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H L Malech

Publications and source records attributed to H L Malech.

At least 73 records · Page 4Linked to original sources
PubMed1994

The requirement of p47 phosphorylation for activation of NADPH oxidase by opsonized zymosan in human neutrophils.

Protein kinase C (PKC) inhibitors, staurosporine or 1,5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited NADPH oxidase activity and phosphorylation of 47 kDa protein (p47) in PMA-stimulated neutrophils in a dose-dependent manner. These PKC inhibitors, at the same doses, did not affect oxidase activity and caused only partial inhibition of p47 phosphorylation in OZ-stimulated neutrophils. There was residual (20%) phosphorylated p47 in the membranes of OZ-stimulated cells in the presence of PKC inhibitors, at concentrations which caused total inhibition of oxidase activity and p47 phosphorylation in PMA-stimulated neutrophils. In the presence of ionomycin, which increased intracellular calcium ion concentrations, staurosporine was less effective in inhibiting both superoxide generation and p47 phosphorylation stimulated by PMA, similar to its effect in OZ-stimulated cells. The results indicate that some phosphorylation of p47 always accompanied oxidase activation induced by PMA or OZ, though the degree of phosphorylation of membrane-bound p47 does not directly correlate with rates of superoxide production.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1994

The requirement for phospholipase A2 for activation of the assembled NADPH oxidase in human neutrophils.

Phospholipase A2 (PLA2) inhibitors suppressed simultaneously, in a dose-dependent manner, the activation of NADPH oxidase and the release of 3H-labelled arachidonic acid ([3H]AA) stimulated by either phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ) in human neutrophils. In spite of total inhibition of superoxide production in the presence of the PLA2 inhibitors, 10 microM bromophenacyl bromide (BPB) or 20 microM quinacrine, a maximal phosphorylation of p47 and translocation of p47 and p67 to the neutrophil membranes induced by PMA or OZ was observed. Addition of 10 microM free AA, which by itself did not stimulate superoxide generation, restored oxidase activity in neutrophils treated with PLA2 inhibitors. These findings indicate that phosphorylation and translocation of the cytosolic factors to the membranes are not sufficient for generating superoxide; a functional PLA2 is also needed to stimulate the oxidase activity. The inhibition of PLA2 activity did not prevent the phosphorylation of p47, suggesting that the location of PLA2 is downstream of and does not activate protein kinase C.

Acetophenones↗
PubMed1993

Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease.

The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.

Adult↗
PubMed1993

Peripheral blood progenitors as a target for genetic correction of p47phox-deficient chronic granulomatous disease.

Peripheral blood contains hematopoietic progenitors (PBHPs), which can be harvested in clinically relevant amounts by apheresis. PBHPs have been used as a source of progenitors alternative to marrow for autologous transplantation following intensive chemotherapy. We have determined culture conditions for growth and differentiation of PBHPs to the mature myeloid phenotype, which in the present study are employed to demonstrate the functional correction of an inherited disorder of myeloid cells in retrovirus-transduced human primary hematopoietic progenitors. Patients with chronic granulomatous disease (CGD) suffer from recurrent life-threatening infections because blood phagocytes fail to produce microbicidal superoxide (O2-.). One-third of the cases of CGD result from defects in the gene encoding p47phox, a cytoplasmic oxidase component required for O2-. generation. In the present study, a replication-defective retrovirus encoding p47phox was used to transduce PBHPs from patients with p47phox-deficient CGD, which resulted in significant correction of O2-. generation when PBHPs were differentiated to mature neutrophils and monocytes. This study provides a model for use of PBHPs in development of gene therapy for diseases affecting bone marrow.

3T3 Cells↗
PubMed1993

Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins.

Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).

Amino Acid Sequence↗
PubMed1993

Recombinant human stem cell factor enhances myeloid colony growth from human peripheral blood progenitors.

Peripheral blood hematopoietic progenitors (PBHP) are capable of colony growth in vitro. The effect of stem cell factor (SCF), interleukin-6 (IL-6), and basic fibroblast growth factor (bFGF) on myeloid colony proliferation of PBHP was determined. PBHP purified by positive selection with CD34-specific antibody were plated in semisolid agarose with reported plateau doses of interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) to enhance myeloid colony growth. Experiments then were done to examine colony growth in response to SCF or with SCF and bFGF and/or IL6. SCF alone in the absence of any other growth factors did not support colony growth. SCF at a determined optimum concentration of 100 ng/mL added to the combination of IL-3, GM-CSF, and G-CSF enhanced colony growth and size relative to proliferation in response to the latter three factors alone (from 78 to 188 total colonies/10(4) PBHP plated and from 10 to 93 large [> 200 cells] colonies/10(4) PBHP plated). Furthermore, addition of bFGF and/or IL-6 to the combination of optimum concentrations of SCF, IL-3, GM-CSF, and G-CSF further enhanced colony number and size in a dose-dependent fashion. Using the optimum combination of all growth factors, we determined that the number of myeloid colony-forming PBHP in whole blood was similar between individuals at about three colonies per milliliter whole blood. We conclude that progenitors capable of responding to the early-acting growth factor, SCF, are represented in PBHP and that the number of circulating myeloid colony-forming PBHP is likely a regulated parameter that may have an important biologic function.

Antigens, CD↗
PubMed1993

Surgical management of pulmonary infections in chronic granulomatous disease of childhood.

Chronic granulomatous disease of childhood is an inheritable disorder of phagocytic cell respiratory burst resulting in recurrent, life-threatening, catalase-positive infections. The lung is the most common site of infection, and pulmonary disease is the primary cause of death in greater than 50% of children with chronic granulomatous disease. Still, the role of surgery in management of this disease remains undefined. Between 1974 and 1990, 19 patients with chronic granulomatous disease required 31 thoracic interventions at our institution. Patients ranged in age from 2.5 to 27 years (mean age, 15 years). Seventeen of 19 patients (89%) had had previous pulmonary infections. Patients presented as toxic (temperature > 38.5 degrees C, chest pain, and cough) in 22 instances before the 31 procedures. Aggressive surgical intervention for diagnosis and extirpation of localized infections was undertaken with lobectomy/pneumonectomy with or without other procedures (5), bisegmentectomy (2), segmentectomy with or without other procedures (5), or wedge with or without other procedures (13). In five instances, an empyema was drained; a chest tube for a sterile collection was placed in one instance. There was one intraoperative death, and 3 patients died 22 to 600 days postoperatively with overwhelming sepsis. The mean hospitalization was 101 days (range, 24 to 600 days). Wound complications occurred in 5 patients, requiring 17 separate anesthetic debridements. A change in therapy was dictated by the results of the procedure in 23 of 31 instances (74%). Thoracic surgeons must be aware of this rare cause of immunosuppression in these children and, due to the unusual nature of the pulmonary infections, should follow an aggressive approach in their diagnosis and management.

Adolescent↗
PubMed1992

Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase.

The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.

Amino Acid Sequence↗
PubMed1992

Retroviral expression of recombinant p47phox protein by Epstein-Barr virus-transformed B lymphocytes from a patient with autosomal chronic granulomatous disease.

Patients with chronic granulomatous disease (CGD) have recurrent infections resulting from a failure of phagocytic cells to produce superoxide. One third of CGD patients have an autosomal gene defect resulting in absence of p47phox protein, a cytoplasmic component critical to superoxide production by phagocytic cells. cDNA encoding p47phox has been cloned and recombinant p47phox (rp47phox) restores superoxide-generating activity to a cell-free assay containing cell membranes and cytosol from p47phox-deficient CGD neutrophils. The goal of the present study was to determine the feasibility of retrovirus mediated expression of rp47phox in the HL60 and U937 human hematopoietic cell lines, and in an Epstein-Barr virus transformed B-lymphocyte cell line (EBV-BCL) derived from a p47phox-deficient CGD patient. Normal EBV-BCL contain p47phox and generate small amounts of superoxide, while this CGD EBV-BCL lacks any detectable p47phox protein. Defective amphotropic retrovirus containing p47phox sequence inserted in the LXSN vector in sense and antisense orientations were used to transduce HL60, U937, and CGD EBV-BCL. p47phox mRNA sequence was detected in cells transduced with either sense or antisense retroviral constructs while rp47phox protein was detected only with the sense construct. The amount of rp47phox protein produced within these cells was greater than the native p47phox present in uninduced HL60 or U937 cells, but substantially less than that present in normal neutrophils, induced HL60 cells, or even normal EBV-BCL. Differentiation of transduced HL60 cells and the associated production of native p47phox in response to dimethyl sulfoxide was not affected. These studies demonstrate that retrovirus constructs can be used to mediate stable expression of rp47phox protein in human hematopoietic cell lines and can restore rp47phox protein within the cytosol of p47phox-deficient EBV-BCL from patients with CGD.

B-Lymphocytes↗
PubMed1992

Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity.

The phagocyte NADPH oxidase is a multicomponent membrane-bound electron transport chain that catalyzes the reduction of O2 to superoxide. Cytochrome b558, the terminal electron donor to O2, is an integral membrane heterodimer containing 91- and 22-kDa subunits (gp91-phox and p22-phox, respectively). Synthetic peptides, whose amino acid sequences correspond to a gp91-phox carboxyl-terminal domain, inhibit superoxide production by blocking assembly of the oxidase from membrane and cytosol components. In this study, we examined the amino acid sequence requirements of a series of synthetic truncated gp91-phox peptides for inhibition of human neutrophil NADPH oxidase activation. RGVHFIF, corresponding to gp91-phox residues 559-565, was the minimum sequence capable of inhibiting superoxide generation. Contributions of individual amino acids to overall RGVHFIF inhibitory activity were determined by comparing the abilities of alanine-substituted RGVHFIF peptides to inhibit superoxide production. Substitution of alanine for arginine, valine, isoleucine, or either of the phenylalanines (but not glycine or histidine) within RGVHFIF resulted in loss of inhibitory activity. Synthetic gp91-phox carboxyl-terminal peptides are likely to be competitive inhibitors of the corresponding carboxyl-terminal domain of native gp91-phox by virtue of amino acid identity. We conclude that properties of arginine valine, isoleucine, and phenylalanine side chains within an RGVHFIF-containing domain of gp91-phox contribute significantly to cytochrome b558-mediated activation of the oxidase.

Amino Acid Sequence↗
PubMed1992

Baculovirus mediated expression of human phagocytic cell oxidase cytochrome b558 in sf9 insect cells.

Chronic granulomatous disease (CGD) results from deficient production of components of the phagocyte NADPH oxidase. Most commonly affected is cytochrome b558, a heterodimer composed of a 22-kDa protein (p22phox) noncovalently bound to a 91-kDa transmembrane glycoprotein (gp91phox). CGD phagocytes lack both p22phox and gp91phox peptides when either gene is affected, suggesting that both peptides must be produced for individual subunit stability. Both genes have been cloned, but eukaryotic expression of recombinant gp91phox has not been reported. To investigate the stability and interaction of cytochrome b558 subunits, we introduced p22phox and gp91phox cDNA into recombinant baculoviruses. Recombinant gp91phox (rgp91phox) and p22phox (rp22phox) were detected individually and together in the same cells by in situ immunofluorescence and by SDS-PAGE immunoblotting of membranes from sf9 cells infected with baculovirus constructs. Formation of rp22phox/rgp91phox complexes was demonstrated by coprecipitation using subunit-specific antibodies. This study demonstrates for the first time that cDNA encoding either subunit is capable of initiating production of stable recombinant cytochrome b558 subunits in eukaryotic cells.

Animals↗
PubMed1992

Delineation of the phagocyte NADPH oxidase through studies of chronic granulomatous diseases of childhood.

The phagocyte NADPH oxidase is a complex system consisting of membrane and cytosolic components that must assemble at the membrane for proper activation. Studies of patients with chronic granulomatous diseases of childhood have enabled the molecular characterization of these components, which has led to studies defining their interaction during NADPH complex assembly. Understanding NADPH oxidase assembly provides an opportunity to develop therapeutics for the regulation of this important reaction of inflammation.

Child↗
PubMed1991

Effect of 1,25-dihydroxyvitamin D3, lipopolysaccharide, or lipoteichoic acid on the expression of NADPH oxidase components in cultured human monocytes.

Human blood monocytes in culture gradually lose their capability to produce superoxide when stimulated. Addition of 10(-8) M 1,25-dihydroxyvitamin D3 [1, 25(OH)2D3], 2.5 ng/ml LPS, or 25 ng/ml lipotechoic acid (LTA) prevented this decrease in monocyte respiratory burst when added at initiation of culture. Monocytes cultured for 4 days in the presence of 1,25 (OH)2D3, LPS, or LTA retained the ability to produce superoxide and 1,25 (OH)2D3 actually increased the oxidative capacity compared with fresh monocytes. Furthermore, addition of 1,25(OH)2D3, LPS, or LTA to monocytes at day 4 of culture restored the oxidase activity by day 7 to the levels seen with fresh monocytes. Cytosol or membrane fractions from monocytes cultured with or without the agents indicated above were tested in a cell-free assay of superoxide production by mixing with fresh monocyte membrane or cytosol fractions, respectively. Membrane oxidase activity from these cultured monocytes showed only minimal changes regardless of the agent present or absent from the culture. However, the activity of cytosol fractions from these same cultured monocytes showed substantial differences depending upon the culture conditions and these changes correlated closely with changes in activity of the intact monocytes from which the cytosols were derived. Immunoblot analysis of monocyte membranes and cytosol was used to assess the amount of the 91-kDa and 22-kDa subunits of membrane oxidase cytochrome 558 (gp91 and p22), and the 47-kDa and 67-kDa cytosol oxidase factors (p47 and p67) in the different culture conditions. The gp91 and p22 decreased during culture of monocytes, although the decrease was only apparent by 7 days in culture. When 1,25 (OH)2D3, LPS, or LTA were present at the initiation of culture, then an increase in gp91 and p22 was seen as compared with fresh monocytes at day 4. When these agents were added at day 4 of culture, the levels of gp91 and p22 at day 7 were similar to fresh monocytes. The p67 cytosol oxidase component showed little change in amount by immunoblot regardless of the time in culture or whether 1,25(OH)2D3, LPS, or LTA were present. In contrast, the p47 cytosol oxidase factor decreased by day 4 of culture and was markedly depressed by day 7. Monocytes cultured in the presence of either 1,25(OH)2D3, LPS, or LTA preserved the expression of p47, whereas addition of these agents to monocytes at day 4 of culture restored the expression of p47 by day 7 to the level of fresh monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcitriol↗
PubMed1991

Kinetics of superoxide production by stimulated neutrophils.

Oxygen-derived active species and superoxide radical in particular are generated and excreted upon granulocyte activation and are instrumental in host defense against bacterial and fungal infections. Associated with the activation of neutrophils is an apparent transitory oxy-radical production. Evidence from independent methods has previously suggested that radical production peaks shortly following neutrophil stimulation and decays within minutes. However, since neutrophil function in the body might reasonably be expected to last beyond the few minutes following stimulation, cessation of the production of oxy-radicals is unexpected. In an attempt to reconcile this discrepancy, the formation kinetics of superoxide by stimulated human neutrophils was reinvestigated by three independent methods: electron spin resonance, chemiluminescence, and ferricytochrome c reduction. The present results demonstrate that under appropriate experimental conditions stimulated neutrophils have the capacity to produce superoxide for several hours. The reasons for the previously reported "apparent" ephemeral nature of oxy-radical formation are discussed.

Electron Spin Resonance Spectroscopy↗
PubMed1991

Unique human neutrophil populations are defined by monoclonal antibody ED12F8C10.

A mouse IgG1 monoclonal antibody ED12F8C10 (C10) binds a constant percentage of peripheral blood neutrophils in the same individual when studied over time, defining a distinct subset of neutrophils in all normal individuals studied to date. Bone marrow studies confirm that the heterogeneity is present to the same degree at all stages of neutrophil development from the myelocyte to the mature neutrophil. Neither in vivo nor in vitro activation of neutrophils explains or significantly alters the relative percentages of C10-positive and -negative neutrophils in the same individual. With both activation and exudation, however, expression of the C10-defined epitope increases in intensity in the C10 binding subpopulation. Studies of NBT reduction, phagocytosis, adherence, light scattering characteristics, and monoclonal antibody surface binding have failed to demonstrate physical or functional differences between the C10-defined populations. We examined C10 binding in patients with different defects of phagocyte function. In two patients with neutrophil-specific granule deficiency, less than 1% of the neutrophils were found to be C10 positive, while neutrophils from a patient with idiopathic leukemoid reaction and recurrent infections demonstrated greater than 99% C10 binding. Although the present study does not delineate the physiologic significance of C10 binding heterogeneity, it firmly supports the concept of neutrophil heterogeneity at the level of surface antigen expression.

Antibodies, Monoclonal↗
PubMed1991

In vivo interferon-gamma therapy augments the in vitro ability of chronic granulomatous disease neutrophils to damage Aspergillus hyphae.

During the recently completed double-blind, placebo-controlled, randomized trial of recombinant interferon-gamma (rIFN-gamma) therapy in chronic granulomatous disease (CGD), a metabolic assay of neutrophil damage to Aspergillus fumigatus hyphae was used to monitor neutrophil function before and during therapy. In this assay, 5 x 10(4) conidia that had germinated into hyphae were exposed to 5 x 10(5), 15 x 10(5), or 50 x 10(5) CGD neutrophils. By analysis of variance, neutrophils from patients on rIFN-gamma were found to produce significantly more damage to hyphae than those from the placebo group (P less than .01). In subgroup analysis, this effect was best seen in the hyphae exposed to 50 x 10(5) CGD neutrophils, where neutrophils from patients receiving rIFN-gamma produced significantly more damage to the hyphae than those from the placebo group (P less than .05). In vivo rIFN-gamma therapy improves the ability of CGD neutrophils to damage Aspergillus fumigatus hyphae in an in vitro assay.

Analysis of Variance↗
PubMed1991

Skeletal involvement in children who have chronic granulomatous disease.

Chronic granulomatous disease is a rare disorder of the oxidative metabolism of the white blood cells that results in immunodeficiency. In a retrospective review of the records of forty-two patients who had chronic granulomatous disease, we identified thirteen patients who had a total of twenty skeletal infections. Two types of infection were noted: Type 1, which resulted from a direct spread of the infection from an adjacent focus, usually of fungus or mycobacteria, and Type 2, which resulted from hematogenous spread, usually of Nocardia or more common bacteria. The thoracic vertebrae, ribs, and metatarsals were the most commonly involved bones. All four patients in whom the vertebrae were involved had a Type-1 fungal infection. Paresis developed in three of these patients, and two of them died. Antibiotics alone effectively eradicated the infection in only one patient. Wide operative débridement combined with administration of antibiotics was the most successful treatment. Comprehensive preoperative imaging and several cultures are necessary to locate the infection in the bone and to identify the organism.

Adolescent↗