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Biomedical subjects

L A Boxer

Publications and source records attributed to L A Boxer.

At least 19 recordsLinked to original sources
PubMed1992

Correlation of messenger RNA levels with protein defects in specific granule deficiency.

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder of unknown cause associated with an impaired inflammatory response and an absence of neutrophil secondary granules. Reduced levels of several neutrophil proteins have led to the suggestion that the defect may lie at the level of transcription, a hypothesis that is supported by abnormally low levels of lactoferrin message in the bone marrow of two SGD patients. We have examined the level of seven granule protein RNAs in one SGD patient and have compared them with reported protein levels. We have found the RNA levels for all of these genes to be reduced in proportion to the decreased levels of their respective proteins. These data further support the hypothesis that the reduced protein levels reflect a defect in transcriptional control.

Blood Proteins↗
PubMed1992

Purification of PKC-I, an endogenous protein kinase C inhibitor, and types II and III protein kinase C isoenzymes from human neutrophils.

Human neutrophil protein kinase C (PKC) activity is inhibited by an endogenous protein found primarily in the pellet fraction from homogenized specific granules, which was both heat- and proteinase-sensitive [Balazovich, Smolen & Boxer (1986) J. Immunol. 137, 1665-1673]. We now report that two PKC isoenzymes and the endogenous PKC inhibitor, which we named PKC-I, were purified from human neutrophils. A neutrophil soluble fraction that was subjected to DEAE-Sephacel chromatography yielded highly enriched PKC because, by definition, enzymic activity was strictly dependent on Ca2+ and phosphatidylserine. Hydroxyapatite chromatography resolved two peaks of PKC activity. Type II and Type III PKC isoenzymes were each identified on Western blots by using isoenzyme-specific monoclonal antibodies. Unlike rat brain, from which PKC isoenzymes were also purified, Type I PKC was not detected in human neutrophils. Western blots indicated that both Type II and Type III PKC isoenzymes had molecular masses near 80 kDa. In agreement with other reports, PKC was autophosphorylated in vitro. PKC-I, an endogenous neutrophil inhibitor of PKC, was purified to apparent homogeneity by DEAE-Sephacel and S-400 Sephacel chromatography. PKC-I had a molecular mass of 41 kDa. PKC-I inhibited purified PKC activity stimulated by 1,2-diacylglycerols in a concentration-dependent manner, and inhibited PKC-dependent phosphorylation of proteins present in neutrophil cytosol.

Animals↗
PubMed1992

Recombinant human granulocyte-colony-stimulating factor in the treatment of patients with neutropenia.

The results of an open-label, randomized, Phase III trial of r-methionyl human granulocyte-colony-stimulating factor (r-metHuG-CSF) in 41 patients with severe chronic neutropenia (SCN) are reported. Patients with diagnoses of congenital, cyclic, and idiopathic neutropenia, with histories of recurrent infections, were evaluated. The primary objective of the trial was to evaluate the ability of r-metHuG-CSF to increase the ANC to greater than 1500/mm3. A secondary objective was to evaluate variables associated with infection-related morbidity in SCN. r-metHuG-CSF treatment consisted of 1 month of dose titration followed by 4 months of treatment at an optimal dose. Patients were randomized to either immediate treatment with r-metHuG-CSF (Group A) or four months of observation followed by r-metHuG-CSF treatment (Group B). r-metHuG-CSF was administered by daily, subcutaneous injection with initial doses of 3 to 10 micrograms/kg/day. Forty of 41 patients who received r-metHuG-CSF had a complete response (median ANC greater than 1500/mm3 during 4 months of r-metHuG-CSF treatment). All cases of gingivitis and severe mouth ulcers resolved upon treatment with r-metHuG-CSF. Serious infections were also eliminated. Only one patient failed to show clinical improvement in response to r-metHuG-CSF treatment. Adverse reactions during the first 5 months of treatment were mild. Splenomegaly (mild) was noted in some patients. The administration of r-metHuG-CSF in patients with SCN significantly increased the ANC (P less than 0.001) and was accompanied by a marked reduction in infectious complications.

Agranulocytosis↗
PubMed1992

Human neutrophil annexin I promotes granule aggregation and modulates Ca(2+)-dependent membrane fusion.

The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.

Annexins↗
PubMed1991

Activation of human neutrophils increases thrombospondin receptor expression.

Thrombospondin (TSP), a 450-kDa extracellular matrix protein secreted by platelets may be instrumental in triggering polymorphonuclear leukocyte (PMN) activation and mediating PMN-endothelial cell interactions. TSP alone had no effect on O-2 generation but caused a significant increase in the chemoattractant FMLP-mediated O-2 generation. Purified HBD, but not the 140-kDa COOH-terminal fragment of TSP, retained the priming activity indicating that the priming effect was mediated through the HBD of TSP. The priming of FMLP-mediated O-2 generation by TSP, and our recent studies demonstrating that TSP stimulates PMN adhesion and motility suggested the presence of specific receptors for TSP on PMN. Binding studies on unactivated PMN, using 125I-TSP and competition with excess unlabeled TSP, demonstrated 2.4 x 10(3) binding sites/cell with an apparent Kd of 7 nM. Heparin did not compete for binding as effectively as unlabeled TSP. There were 1.5 x 10(3) heparin-inhibitable binding sites/cell with an apparent Kd of 8 nM that represented approximately 60% of the TSP-specific sites. Therefore, two distinct TSP receptors appeared to exist on unactivated PMN; one interacting with the heparin-binding domain of TSP and one interacting with a different site. Treating PMN with cytochalasin B followed by FMLP caused a 30-fold increase in TSP receptor expression. Binding studies on activated PMNs revealed 7.6 x 10(4) sites/cell; 60% of which were heparin inhibitable. The majority (5.3 x 10(4) sites/cell) of receptors expressed had an affinity of approximately 20 nM. About 50% of these sites were heparin inhibitable. In addition, there were 2.3 x 10(4) higher affinity sites/cell with an apparent Kd of 6 nM. Heparin-inhibitable sites comprised 70% of the higher affinity sites. The existence of a subset of TSP receptors that were heparin-inhibitable on PMN suggests that binding of TSP may trigger functionally independent responses. Increased receptor expression and expression of two high affinity binding sites following PMN activation may modulate PMN-endothelium or PMN-basement membrane interactions localized at the blood vessel wall.

Amino Acid Sequence↗
PubMed1991

Human neutrophil adherence to thrombospondin occurs through a CD11/CD18-independent mechanism.

Thrombospondin (TSP), a 450-kDa trimeric glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, may play an important role in polymorphonuclear leukocyte (PMN) adherence to blood vessel walls before diapedesis. We have examined the adherence of PMN to TSP and compared it to adherence to other extracellular matrix proteins. PMN adherence to TSP-coated plastic was complete by 60 min with spreading completed by 2 h. The kinetics of adhesion and spreading on TSP were similar to that of vitronectin (VN), laminin (LN), and fibronectin (FN). Activation of PMN with the calcium ionophore A23187 or the chemotactic peptide FMLP increased PMN adherence to LN and FN, but not to TSP or VN, suggesting that PMN activation may differentially regulate expression of TSP and VN receptors as compared to LN and FN receptors. The specificity of PMN adherence to TSP was confirmed by competition with saturating amounts of TSP and inhibition with anti-TSP antibodies. mAb A6.1, which binds to the protease-resistant core of TSP, was the most effective in blocking PMN adherence to TSP. Using TSP proteolytic fragments, we demonstrated that the primary interaction of PMN with TSP was mediated through the 140-kDa COOH-terminal domain. Inasmuch as the 140-kDa fragment of TSP contains an Arg-Gly-Asp sequence similar to the cell recognition site of FN and VN, we determined whether RGDS peptides would inhibit PMN adhesion. RGDS did not significantly inhibit PMN adhesion to TSP, VN, or LN, but reduced PMN adhesion to FN by 50%. To determine if PMN adhesion to TSP was mediated by a beta 2 integrin receptor such as LFA-1, MO-1, or p150,95, we performed adhesion assays using PMN isolated from patients with leukocyte adhesion deficiency that lack beta 2 receptors. Leukocyte adhesion deficiency PMN exhibited normal adherence to TSP. In contrast, adherence to VN, LN, and FN was reduced by 95%. Therefore, adherence to TSP is probably not mediated by a beta 2 integrin receptor. These data contribute to the accumulating evidence that PMN can interact with extracellular matrix proteins through a CD11/CD18-independent process.

Antigens, CD↗
PubMed1991

Histamine inhibits cell spreading and C3bi receptor clustering and diminishes hydrogen peroxide production by adherent human neutrophils.

Cell adherence plays a central role in many host defense mechanisms. Human peripheral blood neutrophils possess cell surface receptors that contribute to cell adherence or detachment. Receptors specific for the C3bi cleavage fragment of the third component of complement (CR3) promote adhesion, whereas histamine receptors promote detachment. In the present study, we tested the ability of histamine to down-regulate the physiological effects of CR3 receptors. Histamine decreased the binding of 51Cr-labeled neutrophils to complement-coated surfaces (C3-coated surfaces) in a dose-dependent fashion. Scanning electron microscopic and optical microscopic observations of neutrophils on C3-coated surfaces revealed polarized or spherical cell morphologies in the absence or presence of histamine, respectively. Histamine inhibited the ability of CR3 to cluster on plasma membranes of neutrophils adherent to C3-coated surfaces as shown by fluorescence microscopy. In addition, histamine diminished but did not abolish the FMLP-stimulated increase in plasma membrane CR3 expression as measured by fluorometry. Histamine did not inhibit the release of marker proteins from specific or gelatinase containing granules by neutrophils in suspension. Histamine also diminished the FMLP-stimulated production of respiratory burst oxidants from cells in suspension or cells allowed to adhere to fibrinogen substrates. We suggest that histamine may modulate selective changes in neutrophil function by diminishing adherence and preventing changes in cell shape following cell activation.

Cell Adhesion↗
PubMed1991

Clinical and biologic effects of granulocyte colony stimulating factor in the treatment of myelokathexis.

Successful treatment of a patient with myelokathexis, a rare form of chronic neutropenia associated with recurrent infections, is described. Rapid mobilization of bone marrow neutrophils and improved myeloid morphologic features were observed after treatment with human granulocyte colony stimulating factor. Transient thrombocytopenia and bone pain were observed during treatment. Although neutrophil chemotaxis, superoxide production, and FcRIII surface expression were reduced, the patient improved clinically after restoration of a normal neutrophil count.

Antigens, CD↗
PubMed1991

Severe congenital neutropenia: clinical effects and neutrophil function during treatment with granulocyte colony-stimulating factor.

We studied neutrophil function and clinical responses in seven patients with severe congenital neutropenia (SCN) after they received treatment with recombinant human granulocyte colony stimulating factor (rhG-CSF). Two subpopulations of patients with SCN were defined by their pattern of absolute neutrophil response, superoxide production, and cytochrome b559 levels. One group had an oscillating absolute neutrophil count and reduced ability to produce superoxide and cytochrome b559 (n = 4), and the second group had a relatively constant absolute neutrophil count response with normal superoxide and cytochrome levels (n = 3). Neutrophils from both groups had decreased surface expression of FcRIII and abnormal upregulation of the C3bi receptor (CR3). All patient neutrophils, however, had normal contents of the primary granule constituent, beta-glucuronidase, and the specific granule constituent, vitamin B 12 binding protein. The clinical response to rhG-CSF was evident by marked improvement in the degree of periodontitis and reduction in the number of oral ulcers in both groups of patients. Although neutrophil function is not completely normal in patients with SCN, it is likely that enough redundancy exists in neutrophil bactericidal capacity to promote normal host response to inflammation.

CD18 Antigens↗
PubMed1991

Recombinant human G-CSF and GM-CSF prime human neutrophils for superoxide production through different signal transduction mechanisms.

Recombinant human granulocyte-colony stimulating factor (G-CSF) and recombinant human granulocyte/macrophage-colony stimulating factor (GM-CSF) stimulate neutrophil production from precursors in the marrow and enhance granulocyte functions in vitro. We studied the effects of G-CSF and GM-CSF on neutrophil superoxide production and secretion. G-CSF and GM-CSF alone stimulated neither superoxide production nor secretion, but both agents primed neutrophils for superoxide production stimulated by either N-formylmethionyl-leucyl-phenylalanine (FMLP) or ionomycin. Optimal priming occurred with G-CSF at 5.3 ng/ml for 20 minutes and for GM-CSF at 1 ng/ml for 60 minutes. Priming by GM-CSF was more readily inhibited by the tyrosine kinase inhibitor ST638 but was unaffected by staurosporine. Conversely, G-CSF priming was inhibited by staurosporine but not by ST638. Neither protein kinase C translocation nor increased protein kinase C activity, however, were observed after G-CSF/GM-CSF treatment. Priming by G-CSF and GM-CSF was sensitive to pertussis toxin, suggesting the involvement of guanine nucleotide-binding proteins (G-proteins). Neutrophils from three siblings with cyclic neutropenia were studied to observe the effects of G-CSF treatment on neutrophil function in vivo; sibling 1 and sibling 2 were treated with G-CSF for 6 months, but sibling 3 was not in the treatment group. Compared with neutrophils from normal donors, neutrophils from sibling 1 and sibling 2 were primed in vivo for superoxide release stimulated by either ionomycin or FMLP. Superoxide released by neutrophils from sibling 3 was similar to control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗
PubMed1990

Iloprost inhibits neutrophil-induced lung injury and neutrophil adherence to endothelial monolayers.

We hypothesized that Iloprost, a long-acting prostacyclin analog, would inhibit neutrophil (PMN)-induced lung injury and decrease PMN adherence to vascular endothelium. Human PMNs infused into isolated buffer-perfused rat lungs subsequently stimulated with phorbol myristate acetate (PMA) resulted in lung injury as assessed by the accumulation of [125I]bovine serum albumin (125I-BSA) in lung parenchyma and alveolar lavage fluid. Addition of Iloprost to the lung perfusate, prior to activation of the PMNs, reduced lung injury as assessed by a decrease in the accumulation of 125I-BSA in the lung. This protective effect was not due to the vasodilatory effect of Iloprost. Protection by Iloprost was not linked to a reduction in PMA-induced PMN superoxide production since Iloprost did not reduce the amount of superoxide released into lung perfusate. In vitro, Iloprost caused a dose-dependent inhibition of PMA-stimulated PMN adherence to endothelial cells. Iloprost did not affect the number of Mo1 adhesion molecules constitutively expressed or the number of receptors expressed on the PMNs following PMA. Addition of cAMP or dibutyryl cAMP to the endothelial cells mimicked the effects of Iloprost, diminishing PMA-stimulated PMN adhesion. In separate experiments, addition of the phosphodiesterase inhibitor IBMX to Iloprost resulted in a greater inhibition of PMA-stimulated PMN adherence, while addition of an adenylate cyclase inhibitor, SQ 22,536, or cAMP antibodies with the Iloprost abolished Iloprost's inhibitory effect on PMN adhesion. Thus, Iloprost inhibits PMA-activated PMN-induced lung injury despite continued superoxide production. Iloprost inhibition of PMN adhesion is dependent on cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1979

Protection of granulocytes by vitamin E in glutathione synthetase deficiency.

The administration of vitamin E (alpha-tocopherol) was found to improve polymorphonuclear leukocyte function in an infant with congenital deficiency of glutathione synthetase activity. Before therapy with vitamin E the abnormal leukocytes exposed to phagocytic challenge showed oxidant damage. They released 60 per cent more hydrogen peroxide than did normal leukocytes, iodinated only 20 to 25 per cent of the normal number of particles, and were unable to kill bacteria as effectively as normal leukocytes although the rates of phagocytosis were normal. These functional abnormalities disappeared when the patient was placed on 400 IU of alpha-tocopherol daily for three months. Associated with the functional improvement was a normalization of microtubule assembly during phagocytic challenge.

Blood Bactericidal Activity↗
PubMed1979

Enhancement of chemotactic response and microtubule assembly in human leukocytes by ascorbic acid.

The incubation of human leukocytes with ascorbic acid increased chemotaxis of the cells. In addition, ascorbic acid promoted the assembly of intracellular polymorphonuclear leukocyte (PMN) with colchicine blocked the effect of ascorbic acid on promoting microtubule assembly. Not only did ascorbic acid promote the assembly of microtubules in vivo, but it enhanced the assembly of bovine brain tubulin into microtubules in vitro as quantitated by a glass-fiber filtration assay and by promotion of viscosity changes. The enhancement in leukocyte mobility by ascorbate at concentrations achievable in normal tissues correlates with its ability to assemble microtubule organelles.

Ascorbic Acid↗
PubMed1979

Infectious mononucleosis with severe neutropenia and opsonic antineutrophil activity.

Severe granulocytopenia is an uncommon complication of infectious mononucleosis, only 18 cases having been reported previously. Our review and experience with this case would indicate that (1) severe granulocytopenia is usually diagnosed at about the third or fourth week of illness, (2) bone marrow examination most commonly reveals lack of mature neutrophil precursors in the myelocytic series, (3) the duration of usually short, lasting about a week, and (4) most patients recover without treatment. The laboratory findings in our case suggests that the neutropenia may be antibody mediated.

Adolescent↗
PubMed1979

Oxidative damage to neutrophils in glutathione synthetase deficiency.

Several episodes of neutropenia were observed in a child with glutathione synthetase deficiency (5-oxoprolinuria). Studies of the patient's glutathione-deficient neutrophils were undertaken to examine the responses of the cells to oxidative stress associated with phagocytosis. The patient's neutrophils contained 10--20% of normal glutathione content. Circulating neutrophils in infection-free periods appeared less mature than normal by morphologic criteria, suggesting increased cell turnover. The cells ingested particles, responded to chemotactic stimuli, and oxidized 1-14C glucose normally. However, following ingestion of particles, the cells accumulated excess hydrogen peroxide compared with normal cells, and showed impaired protein iodination and bacterial killing. Electron micrographs revealed damage to microtubules and membranous structures in the patient's neutrophils during phagocytosis. The level of glutathione in the cells appears inadequate to protect against peroxide generated during normal cell function, and the cells are thus damaged and rendered less effective in bacterial killing. The data provide evidence for a protective role of glutathione in normal neutrophil function.

Chemotaxis, Leukocyte↗
PubMed1979

Impaired microtubule assembly and polymorphonuclear leucocyte function in the Chediak-Higashi syndrome correctable by ascorbic acid.

It was previously shown that the abnormal surface characteristics and defective bactericidal function of polymorphonuclear leucocytes (PMN) in the Chediak-Higashi syndrome (CHS) are correlated with impaired microtubule assembly, and in one patient direct electron microscopic evidence for an anomaly in microtubule assembly following surface membrane activation by concanavalin A (Con A). We show that very few microtubules are visible in CHS leucocytes from two additional patients under conditions where normal PMNs contain abundant microtubules, and that both in vivo and in vitro exposure of the CHS leucocytes to ascorbic acid promotes the assembly of microtubules. This agent, which normalizes chemotaxis and degranulation in CHS leucocytes, is shown also to correct granulocyte adherence in these leucocytes. It is suggested that the improved clinical course of patients with CHS following treatment with ascorbic acid is related at least in part to improvement of microtubule assembly and PMN function by the ascorbic acid.

Ascorbic Acid↗
PubMed1979

Hemoglobin Indianapolis (beta 112[G14] arginine). An unstable beta-chain variant producing the phenotype of severe beta-thalassemia.

Hemoglobin (Hb) Indianapolis is an extremely labile beta-chain variant, present in such small amounts that it was undetectable by usual techniques. Clinically, it produces the phenotype of severe beta-thalassemia. Biosynthetic studies showed a beta:alpha ratio of 0.5 in reticulocytes and about 1.0 in marrow after a 1-h incubation. These results, similar to those seen in typical heterozygous beta-thalassemia, suggested that betaIndianapolis was destroyed so rapidly that its net synthesis was essentially zero. To examine the kinetics of globin synthesis, reticulocyte incubations of 1.25--20 min were performed with [3H]leucine. The betaIndianapolis:beta A ratio at 1.25 min was 0.80 suggesting that beta Indianapolis was synthesized at a near normal rate. At 20 min, this ratio was 0.46 reflecting rapid turnover of beta Indianapolis. The erythrocyte ghosts from these incubations contained only betaIndianapolis and alpha-chains, and the proportion of betaIndianapolis decreased with time, indicating loss of betaIndianapolis. Pulse-chase studies showed little change in beta A:alpha ratio and decreasing betaIndianapolis:alpha and betaIndianapolis:beta A with time. The half-life of betaIndianapolis in the soluble hemoglobin was approximately equal to 7 min. There was also rapid loss of beta Indianapolis from the erythrocyte membrane. From these results, it may be inferred that betaIndianapolis is rapidly precipitated from the soluble cell phase to the membrane, where it is catabolized. Heterozygotes for beta 0-thalassemia usually have minimal hematologic abnormalities, whereas heterozygotes with betaIndianapolis, having a similar net content of beta-chain, have severe disease. The extremely rapid precipitation and catabolism of betaIndianapolis and the resulting excess of alpha-chains, both causing membrane damage, may be responsible for the severe clinical manifestations associated with this variant. It seems likely that other, similar disturbances in the primary sequence of globin polypeptide chains may produce clinical findings similar to those seen with hemoglobin Indianapolis and thus produce the phenotype of severe beta-thalassemia.

Adolescent↗