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Biomedical subjects

R K Root

Publications and source records attributed to R K Root.

At least 37 records · Page 2Linked to original sources
PubMed1977

A randomized clinical trial of granulocyte transfusions for infection in acute leukemia.

In a prospective, controlled, randomized study to evaluate the efficacy of filtration-leukapheresis granulocytes in granulocytopenic, febrile patients with leukemia, 19 patients received antibiotics alone, and 12 received antibiotics plus daily granulocyte transfusions from ABO-matched donors. In skin-chamber studies the granulocytes appeared at sites of inflammation for at least six hours after transfusion. Infected subjects survived longer if they received granulocytes. Differences between control and transfused patients were greatest in patients with persistent bone-marrow failure, the 21-day survival being 20 per cent in controls, and 75 per cent in transfused patients. Granulocytes appeared to have no effect on the outcome of febrile episodes in which infection was not documented, the 21-day survival being 79 per cent for controls and 88 per cent for transfused patients. The transfusion of granulocytes thus appears to offer a survival advantage to infected, persistently granulocytopenic patients.

Acute Disease↗
PubMed1977

Structural analysis of human neutrophil migration. Centriole, microtubule, and microfilament orientation and function during chemotaxis.

Orientation of nucleus, centriole, microtubules, and microfilaments within human neutrophils in a gradient of chemoattractant (5 percent Escherichia coli endotoxin-activated serum) was evaluated by electron microscopy. Purified neutropils (hypaque-Ficoll) were placed in the upper compartment of chemotactic chambers. Use of small pore (0.45 mum) micropore filters permitted pseudopod penetration, but impeded migration. Under conditions of chemotaxis with activated serum beneath the filter, the neutrophil population oriented at the filter surface with nuclei located away from the stimulus, centrioles and associated radial array of microtubules beneath the nuclei, and microfilament-rich pseudopods penetrating the filter pores. Reversal of the direction of the gradient of the stimulus (activated serum above cells) resulted in a reorientation of internal structure which preceded pseudopod formation toward the activated serum and migration off the filter. Coordinated orientation of the entire neutrophil population did not occur in buffer (random migration) or in a uniform concentration of activated serum (activated random migration). Conditions of activated random migration resulted in increased numbers of cells with locomotory morphology, i.e. cellular asymmetry with linear alignment of nucleus, centriole, microtubule array, and pseudopods. Thus, activated serum increased the number of neutrophils exhibiting locomotory morphology, and a gradient of activated serum induced the alignment of neutrophils such that this locomotory morphology was uniform in the observed neutrophil populayion. In related studies, cytochalasin B and colchicines were used to explore the role of microfilaments and microtubules in the neutrophil orientation and migration response to activated serum. Cytochalasin B (3.0 mug/ml) prevented migration and decreased the microfilaments seen, but allowed normal orientation of neutrophil structures. In an activated serum gradient, colchicines, but not lumicolchicine, decreased the orientation of nuclei and centrioles, and caused a decrease in centriole-associated microtubules in concentrations as low as 10(-8) to 10(-7) M. These colchicines effects were associated with the rounding of cells and impairment of pseudopod formation. The impaired pseudopod formation was characterized by an inability to form pseudopods in the absence of a solid substrate, a formation of narrow pseudopods within a substrate, and a defect in pseudopod orientation in an activated serum gradient. Functional studies of migration showed that colchicines, but not lumicolchicine, minimally decreased activated random migration and markedly inhibited directed migration, but had not effect on random migration. These studies show that, although functioning microfilaments are probably necessary for neutrophil migration, intact microtubules are essential for normal pseudopod formation and orientation, and maximal unidirectional migration during chemotaxis.

Blood↗
PubMed1977

Granulocyte function in patients with chronic renal failure: surface adherence, phagocytosis, and bactericidal activity in vitro.

The defects in host defense mechanisms that explain the enhanced susceptibility to infection of patients with chronic renal failure are not understood, and previous studies concerning function of polymorphonuclear leukocytes (PMNL), the major antibacterial defense, conflict. Therefore, the antimicrobial functions of PMNL obtained from chronically uremic patients and serum factors essential for PMNL activity were evaluated. The potential modifying effect of high concentrations of serum from uremic patients on PMNL of uremic patients was determined, and granulocyte adherence, an activity related to recruitment of cells to inflammatory sites, was measured. Phagocytosis of 14C-labeled Staphylococcus aureus and bactericidal activity were normal and unaffected by high concentrations of uremic serum. Serum from uremic patients opsonized staphylococci and yeast normally. Oxidative metabolism of PMNL [14C-1]glucose oxidation, O2 consumption, and quantitative protein iodination) was normal, as was PMNL adherence. If the uremic patient has an increased risk of infection, this risk cannot be ascribed to defects of PMNL responses that have been studied in these patients.

Adult↗
PubMed1977

H2O2 release from human granulocytes during phagocytosis. Relationship to superoxide anion formation and cellular catabolism of H2O2: studies with normal and cytochalasin B-treated cells.

Normal and cytochalasin B-treated human granulocytes have been studied to determine some of the interrelationships between phagocytosis-induced respiration and superoxide and hydrogen peroxide formation and release into the extracellular medium by intact cells. By using the scopoletin fluorescent assay to continuously monitor extracellular hydrogen peroxide concentrations during contact of cells with opsonized staphylococci, it was demonstrated that the superoxide scavengers ferricytochrome c and nitroblue tetrazolium significantly reduced the amount of H(2)O(2) released with time from normal cells but did not abolish it. This inhibitory effect was reversed by the simultaneous addition of superoxide dismutase (SOD), whereas the addition of SOD alone increased the amount of detectable H(2)O(2) in the medium. The addition of sodium azide markedly inhibited myeloperoxidase-H(2)O(2)-dependent protein iodination and more than doubled H(2)O(2) release, including the residual amount remaining after exposure of the cells to ferricytochrome c, suggesting its origin from an intracellular pool shared by several pathways for H(2)O(2) catabolism. When cells were pretreated with cytochalasin B and opsonized bacteria added, reduced oxygen consumption was observed, but this was in parallel to a reduction in specific binding of organisms to the cells when compared to normal. Under the influence of inhibited phagosome formation by cytochalasin B, the cells released an increased amount of superoxide and peroxide into the extracellular medium relative to oxygen consumption, and all detectable peroxide release could be inhibited by the addition of ferricytochrome c. Decreased H(2)O(2) production in the presence of this compound could not be ascribed to diminished bacterial binding, decreased oxidase activity, or increased H(2)O(2) catabolism and was reversed by the simultaneous addition of SOD. Furthermore, SOD and ferricytochrome c had similar effects on another H(2)O(2)-dependent reaction, protein iodination, in both normal and cytochalasin B cells. When oxygen consumption, O(2.) (-), and H(2)O(2) release were compared in the presence of azide under identical incubation conditions, the molar relationships for normal cells were 1.00:0.34:0.51 and for cytochalasin B-treated cells 1.00:0.99:0.40, respectively. Nonopsonized, or opsonized but disrupted, bacteria did not stimulate any of these metabolic functions. The results indicate that with normal cells approximately 50% of H(2)O(2) released during phagocytosis is derived directly from O(2.) (-) by dismutation, the remainder appearing from an (intra)cellular source shared by azide-inhibitable heme enzymes. With cytochalasin B treatment the evidence is consistent with the derivation of all H(2)O(2) from an O(2.) (-) precursor which is released from the cell surface. Furthermore, when activated by phagocytic particle binding, the neutrophil O(2.) (-) generating system appears to make more of this compound than can be accounted for by dismutation to H(2)O(2). This establishes conditions for the direct participation of both compounds in the microbicidal and cytocidal activity of these cells.

Azides↗
PubMed1977

Granulocyte transfusion therapy: experimental basis and clinical applications.

Granulocyte replacement therapy for the infected patient with severe neutropenia is effective in improving the short-term and possibly the long-term prognosis. The best methods of procuring and administering granulocytes still are under investigation, and no reliable method for predicting compatibility has been developed. At present granulocyte transfusion therapy is indicated for severely neutropenic patients with documented infection as an adjunct to antibiotic therapy. It should be used only at institutions that offer the necessary professional and technical skills to minimize danger and discomfort to both donor and recipient as well as the expense of the treatment.

Agranulocytosis↗
PubMed1977

Host-defense mechanisms in hidradenitis suppurativa.

Host-defense mechanisms were studied in seven patients with active hidradenitis suppurativa (HS). Granulocyte phagocytic function was measured by ingestion of Staphylococcus aureus labeled with radioactive carbon 14 and intracellular killing was determined by bactericidal pour plate method. Chemotaxis was measured by radioactive counting of sodium chromate Cr 51 granulocytes migrating in modified Boyden chambers. Granulocyte adherence was estimated in vitro by filtering blood samples through nylon fiber columns. Cell-mediated immunity was measured by intradermal delayed hypersensitivity responses to Candida, mumps. streptokinase/streptodornase, and purified protein derivative antigens. No abnormality was demonstrated in any granulocyte or cell-mediated immune function tests. Moreover, all patients had normal immunoglobulin levels and elevated total hemolytic complement. Therefore, we conclude that HS is a localized chronic infection of apocrine glands without a generalized defect in host defense.

Adult↗
PubMed1975

H2O2 release from human granulocytes during phagocytosis. I. Documentation, quantitation, and some regulating factors.

The extinction of fluorescence of scopoletin during its oxidation by horseradish peroxidase (HPO) provides a highly sensitive and specific assay for small quantities of peroxide in solution. With this assay, the release of free H2O2 into the extracellular medium by phagocytizing human granulocytes has been documented and quantitated, and some of the regulating factors have been determined. Under basal conditions granulocytes released less than 0.01 nmol/ml of H2O2 (2.5 X 10-6 polymorphonuclear leukocytes/ml). Upon the addition of phagocyte particles (latex, opsonized yeast, or staphylococci), an abrupt increase in extracellular peroxide concentration was observed (greater than 50-fold above basal levels) after latencies as short as 10 s. Release reflected increased intracellular H2O2 production during phagocytosis in that it paralleled the respiratory burst and was absent when phagocytosis was prevented or when cells from patients with chronic granulomatous disease were utilized. Evidence that scpoletin oxidation occurred predominantly in the extracellular medium was obtained by demonstrating a marked inhibition when HPO was omitted from the reaction mixture or when exogenous catalase was added. Similarly, it was found that exogenous serum also inhibited scopoletin oxidation, apparently because of the presence of competing hydrogen donors. H2O2 formation and release were observed at rates which closely paralleled those of phagocytosis. With O2 consumption as an approximate index of H2O2 formation, the fractions released during maximal rates of particle uptake were calculated as follows: for latex, 15.7%; for staphylococci, 10.3%; and for yeast, 4.9%. It is postulated that release is due to diffusion of free H2O2 from an expanded intracellular pool of this substance that develops during phagocytosis. This poos represents tha net of increased synthesis versus catabolism by various enxymatic pathways for H2O2 disposal within the cells. The close relationship between rates of H2O2 formation and rates of phagocytosis by human granulocytes suggests a role for specialized areas of the cell membrane, involved in particle ingestion, in the trigger mechanism for H2O2 synthesis. The consequences of H2O2 release to other cells or organisms in the immediate environment of phagocytizing granulocytes remain to be determined.

Catalase↗
PubMed1975

Effects of oxygen exposure on it vitro function of pulmonary alveolar macrophages.

Bacterial infection may complicate pulmonary oxygen (O2) toxicity, and animals exposed to high O2 concentrations show depressed in vivo pulmonary bacterial inactivation. Therefore, in vitro studies were undertaken to define the mechanism by which O2 alters pulmonary antibacterial activity. Normal and BCG pretreated rabbits were exposed to 100% O2 for 24, 48, and 72-h periods. Pulmonary alveolar macrophages (PAM) were obtained from the experimental animals and from nonoxygen exposed controls by bronchopulmonary lavage. O2 exposure did not alter cell yield or morphology. PAMs were suspended in 10% serum-buffer, and phagocytosis of (14C)Staphylococcus aureus 502A and (14C)Pseudomonas aeruginosa was measured. Comparison of the precent uptake of the 14C-labeled S. aureus after a 60-min incubation period demonstrated that normal PAMs exposed to O2 for 48 h showed a statistically significant increase in phagocytosis when compared to their controls (43.5 vs. 29.2%). A similar, but smaller increase was seen after 24-h O2 exposures. 48 and 72-h O2 exposures produced no significant changes in phagocytosis in PAMs from BCG-stimulated rabbits. Normal PAMs also showed an increased phagocytosis of Ps. aeruginosa after 48-h oxygen exposure. No impairment of in vitro bactericidal activity against either S. aureus 502A or Ps. aeruginosa could be demonstrated in PAMs from normal rabbits exposed to O2 for 48 h. These results indicate that the in vitrophagocytic and bactericidal capacity of the rabbit PAM is relatively resistant to the toxic effects of oxygen, and that imparied in vivo activity may possibly be mediated by effects other than irreversible metabolic damage to these cells. The mechanism for the observed stimulation of phagocytosis remains to be determined.

Animals↗
PubMed1974

Myeloperoxidase-mediated iodination by granulocytes. Intracellular site of operation and some regulating factors.

The intracellular site of operation of the myeloperoxidase-H(2)O(2)-halide antibacterial system of granulocytes has been determined by utilizing measurements of the fixation of iodide to trichloracetic acid (TCA) precipitates of subcellular fractions, including intact phagocytic vesicles. Na(125)I was added to suspensions of guinea pig granulocytes in Krebs-Ringer phosphate buffer, and they were then permitted to phagocytize different particles. Phagocytic vesicles were formed by allowing cells to ingest a paraffin oil emulsion (POE) and collected by flotation on sucrose after homogenization. Measurement of (125)I bound to TCA precipitates of the different fractions and the homogenates disclosed that the lysosome-rich fraction obtained by centrifugation from control (nonphagocytizing) cells accounted for a mean 93.1% of the total cellular activity. With phagocytosis of POE, TCA-precipitable iodination increased two- to sevenfold, and the lysosomal contribution fell to a mean 36.9% of the total. The appearance of activity within phagocytic vesicles accounted for almost the entire increase seen with phagocytosis (a mean 75.7%), and iodide was bound within these structures with high specific activity. More iodide was taken up by cells than fixed, regardless of iodide concentration, and was distributed widely throughout the cell rather than selectively trapped within the vesicles. The amount of iodide taken up and fixed varied considerably with the phagocytic particle employed. Yeast particles were found to stimulate iodination to a far greater degree than the ingestion of POE or latex. Such observations are consistent with the concept that the ingested particle is a major recipient of the iodination process. Measurements of metabolic activities related to the formation and utilization of peroxide by cells phagocytizing different particles were made and correlated with iodination. The findings suggest that mechanisms must exist within granulocytes to collect or perhaps even synthesize H(2)O(2) within phagocytic vesicles to serve as substrate for myeloperoxidase. The simultaneous stimulation of other metabolic pathways for peroxide disposal and its release into the medium by phagocytizing cells is consistent with the high diffusability of this important bactericidal substance.

Animals↗
PubMed1972

Abnormal bactericidal, metabolic, and lysosomal functions of Chediak-Higashi Syndrome leukocytes.

Phagocytic, antimicrobial, and metabolic functions were studied in leukocytes obtained from three patients with the Chediak-Higashi syndrome (CHS) and compared to normals, individuals, heterozygous for Chediak-Higashi syndrome, and two subjects with chronic granulomatous disease of childhood (CGD). Chediak-Higashi syndrome leukocytes showed normal ingestion of a variety of bacteria, Candida albicans, and polystyrene latex particles. Intracellular destruction was significantly impaired for Staphylococcus aureus, Group D streptococci, and a rough strain of Type II pneumococci over a 2 hr incubation. Killing of Serattia marcescens was consistently delayed at 1 hr whereas that of Escherichia coli and C. albicans appeared normal, unless the incubations were shortened to 20 min. Examination of the rates of killing indicated that the greatest defect occurred in the first 20 min of contact between Chediak-Higashi syndrome cells and bacteria. Separation of Chediak-Higashi syndrome granulocytes from monocytes revealed that the former were most defective in bactericidal activity. After phagocytosis, Chediak-Higashi syndrome granulocytes displayed a normal burst in oxygen consumption and oxidation of glucose-1-(14)C and glucose-6-(14)C and formate-(14)C. Oxidation of glucose-1-(14)C by non-phagocytizing Chediak-Higashi syndrome granulocytes and monocytes averaged 2-3 times normal, whereas glucose-6-(14)C and formate-(14)C oxidation were not significantly increased by resting cells. Iodination of intracellular protein by Chediak-Higashi syndrome leukocytes was significantly increased above normal in both the resting and phagocytizing state. Electron microscopic histochemistry revealed that almost all peroxidase activity was localized to the giant granules in Chediak-Higashi granulocytes, and after bacterial ingestion there was a failure of delivery of peroxidase to many phagosomes. Upon longer incubation more phagosomes acquired peroxidase activity, presumably through a fusion process, although many giant granules remained intact. The contrasting patterns and kinetics of the killing defects and the differing metabolic properties of Chediak-Higashi syndrome and chronic granulomatous disease leukocytes emphasize the pleiomorphic nature of inherited disorders of leukocyte function.

Adolescent↗