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Biomedical subjects

D J Warren

Publications and source records attributed to D J Warren.

At least 37 records · Page 2Linked to original sources
PubMed1994

Evaluation of methotrexate tissue exposure by in situ microdialysis in a rat model.

The feasibility of using a microdialysis technique to obtain pharmacokinetic data on tissue exposure to methotrexate (MTX) was investigated. Microdialysis probes were implanted in the jugular vein, femoral muscle, and liver of anesthetized male Wistar rats. MTX (100 mg/kg) was given as a bolus injection through an indwelling venous catheter, and blood samples were obtained through a second venous access and by microdialysis for a total of 6 h. Heparinized plasma, ultrafiltered plasma, and microdialysis effluent from tissue and venous probes were analyzed by high-performance liquid chromatography. Centrifugal ultrafiltration of rat plasma spiked in vitro with MTX (1-100 microM) revealed a mean binding to plasma proteins of 21%. In vitro microdialysis of this spiked plasma resulted in 23% relative recovery of the unbound fraction. In rats receiving MTX, plasma protein binding was 23% and the relative drug recovery as assessed with venous microdialysis probes was 18%. Plotting of unbound (i.e., ultrafiltrate) MTX concentrations in the blood against venous microdialysis perfusate values in the blood gave a good linear correlation with a coefficient of correlation (r2) of 0.98. There was also a linear correlation between the total MTX concentrations in venous blood and the drug levels in microdialysis samples from muscle and liver (r2 = 0.93 and 0.74, respectively). Area under the curve estimations were consistent with an MTX exposure of 30% and 46% for the muscle and liver as compared with the circulation. The present study demonstrates that the microdialysis technique can provide reproducible data on tissue exposure to MTX in an animal model and indicates that the methodology is adaptable to clinical settings.

Animals↗
PubMed1993

A high-performance liquid chromatographic method for the determination of 6-thioguanine residues in DNA using precolumn derivatization and fluorescence detection.

An HPLC method is described for the determination of 6-thioguanine (6-TG) residues in DNA. The assay is based on the release of 2'-deoxy-6-thioguanosine 5'-monophosphate (S6dGMP) by P1-nuclease digestion and its derivatization with the thiol-reactive fluorophore monobromobimane (mBBr). Following treatment with alkaline phosphatase, the resultant 2'-deoxy-6-thioguanosine-mBBr adduct is resolved by isocratic elution from a C18 reversed-phase support and quantified fluorometrically. The chromatographic procedure provides good adduct resolution without interference from reagent peaks or endogenous components present in the DNA. Recoveries of S6dGMP following DNA digestion were quantitative and the assay displayed a linear response from 18 pmol 6-TG bases/microgram DNA down to the lowest concentration tested (0.56 pmol 6-TG bases/microgram DNA). Within-run coefficients of variation were 2.6 and 3.1% for samples containing 18 and 0.9 pmol 6-TG bases/microgram DNA, respectively. Between-day coefficients of variation were 3.1% at 18 pmol and 4.4% at 0.9 pmol 6-TG bases/microgram DNA. In the standard procedure, derivatized sample corresponding to 5 micrograms of DNA (approximately 5 x 10(5) cells) was injected per analysis. This allowed the quantification of < 2.8 pmol adduct and permitted an assessment of 6-TG base incorporation into the DNA of cells exposed to 6-mercaptopurine concentrations as low as 30 nM. This method may be useful in clarifying the relationship between drug metabolite uptake into DNA and the anticancer effect mediated by 6-thiopurines. In addition it may form the basis of improved methods for clinical monitoring during pharmacotherapy with these agents.

Chromatography, High Pressure Liquid↗
PubMed1993

A sensitive high-performance liquid chromatographic method for the determination of 6-mercaptopurine in plasma using precolumn derivatization and fluorescence detection.

A sensitive high-performance liquid chromatographic (HPLC) method for measuring plasma concentrations of 6-mercaptopurine (6-MP) is described. After protein precipitation with 5-sulfosalicylic acid, samples are subjected to precolumn derivatization using the thiol-reactive fluorophore monobromobimane (mBrB). The drug-mBrB adduct is then resolved by isocratic elution from a C18 reversed-phase support and quantified by fluorescence detection. Recovery of 6-MP after protein precipitation was consistently > 85% and the drug-mBrB adduct was found to be stable for at least 2 weeks at room temperature. With plasma samples containing 30 nM 6-MP, the assay displayed within-run (n = 6) and between-day (n = 6) coefficients of variation of 2.2 and 10.6%, respectively. The limit of detection for 6-MP in plasma was 3 nM (500 pg/ml) and the standard curve was linear up to 3 microM. Using this method, we have observed that 6-MP is stable in heparinized whole blood for at least 24 h provided samples are maintained on ice. Since this method requires few manipulations during sample preparation and is readily adaptable to automated techniques, it may prove useful in the routine clinical laboratory setting.

Bridged Bicyclo Compounds↗
PubMed1993

A sensitive and simple high-performance liquid chromatographic method for the determination of mitoxantrone in plasma.

A sensitive high-performance liquid chromatographic (HPLC) method for measuring the anthracene derivative, mitoxantrone, in plasma is described. After protein precipitation with 5-sulfosalicylic acid, samples are resolved by isocratic elution from a C18 reverse phase support and quantified by ultraviolet (UV) detection. Recovery of mitoxantrone after deproteinization was > 70%. Within-run and between-day coefficients of variation (CVs) were 5.1 and 13.7%, respectively, at mitoxantrone concentrations of 20 nM (n = 6). The limit of detection was 2.5 nM and the standard curve linear up to 1 microM. Stability studies have shown that mitoxantrone is stable in spiked whole blood for 3-6 h, provided the samples are kept on ice. The drug is stable in plasma and deproteinized plasma samples for at least 24 h. The method requires few manipulations and is readily adaptable to automation.

Blood Specimen Collection↗
PubMed1993

A sensitive and simple high-performance liquid chromatographic method for the determination of doxorubicin and its metabolites in plasma.

A sensitive high-performance liquid chromatographic (HPLC) method for the measurement of doxorubicin and its metabolites in plasma is described. After precipitation with zinc sulphate and methanol, samples are resolved by isocratic elution from a C18 reverse phase support within 20 min and quantified by endogenous fluorescence. Recoveries over a concentration range from 5 to 1,000 nM of doxorubicin, doxorubicinol, doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinolone were 80-110%, while recovery for 7-deoxydoxorubicinone was approximately 60%. At concentrations of 5 nM, within-run and between-day coefficients of variation for each compound were < 8 and < 16%, respectively. Limits of detection for the compounds were 1-2 nM and standard curves were linear up to at least 1,000 nM. The drug and its metabolites are stable in deproteinized plasma samples at room temperature and in the dark for at least 24 h. The method requires few manipulations and is readily adaptable to automated analysis of large series of samples.

Chromatography, High Pressure Liquid↗
PubMed1993

Effect of nitrous oxide on haematopoiesis in vitro: biochemical and functional features.

Prolonged exposure to nitrous oxide (N2O) can induce dyshaematopoiesis by causing a functional cobalamin-deficiency state through oxidation of methyl-cobalamin (vitamin B12). Herein, we demonstrate that N2O exposure of murine bone marrow cells in vitro results in both functional and biochemical perturbations. In haematopoietic and bone marrow stromal cells, N2O exposure results in a decrease in methionine synthetase activity with a corresponding increase in homocysteine efflux. In semisolid cultures, N2O inhibits colony formation by cells belonging to the colony forming unit in culture (CFU-C) compartment. In contrast, N2O-exposed stromal cell layers maintain the ability to bind and support the differentiation of haematopoietic progenitor cells upon reseeding. Together, these data suggest that the dyshaematopoiesis seen after exposure to N2O is mediated through a direct inhibitory effect on haematopoietic cells.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1993

Induction of a reversible block in murine CFU-C differentiation by exposure to nitrous oxide.

Patients subjected to prolonged exposure to nitrous oxide (N2O) often develop megaloblastic bone marrow changes. This toxicity is due to the N2O-mediated inactivation of cobalamin-dependent enzymes with resultant perturbations in cell metabolism. The effect of N2O on the behavior of murine colony-forming units-cytokine (CFU-C) in vitro was studied by incubating granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cultures for 7 days in an atmosphere of either 5% CO2 in air or 50% N2O/5% CO2 in air. Exposure of bone marrow cells in agarose to N2O resulted in an approximately 50% reduction in colony formation when compared with cultures incubated in air. In contrast, when residual CFU-C numbers were determined in bone marrow liquid cultures after 7 days of incubation in the presence of GM-CSF, exposure to N2O was found to dramatically enhance CFU-C recovery. Since these liquid cultures contain a strong differentiation inducer, and are unable to support CFU-C generation, the enhancement of CFU-C recovery in N2O-exposed cultures appears to be related to its ability to induce a reversible block in CFU-C differentiation. The reversible block in CFU-C maturation seen in vitro parallels clinical observations where a rapid hematologic recovery is seen in N2O-exposed patients treated with hydroxycobalamin. These observations would suggest that N2O is not markedly cytotoxic to CFU-C and that its action is, at least in part, cytostatic in nature.

Animals↗
PubMed1990

Protective effects of tumor necrosis factor on murine hematopoiesis during cycle-specific cytotoxic chemotherapy.

Tumor necrosis factor (TNF) is a pleiotropic cytokine which exerts a wide range of effects when administered in vivo. Using a murine model, we have investigated the effect of pretreatment with 1 microgram (2.6 x 10(4) units) per mouse of recombinant murine TNF-alpha on hematopoietic recovery following administration of cyclophosphamide, 5-fluorouracil, methotrexate, or vinblastine. TNF pretreatment results in enhanced regeneration of circulating neutrophils and hematopoietic progenitors, as measured by in vivo and in vitro assays, in animals given cycle-specific chemotherapeutic agents. The results may suggest that TNF affects cycle kinetics in hematopoietic progenitor cell populations, thus making these cells less prone to the cytocidal effects of the chemotherapeutic agents. As myeloablation is a frequent and often critical side effect following cancer treatment, these findings may have clinical implications.

Animals↗
PubMed1990

Cytokine networks involved in the regulation of haemopoietic stem cell proliferation and differentiation.

Additive and synergistic interactions between haemopoietic growth factors and cytokines can be demonstrated in vitro in clonogenic and suspension cultures of murine and human bone marrow. Purification of early stem cells by combinations of purging with cytotoxic agents (5-fluorouracil, 4-hydroperoxy-cyclophosphamide) and selection with monoclonal antibodies for CD34+, CD33- cells permits analysis of factor interactions at the level of the primitive pluripotential stem cell. Interactions between interleukins, tumour necrosis factor and the colony-stimulating factors can be monitored. In vivo, IL-1 alone, or in combination with G-CSF produces accelerated reconstitution of haemopoiesis after chemotherapy, irradiation and bone marrow transplantation in murine and primate systems. IL-1 elicits cytokine cascades that may have positive or negative actions on lymphohaemopoiesis. Induction of products of the cyclooxygenase and lipooxygenase pathways, as well as tumour necrosis factor and TGF-beta, modulate haemopoiesis.

Animals↗
PubMed1990

Tumor-necrosis factor induces cell cycle arrest in multipotential hematopoietic stem cells: a possible radioprotective mechanism.

Tumor-necrosis factor (TNF) and interleukin-1 (IL-1) have been shown to confer protection of hematopoiesis in mice challenged with radiation. Herein, a series of experiments designed to elucidate the underlying mechanism is presented. After TNF administration, colony-stimulating activity, but no IL-1 activity, was detectable in mouse plasma. In endogenous CFU-S assays, TNF enhanced the survival of multipotential progenitors when administered before, but not after, irradiation. In experiments with fractionated irradiation, the radioprotective effect of TNF was distinctly different from that of IL-1. In vivo and in vitro thymidine suicide assays demonstrated that TNF wholly or partially abolished cell cycling of the CFU-S hematopoietic compartment. These data imply that TNF may inhibit the cell cycle in hematopoietic progenitor cell populations.

Animals↗
PubMed1990

Influence of interleukins 3,5, and 6 on the growth of eosinophil progenitors in highly enriched human bone marrow in the absence of serum.

Purified preparations of natural murine interleukin (IL)-5 and recombinant human IL-1 alpha, IL-3, and IL-6 were evaluated alone, and in combination, for effects in vitro on colony formation by eosinophil progenitors (eosinophil colony-forming units, CFU-Eos) in either nonadherent low-density T-lymphocyte depleted (NALT-) or fluorescence-activated cell sorted NALT-HLA-DR+ CD34 (My10) cells from normal human bone marrow, in the absence and presence of serum. Interleukins were assessed on CFU-Eos plated directly in agar, or on CFU-Eos placed in suspension culture for 7 days prior to plating in agar in the presence of IL-5. Eosinophil colonies were identified in situ by staining with Luxol fast blue. IL-3 and IL-5 acted in agar culture as direct stimulators of CFU-Eos, using highly purified cells in the HLA-DR+ My10 fraction of cells, the specificity for CFU-Eos being greatest for IL-5 and most demonstrable in the absence of serum. The IL-3 and IL-5 direct stimulating effects on CFU-Eos were additive. IL-1 alpha and IL-6 had little or no effect, alone or in combination with IL-3 and IL-5; however, IL-3, IL-5, and IL-6 each enhanced the number of IL-5-responsive CFU-Eos found after suspension culture compared to control medium, with the individual effects being additive when the molecules were combined. These results demonstrate that both IL-3 and IL-5, alone and in combination have direct-acting effects on CFU-Eos, with the greatest specificity for stimulation of pure Eos colonies and clusters residing in IL-5, and that IL-3, IL-5, and IL-6, alone and in combination, may play a role in the survival of CFU-Eos.

Antigens, CD↗
PubMed1989

Modulation of colony-stimulating factor-1 receptors on macrophages by tumor necrosis factor.

The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis.

Animals↗
PubMed1989

Effect of recombinant murine tumor necrosis factor on hemopoietic reconstitution in sublethally irradiated mice.

Intravenous bolus administration of a single 2-micrograms dose of murine rTNF-alpha to BALB/c mice 20 h before sublethal total-body irradiation (7.5 Gy) conferred significant protection against radiation-induced leukopenia. Murine rTNF-alpha administration not only reduced the decline of neutrophil and total blood cell counts after radiation, but also accelerated the subsequent normalization of peripheral blood cell counts. This was accompanied by accelerated regeneration of primitive hematopoietic progenitors, as determined by the in vivo spleen CFU assay, and the in vitro assay of the more mature hematopoietic cell compartment. This demonstrates that pretreatment with murine rTNF-alpha enhances hematopoietic reconstitution after sublethal irradiation, and indicates a possible therapeutic potential for this agent in the treatment of radiation-induced myelo-suppression.

Animals↗
PubMed1989

Radioprotection by murine and human tumor-necrosis factor: dose-dependent effects on hematopoiesis in the mouse.

Tumor-necrosis factor (TNF) has been shown to confer significant radioprotection in murine models. Herein, we demonstrate a dose-dependent enhancement of hematological recovery when single doses of either murine or human recombinant TNF are administered prior to irradiation. In addition to its action upon leukocytes and erythrocytes, TNF also alleviates radiation-induced thrombocytopenia in the mouse. These effects on circulating blood constituents are further reflected in increased numbers of both primitive (CFU-S) and more differentiated (CFU-GM, CFU-Mega) hematopoietic progenitors in TNF-treated animals. This suggests that TNF exerts it radioprotective effects on a pool of primitive multi-potential hematopoietic cells.

Animals↗
PubMed1988

Enhancement of release of granulocyte- and granulocyte-macrophage colony-stimulating factors from phytohemagglutinin-stimulated sorted subsets of human T lymphocytes by recombinant human tumor necrosis factor-alpha. Synergism with recombinant human IFN-gamma.

The influence of purified recombinant human TNF-alpha (rhuTNF-alpha) was assessed, alone and in combination with purified recombinant human IFN-gamma (rhuIFN-gamma), for its effects on enhancing release from human T lymphocytes of activities that stimulate colony formation by granulocyte-macrophage, erythroid, and multipotential progenitor cells. rhuTNF-alpha or rhuIFN-gamma enhanced the release of CSF, which were determined to be granulocyte-CSF and granulocyte-macrophage-CSF by human bone marrow colony assays, morphologic assessment of colony types, and neutralization studies with rabbit anti-human granulocyte-CSF and monoclonal mouse anti-human granulocyte-macrophage-CSF. The CSF were released only when PHA was used, whether or not rhuTNF-alpha and/or rhuIFN-gamma were present while the lymphocytes conditioned the medium. T lymphocytes were sorted into subsets by using three-color immunofluorescence and a dye laser flow cytometry system with cells incubated with biotin anti-Leu-4 labeled with Texas Red, FITC-conjugated anti-Leu-3a, and phycoerythrin-conjugated anti-Leu-2a. Both the Leu-4+3a+2a- and the Leu-4+2a+3a- cells released CSF in response to PHA, but the release of CSF from PHA-stimulated lymphocytes was enhanced by rhuTNF-alpha and rhuIFN-gamma only from the Leu-4+3a+2a- subset of cells. Use of the three-color cell sorting made it highly unlikely that NK cells were involved, because both sorted subsets were positive for Leu-4. rhuTNF-alpha and rhuIFN-gamma synergized to enhance release of CSF such that low concentrations of each molecule, which were inactive when used alone, were active when the two molecules were used together. These studies suggest a role, at least in vitro, for TNF-alpha and IFN-gamma in the release of CSF from subsets of T lymphocytes stimulated with PHA.

Adjuvants, Immunologic↗
PubMed1988

Synergism among interleukin 1, interleukin 3, and interleukin 5 in the production of eosinophils from primitive hemopoietic stem cells.

The in vitro production of eosinophils from committed progenitor cells is influenced by interleukin (IL)-5 (eosinophil differentiation factor) and to a lesser extent by IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). In primary suspension cultures of marrow cells taken from eosinophilic mice, IL-3 induced a modest stimulation of eosinophil production compared to IL-5. In contrast, IL-3 was sevenfold more effective than IL-5 in generating eosinophil progenitors (eosinophil colony-forming units (CFU-eo] from more primitive precursors present in the marrow of normal mice. Pre-incubation of marrow cells in suspension culture with IL-3, but not IL-5, increased the recovery of myeloid precursors responsive to G-CSF, GM-CSF, CSF-1, or IL-3 two- to fourfold while eosinophil progenitor cells responsive to IL-5 were increased by more than 70-fold. Similarly, pre-incubation of bone marrow cells under clonal conditions with IL-3, but not IL-5, resulted in a more than 50 fold increase in CFU-eo responsive to IL-5 over input values. Bone marrow from mice pre-treated with 5-fluorouracil is greatly depleted of progenitor cells directly responsive to IL-3 or IL-5. IL-1 which synergistically interacts with various CSF species to confer a clonogenic response by primitive stem cells present in 5-fluorouracil-treated marrow also failed to stimulate eosinophil production. A marked synergism was observed when IL-1 and IL-3 were combined in the suspension pre-culture phase with a more than sixfold recovery of CFU-eo than induced by either factor alone. Furthermore, pre-culture of 5-fluorouracil-treated marrow cells with a combination of IL-1 and IL-3 resulted in a more than 260-fold increase of CFU-eo over input numbers. These data suggest that the concatenate action of IL-1, IL-3, and IL-5 is an absolute requirement for the in vitro generation of eosinophils from primitive hemopoietic stem cells.

Animals↗
PubMed1988

Correction of canine cyclic hematopoiesis with recombinant human granulocyte colony-stimulating factor.

Canine cyclic hematopoiesis (CH) is an autosomal recessive disease of gray collie dogs that is characterized by neutropenic episodes at 14-day intervals. The biochemical basis for CH is not known but may involve a regulatory defect of the response to or production of a hematopoietic growth factor. Administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to two CH and one normal dog caused a marked leukocytosis (greater than 50,000 WBCs) in all three dogs. The leukocytosis was due largely to a greater than tenfold increase in neutrophils. Less pronounced but significant elevations in monocytes occurred during G-CSF treatment. The elevated WBC count was maintained for more than 20 days in all three dogs, and two predicted neutropenic episodes were prevented in both CH dogs during rhG-CSF treatment. A decline in the WBC count occurred simultaneously in all three dogs during the last five treatment days and was presumably associated with the development of neutralizing antibodies to the heterologous rhG-CSF protein. Bone marrow evaluation indicated that the swings in the myeloid/erythroid progenitor cells that are characteristic of CH were eliminated by rhG-CSF treatment in both CH dogs. These results suggest that the regulatory defect in canine CH can be temporarily alleviated by treatment with rhG-CSF and point to the potential treatment of human cyclic neutropenia with this agent.

Agranulocytosis↗