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Biomedical subjects

D G Wright

Publications and source records attributed to D G Wright.

At least 73 records · Page 4Linked to original sources
PubMed1990

Studies of cobalamin as a vehicle for the renal excretion of cyanide anion.

To examine the ability of hydroxocobalamin (vitamin B12a, HO-Cbl) to counteract the toxicity of cyanide by promoting the renal excretion of cyanide anion, we studied the pharmacokinetics of HO-Cbl and cyanocobalamin (vitamin B12, CN-Cbl) given in high doses to dogs. After administration of intravenous bolus doses of HO-Cbl and CN-Cbl (5, 10, 25 mg/kg) to female foxhounds, peak plasma concentrations were attained within 1 minute, after which the cobalamins disappeared from plasma in a manner consistent with two-phase distribution and elimination kinetics. While mean t1/2 measurements for the initial distribution phase were similar for HO-Cbl and CN-Cbl, the secondary excretion phase was more prolonged for HO-Cbl than for CN-Cbl (t1/2, 94.75 minutes vs 62.64 minutes, respectively). Pharmacokinetic data permitted the design of a HO-Cbl infusion schedule that resulted in stable plasma concentrations in test animals. When NaCN was then given intravenously to animals preloaded with HO-Cbl (20 micrograms/ml plasma), conversion of HO-Cbl to CN-Cbl in vivo, was documented by both thin-layer and high-performance liquid chromatographic analysis of successive urine samples. Moreover, in dogs preloaded with HO-Cbl (20 micrograms/ml plasma), up to 10% of the cyanide administered as NaCN infusions was recovered in urine as CN-Cbl by 60 minutes, compared with less than 0.3% in dogs not treated with HO-Cbl. In addition, under these conditions of HO-Cbl loading, the minimum doses of NaCN required to produce sublethal signs of cyanide toxicity (hypotension, tachypnea, tachycardia) were found to be increased to levels that in some cases exceeded normally lethal doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Studies of signal transduction in the respiratory burst-associated stimulation of fMet-Leu-Phe-induced tubulin tyrosinolation and phorbol 12-myristate 13-acetate-induced posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils and HL-60 cells.

A specific stimulation of tubulin tyrosinolation in human neutrophils (PMNs) is induced by the synthetic peptide chemoattractant N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), and this stimulation is closely associated with activation of the NADPH oxidase-mediated respiratory burst (Nath, J., and Gallin, J. I. (1983) J. Clin. Invest. 71, 1273-1281). In contrast, along with tubulin tyrosinolation, a distinctly different respiratory burst-associated random posttranslational incorporation of tyrosine into multiple PMN proteins is observed in PMNs stimulated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or sn-1,2-dioctanoylglycerol (DAG). In studies exploring the mechanism(s) of signal transduction for these distinct neutrophil responses, we found that the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation in PMNs and in differentiated HL-60 cells is completely blocked by pertussis toxin, while the PMA-induced random incorporation of tyrosine is not inhibited. We also found that expression of the fMet-Leu-Phe-mediated stimulation of tubulin tyrosinolation in HL-60 cells is correlated with increases in the specific activity of protein kinase C and with the acquisition of respiratory burst activity which occur during induced myeloid maturation of these cells. Furthermore, both the fMet-Leu-Phe-induced stimulation of tubulin tyrosinolation and the PMA or DAG-induced random posttranslational incorporation of tyrosine into multiple proteins in activated neutrophils, were found to be reversibly inhibited (greater than 70%) by the protein kinase inhibitors 1-(5-isoquinolinesulfonyl)piperazine (C-I) and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), in parallel with inhibition of superoxide (O2-) generation. In related studies, we also found that fMet-Leu-Phe-stimulated O2- production is comparably inhibited by C-I and H-7, but in a highly temperature-dependent manner. Inhibition was observed only when C-I or H-7 is added to PMNs at physiologic temperature, i.e. 37 degrees C. Interestingly, inhibition of the PMA-induced O2- generation by C-I or H-7 was not found to be similarly temperature-dependent. Considered together, these findings argue against the suggestion that there is a protein kinase C-independent pathway for activation of the respiratory burst in neutrophils stimulated with N-formyl peptides.

Cell Line↗
PubMed1989

Agranulocytosis associated with "Mexican aspirin" (dipyrone): evidence for an autoimmune mechanism affecting multipotential hematopoietic progenitors.

A case of acute, transient agranulocytosis and thrombocytopenia associated with ingestion of dipyrone is reported. This once widely used analgesic, which is now banned in the United States, was obtained by the patient as "aspirin" while traveling in Mexico. Studies of the effects of this patient's serum on purified CD34+ marrow cells, which were highly enriched for hematopoietic progenitors, showed not only a drug-dependent suppression of the in vitro growth of myeloid progenitors, as has been reported previously, but also a drug-dependent suppression of primitive multipotential progenitors (CFU-Mix) and erythroid progenitors (BFU-E). These findings indicate that autoimmune, antibody-hapten interactions which have been reported to occur in dipyrone- and aminopyrine-induced agranulocytosis are not restricted to the neutrophil lineage.

Adult↗
PubMed1989

Interactions of cytoplasmic granules with microtubules in human neutrophils.

Ultrastructural and functional studies of degranulation responses by human neutrophils have suggested that microtubules (MTs) have a role in the intracellular transport of neutrophil granules. We have found that granule-MT complexes can be isolated from disrupted taxol-treated (1.0 microM) neutrophils, visualized by electron microscopy, and quantified in terms of granules per MT length. After incubation of neutrophils with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), granule-MT complex formation was found to be increased two- to threefold. Enhanced binding of granules to MTs was detectable within 30 s of fMLP stimulation and was dependent on the concentration of fMLP. Incubation of cells with dibutyryl cAMP inhibited this fMLP-stimulated granule-MT complex formation in a dose-responsive fashion. These granule-MT interactions could be reproduced in a cell-free system with neutrophil granules isolated by density gradient centrifugation and MTs polymerized from phosphocellulose-purified tubulin. Furthermore, reconstituted granule-MT interactions were found to be modulated by ATPase inhibitors. Sodium orthovanadate increased granule-MT interactions in a concentration-dependent manner, while AMP-PNP, a nonhydrolyzable ATP analogue, and N-ethylmaleimide decreased or eliminated these interactions. In addition, we found that a MT-activated ATPase could be recovered from intact neutrophil granules by salt extraction, and that extracts enriched in this ATPase contained a polypeptide of between 115 and 120 kD which binds ATP and is immunologically related to kinesin. These studies demonstrate that cytoplasmic granules interact with MTs in human neutrophils in a regulated stimulus-responsive manner, and they suggest that such interactions may involve an MT-based, ATPase-dependent, vesicle translocation system as has been demonstrated in other types of cells.

Adenosine Triphosphatases↗
PubMed1989

Abnormal responses of myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor in human cyclic neutropenia.

Granulocyte-macrophage progenitors (CFU-GM) from four patients with childhood onset cyclic neutropenia demonstrated abnormal in vitro proliferative responses to purified, recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) when examined in detailed dose-response studies. Marrow aspirate specimens were obtained for these studies from cyclic neutropenia patients (both during neutropenic nadirs and during recovery phases of cycles), from leukemia patients in remission who had received myelosuppressive chemotherapy, and from healthy normal volunteers. Nucleated marrow cells were then isolated by density-gradient centrifugation and cryopreserved to permit studies of CFU-GM from patients and controls to be carried out at the same time and in replicate. Maximum clonal growth of CFU-GM from normal subjects and from individuals recovering from drug-induced myelosuppression was elicited by 20-100 pmol/liter rhGM-CSF, and the CSF concentrations that induced half-maximal responses (ED50) were between 1.0 and 3.0 pmol/liter. In contrast, maximum growth of CFU-GM from the cyclic neutropenia patients required greater than or equal to 1.0 nmol/liter rhGM-CSF and ED50's were greater than 30.0 pmol/liter. These abnormalities in the GM-CSF responsive growth of myeloid progenitors were independent of cycle time and were most apparent with the predominantly neutrophilic 7-d CFU-GM. Moreover, differences in the growth of 14-d CFU-GM could be attributed mostly if not entirely to differences in the generation of neutrophilic colonies. These findings indicate that childhood onset cyclic neutropenia is associated with an underlying disturbance in the GM-CSF responsive growth of myeloid progenitors committed to neutrophilic differentiation.

Adolescent↗
PubMed1989

The Armed Forces medical examiner system: a change for the better.

The armed forces of the United States of America maintain bases throughout the United States as well as in many other countries. The current situation with regard to the investigation of the approximately 2,500 non-combat deaths each year as well as other deaths occurring on military reservations in complex, confusing, and inconsistent at best. Under existing federal law, base commanders can order an autopsy on all active-duty personnel as well as on anyone dying within an area of exclusive federal jurisdiction. Base commanders unfamiliar with the law are often unwilling to order autopsies, and bodies may go to their graves leaving serious questions, such as cause and manner of death, unanswered. In an effort to rectify this situation, as well as to provide consistency in death investigations in the United States military, the Office of the Armed Forces Medical Examiner (AFME) is being established. The AFME will have its headquarters at The Armed Force Institute of Pathology (AFIP) in Washington, DC. All deaths coming under its jurisdiction will be reported in a timely manner and, when required, authorization for autopsy will be granted from the AFME. Regional deputy medical examiners will be appointed to perform autopsies and on-scene investigations. All cases and case materials will be reviewed and retained by the office of the AFME at AFIP. The quality of death investigation will be improved within the United States military by adoption of this system.

Coroners and Medical Examiners↗
PubMed1989

Investigation of incidents of terrorism involving commercial aircraft.

Deaths resulting from terrorism involving aircraft have occurred incident to hijackings as well as bombings. Passengers or groups of passengers have been chosen by terrorists as the recipients of violence based on citizenship, religion, and political beliefs. They have usually been segregated from other passengers and subsequently mistreated and/or murdered. Thorough documentation of the injuries of victims is essential to the investigation of such atrocities; a medicolegal autopsy correlated with a scene investigation is of paramount importance. Aircraft bombings can create extremely sensitive political situations and public demands for quick resolution. The autopsy of victims in such circumstances, if properly conducted, can yield invaluable trace evidence leading to the identification of the explosive device. The examination of any surviving victims as well as the aircraft is also critical in reconstructing the event. Deaths occurring as the result of in-flight aircraft bombings can produce injuries by five different mechanisms, viz. blast, shrapnel, decompression, impact with the aircraft, and ground impact.

Aircraft↗
PubMed1988

Stimulated production of urokinase and plasminogen activator inhibitor-2 by the human promyelocytic leukemia cell line HL-60.

The effects of maturation inducing agents on the production of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) by the human promyelocytic leukemia cell line HL-60 were examined. PA activity, which was calibrated with a urokinase standard, was 3-6 mU/10(6) cells when measured in supernatants from control cells. This activity increased at least two-fold after dimethylformamide (DMF) or retinoic acid (RA) was added to cell cultures, and as much as ten to thirty-fold when cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (PMA), an agent that induces monocytoid differentiation in HL-60 cells. The PA activity produced by control and induced cells had the same molecular weight as urokinase (UK), and was completely inhibited by antibodies to UK. Cells that were induced with PMA but not with RA or DMF also produced an inhibitor to UK that was identified as PAI-2, the plasminogen activator inhibitor that is produced by monocytes. Because of its dual capacity to produce both UK and PAI, the HL-60 cell line represents a useful model for studies of the fibrinolytic mediators that are generated and released by leukemia cells.

Cell Adhesion↗
PubMed1988

ATP induces transient elevations of [Ca2+]i in human neutrophils and primes these cells for enhanced O2- generation.

ATP, when added to human polymorphonuclear neutrophils (PMNs) at concentrations similar to those attained extracellularly at sites of platelet thrombus formation (0.1 to 20 microM), causes an enhancement of N-formyl(methionyl)leucylphenylalanine (FMLP)-stimulated superoxide anion (O2-) generation. However, ATP by itself is an ineffective agonist for O2- generation by PMNs. The ATP-induced enhancement of O2- generation is associated with a shortened lag time in the response of PMNs to FMLP without a change in the median effective dose for FMLP, suggesting that signal transduction, rather than altered receptor affinity, is responsible for the enhanced oxidative response. Maximum enhancement of O2- generation is detected as early as 15 seconds and is maintained for at least 10 minutes. Of various nucleotides and nonhydrolyzable-ATP analogs test d, only ATP, UTP, and ITP were found to cause enhanced O2- generation by PMNs. Addition of ATP to quin2-loaded PMNs, in the absence of other stimuli, elicits a dramatic rise in [Ca2+]i which reaches a maximum of 500 to 800 nM at 30 seconds and slowly returns to baseline over 5 minutes. This ATP-induced rise in intracellular free Ca2+ concentration is correlated with the enhancement of FMLP-stimulated O2- generation both with respect to dose and nucleotide specificity. Stimulated Ca2+ uptake, rather than mobilization of intracellular Ca2+ stores, appears to be primarily responsible for the rise in intracellular free Ca2+ concentration. These studies indicate that an ATP-induced rise in intracellular free Ca2+ concentration, although insufficient by itself to elicit O2- generation by PMNs, is associated with a priming of PMNs for enhanced O2- generation when stimulated by other agonists.

Adenosine Triphosphate↗
PubMed1988

Essential fatty acid deficiency and neutrophil function: studies of lipid-free total parenteral nutrition in monkeys.

Linoleic acid (18:2 omega 6), the obligate precursor of arachidonic acid (20:4 omega 6), is an essential nutrient for humans and other mammals. Because arachidonic acid release and metabolism are components of neutrophil activation by certain stimuli, we questioned whether neutrophils depleted of arachidonate as a result of essential fatty acid deficiency might be functionally impaired. We examined this possibility by producing essential fatty acid deficiency in monkeys with lipid-free total parenteral nutrition (TPN). Rhesus and African green monkeys were given calorically equal TPN for up to 23 days with and without a vegetable fat emulsion rich in linoleic acid. Fatty acids were analyzed in total lipid extracts of serum and isolated blood neutrophils by gas-liquid chromatography. Although fatty acids in the serum and neutrophils of monkeys given TPN with lipid did not change, linoleic acid levels decreased by at least 60% in serum and 50% in neutrophils from animals given TPN with no lipid. Moreover, arachidonate levels in neutrophil lipids decreased by at least 50% within 12 days of lipid-free TPN, and the abnormal fatty acid 20:3 omega 9 (characteristic of essential fatty acid deficiency) appeared and steadily increased with time. These biochemical signs of omega 6 fatty acid deficiency were associated with impaired neutrophil function in vitro. Both migration responses and superoxide generation stimulated by N-formyl-methionyl-leucyl-phenylalanine were significantly decreased by 12 days of lipid-free TPN, as was the capacity of activated cells to synthesize leukotriene B4. In contrast, functional responses of fatty acid-deficient neutrophils to leukotriene B4 and phorbol myristate acetate, which have little or no effect on arachidonate release or metabolism, were not significantly altered. These findings indicate that endogenous supplies of arachidonic acid and other essential omega 6 fatty acids influence the functional responsiveness of neutrophils. These studies also indicate that altered neutrophil function is a feature of essential fatty acid deficiency and that it may contribute to the increased risk of infection and decreased inflammatory responses observed in this condition.

Animals↗
PubMed1988

An analysis of the multilineage production of human hematopoietic progenitors in long-term bone marrow culture: evidence that reactive oxygen intermediates derived from mature phagocytic cells have a role in limiting progenitor cell self-renewal.

To better understand the limited hematopoietic life span of human marrow "Dexter" cultures, we developed a miniaturized, two-stage culture system with which in vitro production of hematopoietic progenitors could be reproducibly detected and quantified. Light-density, gradient-separated human marrow cells were inoculated into Leighton slide tubes, and adherent ("stromal") cell layers were allowed to develop on the removable coverslips within these tubes during an initial 4 weeks of culture. Once stromal cell layers were established, cultures were irradiated (800 cGy) to eliminate all residual hematopoietic progenitors. The cultures were then recharged with autologous, cryopreserved marrow cells (enriched for BFU-E and CFU-GM) to reconstitute stem cell populations and to initiate in vitro hematopoiesis. Most progenitor cells added to irradiated cultures were no longer detectable by clonal assays within one to four days after recharge. Nonetheless, stable populations of adherent BFU-E and CFU-GM became established in these cultures within 24 to 48 hours, and when the total numbers of progenitors (adherent and nonadherent) were measured at weekly intervals thereafter, it was evident that both BFU-E and CFU-GM were generated in vitro. However, progenitor cell production declined as neutrophils and macrophages accumulated in the cultures. Moreover, with this accumulation of mature myeloid cells, increasing levels of O2- and H2O2 could be detected in the cultures, and it was found that the addition of oxidant scavengers (catalase and mannitol) to culture media enhanced the weekly expansions of progenitor cell numbers that could be measured. These findings support the conclusion that reactive O2 intermediates generated by mature myeloid cells have a role in limiting the duration and extent of hematopoietic progenitor cell self-renewal in long-term "Dexter" cultures of human marrow.

Bone Marrow Cells↗
PubMed1987

A role for guanine ribonucleotides in the regulation of myeloid cell maturation.

We have shown previously that induced maturation of the human myeloid leukemia cell line, HL-60, is associated with a selective down-regulation of guanine ribonucleotide synthesis and depletion of intracellular guanosine triphosphate (GTP) and guanosine diphosphate (GDP) pools. We showed, furthermore, that inhibitors of the enzyme, inosine monophosphate (IMP) dehydrogenase, which catalyzes the initial rate-limiting step of guanylate synthesis from the central intermediate IMP, are potent inducers of myeloid maturation in these cells. We now show that induced maturation of HL-60 cells is prevented or impaired if intracellular concentrations of guanine ribonucleotides are maintained at high levels. HL-60 cells can utilize exogenous guanine and guanosine to maintain GTP and GDP pools through a salvage pathway that bypasses guanylate synthesis from IMP. Moreover, incubation of HL-60 cells with guanosine or guanine (10(-6) to 10(-4) mol/L) prevents both the depletion of intracellular guanine ribonucleotides and the induction of myeloid maturation caused by the IMP dehydrogenase inhibitor, tiazofurin. These findings provide strong additional support for the concept that terminal myeloid differentiation is influenced by a guanine ribonucleotide-dependent regulatory system.

Cell Differentiation↗
PubMed1987

Inosine monophosphate dehydrogenase and myeloid cell maturation.

In previous studies of purine ribonucleotide metabolism in the human myeloid leukemia cell line HL-60, we observed that there is a down-regulation of guanine ribonucleotide biosynthesis from the central intermediate, inosine monophosphate (IMP) and a depletion of intracellular guanosine triphosphate (GTP) and guanosine diphosphate (GDP) pools that occur during the induced maturation of these cells. We also found that inhibitors of IMP dehydrogenase, the enzyme that catalyzes the first step of guanylate synthesis from IMP, are potent inducers of HL-60 maturation. Because of these observations we specifically investigated the activity of IMP dehydrogenase in HL-60 cells and in a new inducible human myeloid leukemia cell line, RDFD2-25, both during maintenance culture and during induced maturation of the cells. Enzyme activity was examined directly in cell extracts with a radiometric assay that measures free 3H2O formed from [2-3H] IMP during the conversion of IMP to XMP. Uninduced HL-60 and RDFD2 cells in maintenance culture were found to have high levels of IMPD activity (5.2 to 5.7 pmol IMP metabolized/10(7) cells/min) compared with normal neutrophils and monocytes that had been purified from blood (less than 1.5 pmol IMP metabolized/10(7) cells/min). However, when HL-60 and RDFD2-25 cells were induced to mature with retinoic acid (10(-6) mol/L), dimethylformamide (6 X 10(-2) mol/L), or a known IMPD inhibitor, tiazofurin (10(-6) mol/L), IMPD activity in the cells fell by 51% to 80% within three to six hours. These changes in IMPD activity preceded detectable functional and antigenic maturation of the cells by at least 12 hours and were not temporally related to changes in cellular proliferation. These findings are consistent with the concept that the regulation of myeloid cell maturation may be influenced by intracellular concentrations of guanine ribonucleotides because IMP dehydrogenase activity is known to be rate limiting for the production of these nucleotides.

Cell Division↗
PubMed1986

Assessing the delivery of neutrophils to tissues in neutropenia.

Studies of neutrophil kinetics in neutropenic individuals, as well as clinical observations of variability in the occurrence of infection among patients with neutropenia, have suggested that blood neutrophil counts may not uniformly reflect the effective delivery of neutrophils to extravascular tissues where the cells perform their principal host defense functions. To evaluate this possibility we developed a sensitive, reproducible method of measuring the extravascular delivery of neutrophils to a normal mucosal site of neutrophil turnover. This method is based upon the quantification of neutrophils recoverable from saline mouth wash specimens. Twenty-five mL specimens, obtained in a controlled manner from neutropenic patients and normal subjects, were centrifuged and the sediments resuspended in 1.0 mL Hank's buffer with 2 micrograms acridine orange, incubated at 37 degrees C for 15 minutes, and then examined in a hemocytometer chamber by fluorescence microscopy. Neutrophils could be clearly distinguished by their characteristic fluorescence and were counted. With this method as few as 1,500 neutrophils were detected reliably in mouth wash specimens. Mucosal neutrophil counts varied less than 10% with repeated sampling of individual subjects over 5-day periods and were consistently greater than 1.3 X 10(5)/specimen in non-neutropenic individuals. Although profound neutropenia was generally reflected by lower than normal oral mucosal neutrophil counts, these counts were significantly higher in individuals with chronic severe neutropenia (blood neutrophils less than 300/mm3) than in patients with acute neutropenia of comparable severity that had developed following chemotherapy. Also, in individuals recovering from profound neutropenia, neutrophils usually reappeared earlier in mouth wash specimens than in blood, and oral mucosal neutrophil counts attained recovery levels more rapidly than did blood counts. This phenomenon was particularly evident in an individual with cyclic neutropenia. Moreover, mucosal neutrophils could occasionally be detected in profoundly neutropenic patients when neutrophils were not present in blood samples. These findings indicate that mucosal neutrophil counts in individuals with neutropenia provide information about the delivery of neutrophils to tissues that may not be apparent from blood neutrophil counts alone.

Acute Disease↗