PubMed Health⌕ Search

Biomedical subjects

R A Ezekowitz

Publications and source records attributed to R A Ezekowitz.

At least 91 records · Page 5Linked to original sources
PubMed1990

Molecular characterization of the human macrophage mannose receptor: demonstration of multiple carbohydrate recognition-like domains and phagocytosis of yeasts in Cos-1 cells.

The macrophage mannose receptor is an integral membrane protein expressed on the surface of tissue macrophages. After ligation of mannose-rich glycoconjugates or pathogens, the receptor mediates endocytosis and phagocytosis of the bound ligands by macrophages. The cDNA-derived primary structure of the mannose receptor predicts a cysteine-rich NH2-terminal domain, followed by a fibronectin type II region. The remainder of the ectodomain is comprised of eight carbohydrate recognition-like domains, followed by a transmembrane region, and a cytoplasmic tail. Transfection of the mannose receptor cDNA into Cos-I cells is necessary for receptor-mediated endocytosis of mannose-rich glycoconjugate as well as phagocytosis of yeasts. Deletion of the cytoplasmic tail results in a mutant receptor that is able to bind but not ingest the ligated pathogens, suggesting that the signal for phagocytosis is contained in the cytoplasmic tail.

Amino Acid Sequence↗
PubMed1989

The human mannose-binding protein gene. Exon structure reveals its evolutionary relationship to a human pulmonary surfactant gene and localization to chromosome 10.

The human mannose-binding protein (MBP) plays a role in first line host defense against certain pathogens. It is an acute phase protein that exists in serum as a multimer of a 32-kD subunit. The NH2 terminus is rich in cysteines that mediate interchain disulphide bonds and stabilize the second collagen-like region. This is followed by a short intervening region, and the carbohydrate recognition domain is found in the COOH-terminal region. Analysis of the human MBP gene reveals that the coding region is interrupted by three introns, and all four exons appear to encode a distinct domain of the protein. It appears that the human MBP gene has evolved by recombination of an ancestral nonfibrillar collagen gene with a gene that encodes carbohydrate recognition, and is therefore similar to the human surfactant SP-A gene and the rat MBP gene. The gene for MBP is located on the long arm of chromosome 10 at 10q11.2-q21, a region that is included in the assignment for the gene for multiple endocrine neoplasia type 2A.

Acute-Phase Proteins↗
PubMed1989

The human mannose-binding protein functions as an opsonin.

The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains: a cysteine-rich NH2-terminal domain that stabilizes the alpha-helix of the second collagen-like domain, and a third COOH-terminal carbohydrate binding region. The function of MBP is unknown, although a role in host defense is suggested by its ability to bind yeast mannans. In this report we show that native and recombinant human MBP can serve in an opsonic role in serum and thereby enhance clearance of mannose rich pathogens by phagocytes. MBP binds to wild-type virulent Salmonella montevideo that express a mannose-rich O-polysaccharide. Interaction of MBP with these organisms results in attachment, uptake, and killing of the opsonized bacteria by phagocytes. These results demonstrate that MBP plays a role in first line host defense against certain pathogenic organisms.

Antigens, Bacterial↗
PubMed1989

A human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus.

In vitro infection by the human immunodeficiency virus (HIV) of CD4+ H9 lymphoblasts is inhibited by a mannose-binding protein (MBP) purified from human serum. In addition, MBP is able to selectively bind to HIV-infected H9 cells and HIV-infected cells from the monocyte cell line U937. These results indicate MBP most likely recognizes high mannose glycans known to be present on gp120 in the domain that is recognized by CD4 and thereby inhibits viral entry to susceptible cells. In support of this contention, recombinant gp120 binds directly to MBP; the binding is saturable, mannan inhibitable, removed by N-glycanase treatment, and dependent on divalent cations.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Human mannose-binding protein activates the alternative complement pathway and enhances serum bactericidal activity on a mannose-rich isolate of Salmonella.

The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains, a cysteine-rich NH2-terminal domain that stabilizes the collagen alpha helix of the second domain and a third COOH-terminal carbohydrate recognition domain. Previous studies have shown that both native and recombinant human MBP bind to wild-type virulent Salmonella montevideo that expresses a mannose-rich lipopolysaccharide. Interaction with MBP results in opsonization and killing by phagocytes. In this report we show that low concentration of MBP (less than 10 micrograms/ml) markedly enhance complement deposition via the alternative complement pathway on S. montevideo. Despite structural similarities between MBP and the C1q subcomponent of the first complement component, MBP did not restore classical pathway activity to C1q-deficient serum, nor did it activate C1s when added to a mixture of C1r and C1s. In the presence of MBP the C3 bound to S. montevideo during incubation in serum was in the form of C3b and iC3b at a ratio of 1:2. Presensitization of S. montevideo with MBP rendered this normally serum resistant organism susceptible to complement-mediated killing. These results emphasize that MBP and complement cooperate in first line defense of the nonimmune host.

Amino Acid Sequence↗
PubMed1989

Early detection of hereditary medullary thyroid cancer with polymorphic DNA probes. Groupe d'Etude des Tumeurs a Calcitonine.

We have performed linkage analysis on 32 families with hereditary medullary thyroid cancer (MTC) with seven polymorphic DNA probes situated near the centromere of chromosome 10. Nineteen of these families were affected with multiple endocrine neoplasia type 2A (MEN 2A), and the remainder had MTC without pheochromocytoma. There were no instances of recombination between the MEN 2A susceptibility gene and the IRBP.H4 marker. Two other probes, TB14.34 and MCK2, are also tightly linked (theta = 0.00 and theta = 0.02, respectively). Because TB14.34 and IRBP.H4/MCK2 are situated on opposite sides of the MEN 2A gene, screening with flanking DNA markers is now feasible.

Adrenal Gland Neoplasms↗
PubMed1989

Hereditary medullary thyroid carcinoma: genetic annalysis of three related syndromes. Groupe d'Etude des Tumeurs a Calcitonine.

Hereditary medullary thyroid carcinoma (MTC) appears in three forms: 1) in association with pheochromocytomas and parathyroid hyperplasia (multiple endocrine neoplasia type 2A [MEN 2A]); 2) with pheochromocytomas, neuromas of the mucous membranes, and a marfanoid appearance (MEN 2B); and 3) without pheochromocytoma. Despite these differences in presentation, age of onset, and clinical severity, limited genetic studies suggest that the three MTC variants may be due to inherited mutations at the same gene locus. We present further data in support of the hypothesis that allelic variation may underlie the diversity of these endocrine neoplasia syndromes.

Adrenal Gland Neoplasms↗
PubMed1988

Partial correction of the phagocyte defect in patients with X-linked chronic granulomatous disease by subcutaneous interferon gamma.

Chronic granulomatous disease, a disorder of host defense, is characterized by an impairment in the killing of microbes that results from a defect in the production of superoxide anion by phagocytes. We examined the efficacy of interferon gamma, a physiologic activator of phagocytic-cell function, in the treatment of the disease. Two subcutaneous injections of recombinant interferon gamma (0.1 mg per square meter of body-surface area per dose) were administered on consecutive days to four patients with the X-linked form of the disease. Treatment resulted in 5- to 10-fold increases in superoxide production by granulocytes and monocytes; the improvement was sustained for more than two weeks. Granulocyte bactericidal activity rose proportionally. In the two most responsive patients, both phagocytic functions reached the normal range of activity. In association with these functional changes, we observed an increase in cellular contents of phagocyte cytochrome b (a critical component of the superoxide-producing oxidase) and immunoreactive cytochrome b heavy chain (the product of the gene that is defective in X-linked chronic granulomatous disease). Levels of cytochrome b detected by spectrophotometry rose from near zero to 10 to 50 percent of normal values. This study demonstrates partial correction of the cellular defects in chronic granulomatous disease by interferon gamma and provides a basis for clinical trials of the agent.

Adult↗
PubMed1988

A human mannose-binding protein is an acute-phase reactant that shares sequence homology with other vertebrate lectins.

Mannose-binding proteins have been isolated from the liver of rats and humans and subsequently been found in the serum of rats, rabbits, and humans. We report the isolation of cDNA clones isolated from a human liver cDNA library that encodes a human mannose-binding protein. The primary structure has three domains: (a) an NH2-terminal cysteine-rich segment of 19 amino acids which appears to be involved in the formation of interchain disulfide bonds that would stabilize multimeric forms of the protein; (b) a collagen-like region consisting of 19 repeats of the sequence Gly-x-y; and (c) a COOH-terminal putative carbohydrate-binding domain consisting of 148 residues. This human mannose-binding protein bears 51% overall homology (allowing three gaps) with a rat mannose-binding protein C and 48% homology (allowing seven gaps) with a rat mannose-binding protein A. Like these homologous rat proteins, the human mannose-binding protein COOH-terminal sequences are homologous to the carbohydrate recognition portion of several other lectin-like proteins including mammalian hepatic receptors, an insect-soluble hemolymph, and a sea urchin lectin found in coelomic fluid. The apoproteins of dog and human surfactant and the human lymphocyte IgE Ec receptor have not been shown to have lectin-like properties, yet by homology are members of this family of lectin-like proteins. The human mannose-binding protein is preceded by a typical hydrophobic signal sequence and its hepatic secretion is induced as part of the acute-phase response consistent with its probable role in host defense.

Acute-Phase Proteins↗
PubMed1988

Induction of phagocyte cytochrome b heavy chain gene expression by interferon gamma.

Phagocytic cells, such as macrophages and polymorphonuclear leukocytes, produce a "respiratory burst" in which oxygen is reduced to superoxide and other active oxygen species responsible for many of the microbicidal, tumoricidal, and inflammatory activities of these cells. Interferon gamma has been shown to augment phagocyte superoxide production, but the molecular mechanisms underlying this effect have remained unknown. Recently a key component of the oxidase, phagocyte cytochrome b, has been characterized as a heterodimer of a 91-kDa glycoprotein and a 22-kDa polypeptide. The present studies examined the effects of human recombinant interferon gamma on the expression of the genes for these components of the cytochrome b. In vitro treatment with interferon gamma substantially increases the level of phagocyte cytochrome b heavy chain gene transcripts in normal polymorphonuclear leukocytes, normal monocyte-derived macrophages, and the monocytic leukemia cell line THP-1. Light chain gene transcripts are less affected. In monocyte-derived macrophages and THP-1 cells, the enhanced expression of the heavy chain gene appears in large part attributable to increased rates of transcription. Treatment of monocyte-derived macrophages with human recombinant interferon alpha (a down-regulator of the respiratory burst) decreased the heavy chain transcript levels; interferon beta produced no detectable change. These findings demonstrate the responsiveness of one essential component of the phagocyte oxidase system to activation by interferon gamma and provide a rationale for its use to augment phagocytic function in chronic granulomatous disease.

Cytochrome b Group↗
PubMed1988

The structure and function of vertebrate mannose lectin-like proteins.

Sugar-specific recognition is now well established as an important determinant of cell-cell interactions and host defence mechanisms. Macrophages, in particular, are known to express a variety of lectin-like proteins that are specific for oligosaccharides terminating in mannose, fucose, galactose and sialic acid. This review focuses on mannose recognition systems. Terminal mannose is rarely found on mammalian cell surfaces whereas it is ubiquitous on the surfaces of lower organisms. Mammals have evolved at least two mechanisms to recognize terminal mannose residues. These are, first, a mannose receptor located on the surface of macrophages and second, a mannose-binding protein found in blood plasma and secreted by hepatocytes. The mannose receptor is a 175K (K = 10(3) Mr) membrane glycoprotein. The receptor binds glycoproteins bearing high-mannose chains avidly at neutral pH but poorly at pH 5-6. The receptor recycles rapidly between the cell surface, where ligand binding occurs, and various acid intracellular compartments, where the ligand is discharged. The pH dependency of binding and the rapid recycling of receptor allow cells to accumulate many ligand molecules over an extended time. Endocytosis via this pathway appears to be regulated since mannose receptor expression is closely regulated. For example, the receptor is absent from monocytes but strongly expressed on 3- to 4-day-old monocyte-derived macrophages. Receptor expression can be up- and down-regulated by anti-inflammatory steroids and gamma interferon, respectively. The mechanisms are poorly understood as is the physiological basis for modulation. The mannose-binding protein is a large oligomeric plasma protein secreted by liver and present in significant amounts in serum. The basic subunit of the mannose-binding protein is a 32K protein, which shows homology with other lectin-like proteins, notably certain surfactant proteins of the lung. The structure of the mannose-binding protein has been deduced from sequence information and DNA cloning experiments. The elaboration of the mannose-binding protein by the liver is regulated; it appears to be an acute phase reactant. Recent evidence suggests that the mannose-binding protein may play some opsonic role in the recognition and killing of organisms. In this review, it is suggested that the mannose receptor and the mannose-binding protein operate 'hand in glove' to deal with infectious agents that express mannose on their surfaces.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Cellular and molecular effects of recombinant interferon gamma in chronic granulomatous disease.

We have examined the potential of IFN-gamma to ameliorate the physiologic defect of CGD by studying its effects on CGD phagocyte superoxide generation, NADPH-oxidase kinetics, and expression of the gene for the phagocyte cytochrome b heavy chain. In vitro treatment with IFN-gamma increased the respiratory burst activity of PMN and macrophages from three patients in two kindreds with type IA (variant, X-linked). Phagocytes from type I (classic, X-linked) and types IIA and III (autosomal recessive) CGD did not respond to IFN-gamma in vitro. Preliminary studies of in vivo treatment of several of the same patients with subcutaneous IFN-gamma demonstrated similar responses. All subjects whose phagocytes had responded in vitro showed complete or partial correction of the CGD defect in superoxide generation for up to 1 month after IFN-gamma administration. One patient with type I CGD with no detectable in vitro response also showed improved phagocyte respiratory burst activity after in vivo IFN-gamma treatment. These studies establish the potential efficacy of IFN-gamma in the treatment of patients with X-linked CGD and provide an example of pharmacologic modulation of gene expression in human disease. The ease of administration and absence of toxicity suggest a place for IFN-gamma as an adjunct to more conventional antimicrobial therapy during acute infections in CGD and perhaps even other congenital and acquired immunodeficiency states.

Cytochrome b Group↗
PubMed1987

Recombinant interferon gamma augments phagocyte superoxide production and X-chronic granulomatous disease gene expression in X-linked variant chronic granulomatous disease.

We examined the potential of interferon gamma (IFN-gamma) to ameliorate the physiologic defect of chronic granulomatous disease (CGD) by studying its effects on CGD phagocyte superoxide generation, NADPH oxidase kinetics, cytochrome b559 content, and expression of X-CGD (the gene for the X-linked disease). Granulocytes and macrophages from three patients in two kindreds with "variant" X-linked CGD (i.e., with very low, but detectable, baseline superoxide-generating activity) responded to IFN-gamma with enhanced nitroblue tetrazolium reduction and two- to eightfold increases in superoxide generation. IFN-gamma did not augment the respiratory burst activity of phagocytes from patients with "classic" CGD (i.e., no detectable baseline superoxide generation) or autosomal variant CGD. Incubation of a responding patient's granulocytes with IFN-gamma nearly doubled the maximal velocity for the NADPH oxidase, but did not change its abnormal Michaelis constant. Although the interferon-treated CGD granulocytes produced superoxide at a rate 40% of normal, the cytochrome b spectrum remained undetectable. IFN-gamma treatment of cultured monocytes from an IFN-gamma-responsive CGD patient increased the steady state level of RNA transcripts from the X-CGD gene from barely detectable up to approximately 5% of normal.

Cytochrome b Group↗
PubMed1986

Interferon alpha/beta selectively antagonises down-regulation of mannosyl-fucosyl receptors on activated macrophages by interferon gamma.

Previous reports have described synergism of various interferon preparations in anticellular and antiviral activity. We report that recombinant interferon (rIFN gamma) and IFN alpha/beta mediate distinct, antagonistic effects on expression of a lectin-like receptor for mannose and fucose (MFR) on mouse peritoneal macrophages (M phi). IFN gamma down-regulates MFR activity, a highly reproducible change in mouse M phi activated to secrete enhanced levels of o-2/H2o2. IFN alpha/beta enhances MFR activity and prevents the action of IFN gamma when added in combination. Antagonism is selective for this M phi activation marker and requires a minimum 4 h exposure period to rIFN gamma, during which IFN alpha/beta can prevent its action.

Animals↗
PubMed1986

Interaction and regulation of macrophage receptors.

Macrophages express distinct plasma membrane receptors for different isotypes of immunoglobulin, bear at least two receptors for cleaved third complement component (CR1 and CR3) and have a lectin-like receptor that mediates endocytosis of glycoproteins or glycoconjugates with terminal mannose or fucose residues (MFR). Interferon-gamma, a macrophage-activating factor, induces effects common to other interferons as well as having unique effects on cell function. The down-regulation of MFR, induction of IgG2a Fc receptors and Class II antigens and enhanced production of superoxide and hydrogen peroxide can be considered interferon-gamma-specific effects on macrophages. Previous reports described synergism of various interferon preparations in anticellular and antiviral effects. However, interferon-alpha/beta can selectively antagonize the down-regulation of macrophage MFR by interferon-gamma. The macrophage MFR and CR3 also play a synergistic role in the uptake of zymosan and Leishmania donovani in the absence of serum. The receptors may act independently or in concert. Cleaved third complement components can be specifically eluted from zymosan particles in the absence of exogenous complement and are derived from the macrophages themselves. These studies indicate a role for macrophage complement in local opsonization of pathogens at extravascular sites and focus on the role of the tissue macrophage in first-line host defence.

Animals↗
PubMed1986

Monoclonal antibodies to a particulate superoxide-forming system stimulate a respiratory burst in intact guinea pig neutrophils.

Monoclonal rat antibodies were produced against a subcellular preparation of phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig neutrophils that retains NADPH-oxidase activity. Two antibodies, 1A10.4 and IG4, were isolated that bind to a surface antigen restricted to guinea pig neutrophils from bone marrow and peritoneal exudate and to macrophages and that trigger a respiratory burst in neutrophils in the presence of cytochalasin B. Intact antibody 1A10.4, subclass IgG2c, can trigger superoxide anion release directly; F(ab')2 fragments of 1A10.4 and intact IG4 require further cross-linking by F(ab')2 fragments of anti-rat immunoglobulin antibody. Both antibodies recognize the same antigen, a proteolipid of apparent molecular mass 10 kDa. Immunoprecipitation of solubilized oxidase activity with 1A10.4 brings down this activity as part of a macromolecular complex. Surface expression of the antigen is increased on treatment of cells with both PMA and cytochalasin B. 1A10.4 also triggers release of the granule enzyme beta-glucuronidase. Triggering of a respiratory burst by the antibodies appears distinct from the PMA and fMet-Leu-Phe signalling systems. These studies indicate that the antigen defined by antibodies 1A10.4 and IG4 becomes associated with the superoxide anion-generating system of neutrophils but may play a more general role in signal transduction in phagocytic cells.

Animals↗
PubMed1986

Neutrophil defect associated with malignant infantile osteopetrosis.

We studied the phagocytes from three infants with malignant osteopetrosis and from their families in an attempt to define further the phagocyte abnormalities associated with this disorder. The rapid membrane potential depolarization in response to the soluble stimuli formylmethionylleucylphenylalanine (FMLP) and phorbol myristate acetate (PMA) served as a measure of neutrophil activation, with 3,3-dipentyloxacarbocyanine (diOC5 [3]) used as the probe. A fluorescence-activated cell sorter (FACS) allowed us to use small volumes of blood in a new quantitative evaluation of neutrophil response. The neutrophils from the infants with malignant osteopetrosis were very minimally activated with either stimulus, as demonstrated by incomplete membrane potential depolarization (10% to 15% of normal controls). This finding indicates that malignant osteopetrosis is accompanied by a significantly reduced neutrophil response to stimulation. The abnormal activation process was also reflected in the respiratory burst response of the patients' neutrophils and monocytes. Fifty percent to 60% of the infants' neutrophils totally failed to reduce nitro blue tetrazolium dye (NBT), 30% to 40% of the cells showed only slight reduction after PMA or FMLP stimulation, and only 5% to 10% demonstrated normal reduction. Peripheral blood monocytes failed to reduce NBT in 35% to 70% of the cells tested. Similar testing of granulocyte-macrophage colonies grown in vitro from circulating progenitor cells also showed an abnormal distribution of response to PMA, with a majority of colonies showing a decrease or absence of NBT reduction. Thus control of expression of the osteopetrotic defect occurs at or before the progenitor cell level.

Flow Cytometry↗