combinations in a novel perfusion bioreactor system, which continuously maintained culture conditions within desired ranges. Interleukin-3 (IL-3) and interleukin-6 (IL-6) in perfusion culture resulted in rapid 7-day expansion of granulocyte-macrophage colony forming units (CFU-GM, 11-fold), erythroid burst-forming units (BFU-E, 2.5-fold), and granulocyte-erythroid-macrophage colony forming units (CFU-Mix, 2.4-fold), compared to 6-fold, 1.4-fold, and no expansion, respectively, in static cultures. Addition of stem cell factor (SCF) to IL-3/IL-6 in static culture increased the extent of CFU-GM expansion (to 9-fold), but did not result in BFU-E or CFU-Mix expansion. In perfusion cultures with IL-3/IL-6/SCF, much greater expansions of CFU-GM (18-fold) and CFU-Mix (5.3-fold) were obtained. More importantly, expansion of LTC-IC (nearly 3-fold in two of three experiments) was only obtained with IL-3/IL-6/SCF and perfusion. The ability to expand hematopoietic cells while maintaining or expanding primitive progenitors has potential clinical applications in bone marrow transplantation and gene therapy.

Cell Count↗
PubMed1992

Beneficial effects of reduced oxygen tension and perfusion in long-term hematopoietic cultures.

Growth of hematopoietic stem and progenitor cells found in the MNC fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Reduced oxygen tension increased total cell numbers by as much as 5-fold in cord blood suspension cultures, but this effect was less pronounced in cultures containing an irradiated bone marrow stromal cell layer. However, reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in both types of LTHC studied. Under low oxygen, CFU-C progenitor cell numbers were as much as 10-fold higher. Finally, reduced oxygen tension slowed the rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of human stromal and progenitor cells in vitro. These low oxygen findings were then applied to a murine model LTHC perfusion system. In this system, irradiated 3T3 stromal layer integrity was improved under low oxygen and was substantially further improved with continuous medium perfusion. Cell counts and flow cytometry analysis indicated that the total cell production and the production of immature cells from murine bone marrow MNC on irradiated 3T3 cells were significantly enhanced under low oxygen with perfusion. After three weeks of culture, a 24-fold higher number of Thy1.2lo F4/80- MAC1- cells (indicative of murine stem and progenitor cells) was observed in the perfusion system as compared with static culture under ambient oxygen.

Animals↗
PubMed1992

Modulation of glutathione level in CHO cells. Effects of oxygen concentration and prior exposure to hypoxia.

A microtiter-plate assay has been developed for total intracellular glutathione that facilitates multiple-sample analysis and reduces the amount of time and chemicals required. Sonication time, pH, and storage conditions were identified as key parameters that affect the accuracy of the assay. Using this assay, it was found that CHO cells increase their glutathione level under higher oxygen tension. This adaptive response suggests that a rise in glutathione may be used as an indicator of oxidative stress. Based on this criterion, it was found that hypoxic and anoxic cells are sensitized to reoxygenation. This sensitization could not be attributed to a drop in glutathione during low oxygen exposure because the glutathione content reached a basal level at a PO2 of about 40 torr.

Animals↗
PubMed1992

Effects of synergistic cytokine combinations, low oxygen, and irradiated stroma on the expansion of human cord blood progenitors.

Expansion of hematopoietic progenitor cells in the mononuclear cell (MNC) fraction of human cord blood was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tensions. Cells were cultured with synergistic cytokine combinations in suspension (without stroma) and on irradiated bone marrow stroma. Addition of interleukin (IL)-3 and IL-6 (IL-3/IL-6) provided a greater expansion of both total and progenitor cells than IL-1 and IL-3 (IL-1/IL-3). IL-3/IL-6 maintained a higher level of progenitors throughout the 8-week culture period, whereas progenitors disappeared earlier from cultures with IL-1/IL-3. This indicates that an earlier cell type was affected by IL-3/IL-6, and/or that IL-3/IL-6 favored self-renewal while IL-1/IL-3 induced differentiation. Reduced oxygen tension enhanced the productivity of these long-term hematopoietic cultures (LTHC) under all conditions tested. In suspension cultures, reduced oxygen increased cumulative total cell production by 125% and 167%, and cumulative progenitor production by 68% and 21%, with IL-1/IL-3 and IL-3/IL-6, respectively. The presence of irradiated stroma increased cumulative progenitor cell production almost threefold in cultures without cytokines. In cultures with cytokines, the beneficial effect of stroma was less significant, but was greater under 20% O2 than 5% O2. Cultures under 5% O2 provided more progenitors and often maintained progenitors for 1 to 2 weeks longer than those under 20% O2. To quantitate more precisely the shift in cell populations induced by IL-3/IL-6 and stroma in cultures under 5% O2, flow cytometry analysis was used. By week 3, the addition of IL-3/IL-6 stimulated a 15-fold and 25-fold expansion of promyelocytes (CD15+CD11b-) in suspension and stromal cultures, respectively. Addition of IL-3/IL-6 also increased mature granulocyte (CD15hiCD11b+) and monocyte (CD15loCD11b+) numbers, while no effect was seen on T-(CD3+) or B- (CD19+) lymphocytes. Endogenous production of IL-6 was significantly higher under 5% O2 in both suspension and stromal cultures, and IL-6 production was increased threefold by the addition of IL-1/IL-3. Very little IL-1 beta was produced in these cultures, and endogenous IL-3 and tumor necrosis factor (TNF)-alpha were undetectable by enzyme-linked immunosorbent assay (ELISA) analysis.

Cell Division↗
PubMed1992

UDP-N-acetylhexosamine modulation by glucosamine and uridine in NCI N-417 variant small cell lung cancer cells: 31P nuclear magnetic resonance results.

Small cell lung cancer (SCLC) occurs as two neuroendocrine subtypes, SCLC-C (classic) and SCLC-V (variant). One reported difference is elevated levels of diphosphodiesters (DPDE) in the more differentiated SCLC-C subtype. DPDE have been identified as primarily UDP-N-acetylhexosamines (UDP-NAH) in a variety of tumors, and changes in DPDE levels have been observed during experiments designed to induce cell differentiation. UDP-NAH synthesis is controlled by negative feedback regulation of glutamine:fructose-6-P amidotransferase (EC 2.6.1.16), which can be circumvented by glucosamine. Using 31P nuclear magnetic resonance analysis of extracts and perfused cells, we have identified UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine as the primary metabolites in the DPDE spectral region of SCLC-V N-417 cells. Glucosamine addition causes a rapid increase in UDP-NAH levels. At glucosamine: glucose ratios of 1:1 and 10:1 formation of the UDP-NAH intermediates N-acetylglucosamine 6-phosphate and UDP-N-acetylglucosamine 1-phosphate is also observed, indicating UTP limitation. Subsequent uridine addition results in depletion of the intermediates and increased UDP-NAH formation. Moreover, N-417 cells retain the capacity to rapidly convert uridine to UTP despite low ATP and phosphocreatine levels. This expansion of the uridine pool may represent an additional metabolic reserve not yet addressed in the design of therapy options.

Carcinoma, Small Cell↗
PubMed1992

Reduced oxygen tension increases hematopoiesis in long-term culture of human stem and progenitor cells from cord blood and bone marrow.

Growth of hematopoietic stem and progenitor cells found in the mononuclear cell (MNC) fraction of human cord blood and bone marrow was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Cord blood MNC were grown in suspension and on preestablished irradiated bone marrow stromal layers, whereas bone marrow MNC were used to initiate one-step long-term bone marrow cultures (LTBMC). Reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in all three types of long-term hematopoietic cultures (LTHC) studied. At various time points under low oxygen, progenitor cell numbers were as much as 12-fold, 3-fold, and 4-fold higher for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU-GEMM), respectively. In addition to these numerical increases, progenitor cells were maintained for 1-2 weeks longer under low oxygen conditions. Reduced oxygen tension also increased total cell numbers by as much as fivefold in cord blood suspension cultures, but this effect on total cell numbers was less pronounced in cultures containing a stromal layer. The rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination, was reduced under low oxygen. Finally, the beneficial effect of reduced oxygen was comparable to the effect of an irradiated stromal layer for maintaining cord blood progenitor cells in LTHC. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of both stromal and progenitor cells for a longer period of time in vitro.

Bone Marrow↗
PubMed1991

Lack of induced increase in sister chromatid exchanges in human lymphocytes exposed to in vivo therapeutic ultrasound.

In 1984 Stella et al. reported that each of 10 patients exposed to therapeutic ultrasound had a statistically significant increase in sister chromatid exchanges (SCEs) at mid- and end of therapy; there was a wide range of clinical symptoms (e.g., elbow epicondylitis to knee arthrosis) in this set of patients. In the present study, a set of 4 patients each with some diagnosis recommending therapeutic ultrasound was similarly tested for SCE induction; an additional set of 4 healthy persons underwent sham-therapeutic ultrasound exposures. For both sets of subjects (patients, nonpatients) there was no increase in the frequency of SCEs.

Adult↗
PubMed1991

Effect of wet or dry corn gluten feed on nutrient digestibility and milk yield and composition.

A 22-wk trial was conducted to determine variations in nutrient concentrations of wet and dry corn gluten feed and their effect on nutrient digestibility and milk yield and composition. Holstein cows (n = 48) were blocked at parturition by parity and assigned randomly within block to one of three diets. All cows were fed a control TMR containing corn silage, ground corn, and commercial concentrate plus 2.3 kg/d of alfalfa hay during the 2-wk adjustment period. Thereafter, control diet was fed as wet or dry corn gluten feed substituted for 27% of dietary DM supplied by the control diet. Each cow received TMR for ad libitum consumption. Variations in nutrient concentrations of corn gluten feed were observed throughout the trial. The coefficient of variation was highest for ADIN, and standard deviation was highest for NDF. Intake of CP and NDF and apparent digestibility of DM were greatest when wet or dry corn gluten feed was fed. No differences in DMI, milk yield, and percentages of milk protein, lactose, or SNF were observed, but milk fat percentage was lowest when dry corn gluten feed was fed. Results indicate that corn gluten feed can replace 27% of dietary DM without altering milk yield, but new deliveries should be sampled regularly and amounts fed adjusted to compensate for varying nutrient concentrations.

Animal Feed↗
PubMed1987

Effects of dissolved oxygen concentration on hybridoma growth and metabolism in continuous culture.

Oxygen transport is a major limitation in large-scale mammalian cell culture. The effects of the dissolved oxygen concentration (DO; from 0.1 to 100% saturation with air) on Sp2/0-derived mouse hybridomas were investigated using continuous culture. The steady-state concentration of viable cells increased with decreasing DO until a critical dissolved oxygen concentration of 0.5% of air saturation was reached. The cell concentration declined at lower DO because of incomplete glutamine oxidation, and the specific lactate production from glucose increased to offset the reduced energy production from glutamine. Cell viability increased as the DO was decreased; the viability continued to increase even when the DO was reduced below 0.5%. The specific oxygen uptake rate was essentially constant for DO greater than or equal to 10% of air saturation and then decreased with decreasing DO. The P/O ratio (ATP molecules produced per O atom consumed) appears to change from 2 to 3 between 10 and 0.5% DO. The specific ATP production rate calculated using this assumption decreases only slightly with decreasing DO. The optimum DO of 50% for antibody production is different than the optimum (approximately 0.5% DO) for cell growth.

Adenosine Triphosphate↗
PubMed1987

Radiation hormesis in plants.

Many reports discuss plant growth stimulation by exposure of seeds or plants to low doses of ionizing radiation. The reported effects include increased height, weight, growth rate, flowering and yield. The magnitude of the effect(s) is usually small, being about 10% of control values; and the effects often are not reproducible. None has been independently confirmed. The exposure level reported to induce such effects is about an order of magnitude greater than that reported for similar hormetic responses in animals. There is no understanding of the mechanism(s) of such responses or of the cellular and physical factor(s) pertinent to the induction of such effect(s). However, observations that certain low-level exposures result in increased yield (e.g. more branches, flowers and fruits) are consistent with known mechanisms of cellular damage and known responses of plants to a compromise in apical dominance.

Dose-Response Relationship, Radiation↗
PubMed1987

Species specificity in the metabolism of nabilone. Relationship between toxicity and metabolic routes.

The disposition of 14C-nabilone has been examined in dogs and monkeys; it is rapidly absorbed and extensively metabolized in both species. A metabolic pathway initiated by the stereoselective enzymic reduction of the 9-keto moiety of nabilone was of major importance in the biotransformation of nabilone in the dog but was only a minor pathway in the monkey. The resulting long half-lived carbinol metabolites accounted for 77% of the circulating 14C in dog but for only 19% in monkey, 48 h after drug administration. Accumulation of carbinol metabolites in brain tissue was observed in dogs, with 24 h brain concentrations being five to six times higher than the plasma concentrations. No accumulation of carbinol metabolites, nabilone or of 14C occurred in the brain of monkeys. Accumulation of the carbinol metabolites in brain tissue has been implicated as the causative factor for the CNS toxicity observed in dogs treated chronically with nabilone. Comparison of nabilone pharmacokinetics in dog and humans showed the dog to be unique in producing high levels of carbinol metabolites, so that monkey appeared to be a more appropriate model than dog for pre-clinical toxicological and safety studies. Studies with liver 15,000 g supernatants indicated that the enzymic reduction of nabilone to its carbinol metabolite was a viable metabolic pathway in rat, dog and monkey, so that a distinct species difference exists between dog and monkey in the subsequent metabolism and/or elimination of these carbinol metabolites.

Animals↗
PubMed1983

Immunoblastic lymphoma with extracellular and intravascular immunoglobulin deposits: immunocytochemical and electron microscopic studies.

Immunoblastic lymphoma (IBL) showing features of angioimmunoblastic lymphadenopathy (AIBL) is described. Abundant eosinophilic extracellular deposits were present in lymph nodes. Electron microscopic and immunoperoxidase studies showed this material was composed of degenerating tumor cells and polyclonal immunoglobulins (Mu and Alpha heavy chains and Kappa and Lambda light chains). Numerous arborizing vessels were thickened with monoclonal IgM(K) immunoglobulins. The tumor cell lysis and degeneration are though to be due to the high cell turnover and immune reaction to unrecognized substances.

Antibodies, Monoclonal↗
PubMed1981

Terminal-deoxynucleotidyl-transferase immunofluorescence on bone marrow smears: serial studies on 28 patients.

Terminal-deoxynucleotidyl-transferase (TdT) activity determinations, using the indirect immunofluorescence technique, were performed on 28 consecutive leukemia patients. Fifteen patients with acute lymphoblastic leukemia had 38 bone marrow smears examined. TdT-positive cells were a good indicator of disease activity in acute lymphoblastic leukemia in that numbers were increased at the time of diagnosis, normal during remission, and increased during relapse. Ten patients with acute myeloid leukemias had 42 smears studied. The number of TdT-positive cells did not reflect the activity of the disease, except for two patients with acute myelomonocytic leukemia in which TdT-positive cells were mildly increased during relapse. Three patients with chronic myelogenous leukemia had eight smears studied. The number of positive cells seemed meaningful only in following the course of lymphoid blast crisis. It would therefore appear that TdT determinations seem to be of value in lymphoblastic leukemia, but of limited value in myeloid leukemias.

Adolescent↗
PubMed1981

Absorption and metabolism of anitrazafen, a topically effective anti-inflammatory agent, in the rat.

1. The metabolism and pharmacokinetics of anitrazafen, a topically effective anti-inflammatory agent, have been investigated in the rat after oral, subcutaneous and topical administration. 2. [14C]Anitrazafen is rapidly absorbed from the gastro-intestinal tract and subsequent metabolism is rapid and extensive; biliary excretion is the major route of elimination. After subcutaneous or topical administration, elimination of [14C]anitrazafen was delayed due to a slower rate of systemic absorption. 3. Pharmacokinetic studies confirmed these results. After oral administration peak concn. of parent drug were attained within 1 h; plasma concn. of 14C were an order of magnitude greater than those of unchanged drug. The apparent volume of distribution of anitrazafen was high (112l/kg) consistent with observed tissue 14C concentrations. Subcutaneous administration resulted in delayed absorption but with maximum bioavailability of parent drug. Absorption was slowest following topical application. 4. Anitrazafen was extensively metabolized in rats. No unchanged drug was found in excreta. The most important mechanism of biotransformation was oxidative O-demethylation, with glucuronide or sulphate conjugates of two isomeric mono-O-demethylated and the di-O-demethylated analogues of anitrazafen, as metabolites.

Absorption↗