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Biomedical subjects

B H Gray

Publications and source records attributed to B H Gray.

At least 55 records · Page 3Linked to original sources
PubMed1991

Safety and efficacy of thrombolytic therapy for superior vena cava syndrome.

The experience at the Cleveland Clinic from 1982 to 1990 using thrombolytic therapy for superior vena cava (SVC) syndrome was retrospectively reviewed. Sixteen patients, 11 of whom had indwelling central venous catheters, were treated with either urokinase (n = 11) or streptokinase (n = 5). Either urokinase (4,400 U/kg bolus followed by 4,400 U/kg/h) or streptokinase (250,000 U bolus followed by 100,000 U/h) was used, and venograms were performed before and after. Overall, 56 percent of patients had complete clot lysis and relief of symptoms. Thrombolytic therapy was effective in eight (73 percent) of 11 patients receiving urokinase and one (20 percent) of five patients receiving streptokinase. Of those with a central venous catheter, eight (73 percent) of 11 patients were successfully lysed, whereas only one (20 percent) of five patients was successfully lysed if no catheter was present. If thrombolytic therapy was performed less than or equal to five days of symptom onset, seven (88 percent) of eight patients were successful, if thrombolytic therapy was performed greater than five days after symptom onset, two (25 percent) of eight patients were successful. Symptoms were relieved and the catheter was preserved in patients in whom thrombolytic therapy was effective. Factors predicting success were as follows: (1) the use of urokinase compared with streptokinase; (2) the presence of a central venous catheter; and (3) a duration of symptoms less than or equal to five days.

Catheterization, Central Venous↗
PubMed1990

The neutrophil and chronic allergic inflammation. Immunochemical localization of neutrophil elastase.

To test whether neutrophils infiltrate and degranulate in areas of chronic respiratory allergic inflammation, we developed an indirect immunofluorescence technique to localize neutrophil elastase in formalin-fixed, paraffin-embedded tissues. The affinity-purified antielastase stained only neutrophils on peripheral blood buffy coat smears, and in lung tissue from patients with pneumonia. We examined tissue specimens from four patients with fatal asthma, 10 patients with chronic sinusitis, and 10 patients with nasal polyposis for the presence of elastase, as well as eosinophil granule major basic protein (MBP). Neutrophil infiltration and extracellular elastase deposition in association with damage to respiratory epithelium were generally sparse in most specimens; the exceptions were one patient with asthma, one patient with chronic sinusitis, and two patients with nasal polyposis. In contrast, eosinophil infiltration and extracellular MBP deposition were generally marked in most specimens; the exceptions were one patient with asthma and one patient with nasal polyps where extracellular MBP deposition did not coincide with damage to respiratory epithelium. The results suggest that the neutrophil does not usually infiltrate tissues showing allergic inflammation; however, on occasion, it may participate in these inflammatory reactions.

Adult↗
PubMed1990

Extracellular deposition of eosinophil and neutrophil granule proteins in the IgE-mediated cutaneous late phase reaction.

Intradermal injection of allergens in sensitive subjects produces an IgE-dependent prolonged inflammatory reaction, the late phase reaction (LPR). Histologically, eosinophils are present in the LPR but are not as numerous as neutrophils or mononuclear cells. We determined whether extracellular deposition of eosinophil and neutrophil granule proteins occurs in the LPR by immunofluorescent localization of eosinophil granule major basic protein (MBP), eosinophil-derived neurotoxin (EDN), and neutrophil elastase. Before intradermal challenge, eosinophils and neutrophils were present only in blood vessels, and MBP, EDN, and elastase were localized to cells. At 15 minutes, small amounts of MBP, EDN and elastase were found outside of cells in focal areas. By 1 to 3 hours, MBP, EDN and elastase were extensively deposited throughout the dermis in a granular and diffuse manner; these deposits persisted up to 56 hours. Both actively and passively sensitized subjects showed similar MBP and elastase deposition. Skin sites passively sensitized by sera depleted of IgE showed essentially no MBP or elastase deposition. Electron microscopy showed degenerating eosinophils and free eosinophil granules in the dermis. Mast cell numbers diminished during the LPR when extracellular eosinophil and neutrophil granule protein deposition was maximal. These results demonstrate that striking dermal eosinophil and neutrophil granule protein deposits are prominent features of the cutaneous LPR, are IgE-dependent and precede the maximal clinical expression of the LPR. The possible significance of these findings in the pathophysiology of the LPR is discussed.

Administration, Cutaneous↗
PubMed1988

Preparation and characterization of a monoclonal antibody that inhibits human neutrophil elastase activity.

Neutrophil proteases are believed to play a role in the pathogenesis of a variety of human diseases. While many studies of proteases in models of disease have focused on elastase, neutrophils contain several proteases some of which share a high degree of homology. This report describes the production and characterization of an IgG1 murine monoclonal antibody (AHN-10) that reacts with human neutrophil elastase but not with the other major neutrophil neutral proteases: cathepsin G, proteinase 3, collagenase, or the newly purified neutral protease, esterase N. AHN-10 inhibited the elastinolytic activity of purified human neutrophil elastase and could detect elastase in alcohol-fixed cytospin preparations. The epitope recognized by AHN-10 was resistant to treatment with NaIO4, suggesting that the epitope is not a carbohydrate. AHN-10 should be useful for the immunolocalization of neutrophil elastase in tissue specimens and as a stable source of characterized antibody for quantitative identification of neutrophil elastase.

Animals↗
PubMed1988

Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters.

Studies were designed to explore the possibility that human polymorphonuclear leukocyte granule constituents in addition to elastase (HLE) had the potential to cause emphysema. A two-step purification of three serine proteinases was developed. Granule extract proteins were initially separated by dye-ligand affinity chromatography. Fractions eluted were divided into four pools. Hamsters were given a single intratracheal instillation of saline +/- 0.1 mg protein of each pool. While pool 2 contained HLE and cathepsin G, the most dramatic bullous emphysema developed in animals treated with pool 4. The esterase from pool 4, designated proteinase 3 (PR-3) was purified, characterized in vitro, and tested for its ability to cause emphysema. PR-3 is a neutral serine proteinase with isoenzyme forms. Its ability to degrade elastin at pH 6.5 is slightly greater than that of HLE, but it is less active than HLE at pH 7.4 or 8.9. PR-3 has weak activity against azocasein. Its ability to degrade hemoglobin is intermediate to that of HLE and cathepsin G at pH 7.4. PR-3 has no activity against chromogenic substrates specific for HLE or cathepsin G. Its pI is substantially less than HLE or cathepsin G. It is also immunologically distinct from HLE. It induces emphysema in hamsters commensurate with that of HLE. We conclude that PR-3 may be important in the pathogenesis of human emphysema.

Animals↗
PubMed1987

tri-n-Butyltin: a membrane toxicant.

Increased use of the biocidal compound tri-n-butyltin (TBT) in antifouling paints has prompted research aimed at determining the mechanism for TBT toxicity. Past investigations indicate that the primary cellular target for TBT is the cell membrane. Erythrocyte suspensions treated with TBT concentrations 2 greater than or equal to 5 microM undergo hemolysis described by a sigmoidal kinetic pattern. Transformation of cell shape from discocyte to echinocyte occurs at TBT concentrations greater than or equal to 0.1 microM, indicating that the compound enters the outer membrane bilayer. TBT at concentrations greater than or equal to 10 microM forms electron-dense aggregates that are intercalated within plasma membranes as viewed in ultrathin sections by transmission electron microscopy. Qualitative X-ray microanalysis of these aggregates confirms the presence of tin. The size of these structures can be modified by either 10 mM cyanide or 2,3-dimercaptopropanol (British Anti-Lewisite, BAL). Adding 10 mM cyanide to hemolytic TBT concentrations resulted in a synergistic stimulation of hemolysis attributable to high cyanide anion concentrations in or near the cell membrane. The elevated cyanide anion levels are thought to contribute to membrane lysis. The lipophilic dimercapto compounds BAL, dithiothreitol, and 2,3-dimercaptosuccinate are effective inhibitors of TBT-induced lysis. Water-soluble 2,3-dimercapto-1-propane sulfonate, a BAL analog, was largely ineffective as an inhibitor. The detailed molecular mechanism for TBT-induced membrane lysis is not yet clear. Cellular ATP depletion could be induced by TBT as well as by delipidation of anionic phospholipids or even formation of tributylstannylperoxy radicals, resulting in lipid peroxidation.

Chemical Phenomena↗
PubMed1987

Organotin-induced hemolysis, shape transformation and intramembranous aggregates in human erythrocytes.

Organotin compounds examined in this study exhibited a relative order of potency for induction of in vitro hemolysis in human erythrocytes as follows: tri-n-butyltin greater than tri-n-propyltin greater than tetra-n-butyltin greater than triphenyltin chloride greater than tri-n-ethyltin bromide greater than dibutyltin dichloride greater than stannous chloride greater than tri-n-methyltin chloride = butyltin chloride dihydroxide. All of the organotin compounds induced erythrocyte shape transformation from the normal discocyte to an echinocyte and, in addition, triphenyltin chloride, tetra-n-butyltin and tri-n-ethyltin bromide also elicited stomatocyte formation at higher concentrations. Select organotin compounds also formed tin-containing aggregates within the plasma membrane. The relative order of effectiveness for organotin induction of intramembranous aggregates was tri-n-butyltin greater than tri-n-propyltin greater than tetra-n-butyltin greater than tri-n-ethyltin bromide, which was based upon the lowest concentration at which they were observed. These results support the previously suggested theory that organotins are membrane effectors because of their comparatively high hydrophobic, lipid partitioning properties. The relatively lipophilic compound, triphenyltin chloride, appeared to be anomalous because it did not readily promote hemolysis or induce the formation of intramembranous aggregates in human erythrocytes. A log-linear statistical model demonstrated an association of hemolysis with both tri-n-butyltin aggregate formation and shape transformation. Select organotin compounds should be useful probes in membrane studies because of their numerous effects.

Erythrocyte Aggregation↗
PubMed1987

Shaky basis for report's sweeping recommendations.

The Herzlinger-Krasker report bases its sweeping conclusions on a methodologically flawed study that fails to account for differences between system hospitals and independent hospitals, includes contract-managed facilities and a large health maintenance organization in the sample, and ignores the effects of differences in state Medicaid programs and the availability of public hospitals on the provision of uncompensated care. These methodological problems undoubtedly help explain why the authors' assertions and conclusions are at variance with most of the other literature on this topic. Perhaps the tax-exempt status of not-for-profit hospitals should be reexamined, as Herzlinger and Krasker suggest. The need for reexamination, however, lies in the extent to which hospitals meet expectations of social benefit in exchange for tax exemptions, not in the hope of creating a level playing field. Justification for tax exemptions could be based on an organization's research and educational activities and its provision of needed but unprofitable services, in addition to its service to poor patients.

Data Collection↗
PubMed1986

Cyanide stimulation of tri-N-butyltin mediated hemolysis.

The effects of tributyltin and sodium cyanide on hemolysis in human erythrocytes are described. Tributyltin has a sharp cut-off concentration for induction of hemolysis. A 5 microM concentration of tributyltin induces hemolysis and 1 microM or less does not in erythrocyte suspensions with 2.3 X 10(8) cells per ml. The kinetics for tributyltin-induced hemolysis are sigmoidal indicating a complex molecular mechanism leading to lysis. Ten mM sodium cyanide plus 1 microM tributyltin does not stimulate hemolysis rates above levels observed with 10 mM sodium cyanide alone. Five mM sodium cyanide plus hemolytic concentrations of tributyltin stimulates hemolysis rates synergistically compared with either cyanide or tributyltin alone. Ultrastructurally, hemolytic concentrations of tributyltin can be visualized in the electron microscope by osmium staining during fixation as electron-dense spheres penetrating the lipid bilayer of the erythrocyte plasma membrane. Ten mM sodium cyanide plus 25 microM tributyltin increases slightly the size of osmiophilic structures in erythrocyte membranes compared with those spheres seen in cells exposed to 25 microM tributyltin alone. Sodium cyanide is the only compound tested that stimulates tributyltin-induced hemolysis. Sodium thiocyanate, previously shown to be rapidly exchanged across erythrocyte membranes by tributyltin, reduces the rate of hemolysis. It is likely that sodium cyanide stimulates hemolysis in the presence of hemolytic tributyltin concentrations because the multimolecular, membrane-intercalated, tributyltin aggregates are transporting cyanide anion by the electrically silent, anion exchange mechanism demonstrated for other anions. It is suggested that this mechanism delivers cyanide anion to or near critical membrane sites where it is available for enzyme inhibition or cross-linking of protein sulfhydryl groups.

Cyanides↗
PubMed1986

Inhibition of tributyltin mediated hemolysis by mercapto compounds.

Hydrophobic tributyltin (TBT) compounds at concentrations greater than 10 microM caused hemolysis of human erythrocytes and formed structures in plasma membranes. The mercapto compounds, beta-mercaptoethanol (beta MER), 2,3-dimercaptopropanol (BAL), 2,3-dimercapto-1-propane sulfonate (DMPS), DL-dithiothreitol (DTT), and meso-2,3-dimercaptosuccinic acid (DMSA) were examined for their ability to inhibit TBT mediated hemolysis. The relative order of effectiveness for inhibition of TBT mediated hemolysis was BAL greater than DTT greater than DMSA greater than DMPS greater than beta MER. A four-fold excess of BAL over TBT prevented hemolysis for 4 hrs and addition of BAL 0.5 hr after TBT reduced the rate of hemolysis. The number of membrane associated TBT aggregates observed per cell profile decreased as the BAL concentration increased from 0 to 100 microM. However, the mean diameter of TBT aggregates nearly doubled in erythrocyte suspensions at 100 microM BAL. Reactions of dimercapto compounds with lipophilic TBT aggregates may depend on their relative lipid solubilities. Also, conversion of the weak Lewis acid, TBT, from a four to a five or six-coordinate tin adduct by the dimercapto Lewis bases used could also be a factor slowing hemolysis rates.

Erythrocytes↗
PubMed1986

Physiological effects of a bactericidal protein from human polymorphonuclear leukocytes on Pseudomonas aeruginosa.

The physiological changes seen in Pseudomonas aeruginosa after exposure to a bactericidal protein (BP) from the granules of human polymorphonuclear leukocytes were studied. It was demonstrated, using radiolabeled proline or leucine, that both the rate of cellular uptake and amino acid incorporation into trichloroacetic acid-insoluble material were markedly decreased immediately after exposure to BP. The rate of O2 consumption by P. aeruginosa was decreased immediately after exposure to BP and continued to decline exponentially until it ceased completely 30 min after exposure to BP. In the presence of 30 mM CaCl2 or MgCl2, bacteria were protected from death due to BP and respiration rates were unaffected. The cellular ATP pool of P. aeruginosa remained constant for up to 2 h after exposure to BP. Membrane depolarization was measured by the influx of the lipophilic anion thiocyanate. It was shown that the cytoplasmic membrane of P. aeruginosa was partially depolarized after exposure to BP. Purified BP killed 95% of 5 X 10(6) CFU of P. aeruginosa at a concentration of 60 to 100 ng of protein per ml. Although the concentration of bacteria and BP varied with each type of experiment, the BP/bacteria ratio required to cause a 95 to 99% loss in viability remained constant. We propose that cytoplasmic membrane depolarization is the biochemical lesion responsible for the other physiological changes seen and ultimately for the death of P. aeruginosa induced by BP.

Adenosine Triphosphate↗
PubMed1986

Characterization of a protein from normal human polymorphonuclear leukocytes with bactericidal activity against Pseudomonas aeruginosa.

Purification of a bactericidal protein (BP) from the cytoplasmic granules of normal human polymorphonuclear leukocytes (PMN) with activity against Pseudomonas aeruginosa is described. Bactericidal activity from acid extracts of a mixed granule population was purified 175-fold by a two-step chromatographic procedure. The first step, dye-ligand affinity chromatography with Matrex-Gel Orange A, was followed by cation-exchange chromatography with Bio-Rex 70 resin, and this combination routinely gave a yield near 80%. Only one peak of bactericidal activity against P. aeruginosa type I was found after each chromatographic step. Characterization of BP showed a single band with an apparent molecular weight of 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified BP was most active against the six strains of P. aeruginosa tested and against Escherichia coli B (a deep rough mutant). Purified BP killed 5 X 10(6) CFU of P. aeruginosa type I per ml at a concentration of 60 to 80 ng/ml. Proteus mirabilis and Staphylococcus aureus were both resistant to the bactericidal activity of BP. BP was shown to be glycosylated by periodic acid staining after gel electrophoresis and to have an isoelectric point near 7.5 by chromatofocusing. The amino acid composition of BP is presented.

Amino Acids↗
PubMed1986

The ultrastructural localization of tri-n-butyltin in human erythrocyte membranes during shape transformation leading to hemolysis.

Scanning electron microscopy, transmission electron microscopy, freeze-fracture, and x-ray energy dispersive spectrometry were used to localize tri-n-butyltin (TBT) in human erythrocytes (RBC's). TBT induced a rapid shape transformation of the RBC discocyte to an echinocyte, which led to hemolysis at concentrations at or above 10 microM TBT. Electron dense spheres or ellipsoids were observed in association with blood cell membranes at or above 10 microM TBT. These structures were visualized initially in thin sections when postfixed with osmium tetroxide. Control cell preparations without TBT did not exhibit these structural densities when fixed with osmium. Freeze-fracture replicas confirmed the presence of TBT aggregates associated with cell membranes as intercalations in the lipid bilayer. In thin sections, these structures measured 71.5 +/- 18.2 nm in diameter. In freeze-fracture replicas of TBT-treated RBC's, particulate structures measuring 60 +/- 18.5 nm in diameter were present on membrane exoplasmic fracture faces and 59.6 +/- 10.8-nm depressions on membrane protoplasmic fracture faces. Qualitative x-ray energy dispersive spectrometry analysis of ultrathin sections of glutaraldehyde-carbohydrazide-embedded samples revealed that the membrane-associated aggregates contained tin. TBT-treated RBC's that were washed with normal saline resulted in a paucity of TBT aggregates associated with the membranes and a reduction in the RBC hemolysis rate. RBC shape transformation occurred at each concentration examined from 0.1 to 100 microM TBT, but was reversible below 1 microM TBT.

Electron Probe Microanalysis↗
PubMed1985

Interspecies variability in propylene glycol dinitrate-induced methemoglobin formation.

Interspecies variability of propylene glycol dinitrate (PGDN)-induced methemoglobin formation was studied in vitro employing erythrocytes from four separate species. The net rate of methemoglobin formation was significantly different among species with dog greater than guinea pig greater than rat greater than or equal to human. This order of susceptibility was maintained in stroma-free hemolysates, indicating that interspecies variability was not a reflection of differences in red cell membrane permeability or intracellular transport of PGDN. The erythrocytic enzymes, catalase, superoxide dismutase, glutathione peroxidase, 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, methemoglobin reductase, and glutathione-S-transferase, were assayed by adaptation of existing methods to a centrifugal analyzer. The above enzymes were removed from hemoglobin derived from each species and the order of susceptibility to PGDN-induced methemoglobin formation remained essentially the same with dog greater than guinea pig greater than human = rat. However, the net rate of PGDN-mediated oxidation of hemoglobin to methemoglobin increased in purified hemoglobin preparations from each species. These results demonstrate that there is species variability in the net rate of PGDN-mediated methemoglobin formation. Total enzyme activity in erythrocytes may contribute to reduction in the net rate of methemoglobin formation. However, the primary determinant of the net rate of methemoglobin formation induced by PGDN appears to be the structure of each hemoglobin molecule.

Animals↗