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Biomedical subjects

T Ganz

Publications and source records attributed to T Ganz.

126 records · Page 7Linked to original sources
PubMed1988

Microbicidal/cytotoxic proteins of neutrophils are deficient in two disorders: Chediak-Higashi syndrome and "specific" granule deficiency.

Although several genetic defects are known to impair oxidative microbicidal/cytotoxic mechanisms in human PMN, no deficiencies of PMN granule components that mediate oxygen-independent microbicidal activity have yet been reported. We analyzed PMN from patients with various granulocyte disorders for their content of two azurophil granule constituents, defensins and cathepsin G, that exert microbicidal/cytotoxic activity in vitro, and one component, elastase, that has ancillary microbicidal/cytotoxic activity. PMN from two (of two) patients with specific granule deficiency (SGD) displayed an almost complete deficiency of defensins, which in normal cells constitute greater than 30% of the protein content of azurophil granules. The SGD PMN contained normal or mildly decreased amounts of cathepsin G and elastase. Conversely, the PMN of three (of three) patients with Chediak-Higashi syndrome (CHS) substantially lacked cathepsin G and elastase, but their defensin content was normal or mildly decreased. Both CHS and SGD patients suffer from frequent and severe bacterial infections, and CHS patients frequently develop an atypical lymphoproliferative syndrome. The profound deficiency of PMN components with microbicidal/cytotoxic activity in SGD and CHS may contribute to the clinical manifestations of these disorders.

Blood Bactericidal Activity↗
PubMed1988

Oxygen-independent bactericidal systems. Mechanisms and disorders.

Neutrophils contain microbicidal proteins packaged in numerous cytoplasmic granules. During phagocytosis, granules fuse to phagolysosomes where the granule contents contribute to the destruction and dissolution of ingested bacteria. The properties of the microbicidal proteins cathepsin G, BPI, and defensins are described and contrasted.

Animals↗
PubMed1987

Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMN) contain three antimicrobial and cytotoxic peptides which belong to a family of mammalian granulocyte peptides named defensins. To determine their potential availability for extracellular microbicidal or cytotoxic events, we quantified the extracellular release of defensins after stimulation of human PMN with phorbol myristate acetate and opsonized zymosan. As determined by enzyme immunoassay and confirmed by polyacrylamide gel electrophoresis and densitometry, 10(6) human PMN contained 4 to 5 micrograms of defensins. After stimulation with a high concentration of phorbol myristate acetate (1 microgram/ml), about 8% of PMN defensins were found in the media. Release of defensins correlated best with the release of azurophil granule marker beta-glucuronidase or elastase and poorly with the release of either the specific granule marker lactoferrin or cytoplasmic lactate dehydrogenase. Phagocytosis of opsonized zymosan resulted in the extracellular release of less than 3% of PMN defensins. The factors responsible for less release of defensins into media relative to the release of other azurophil granule proteins may include heterogeneity of azurophil granules and the affinity of defensins for cellular surfaces and opsonized particles. In vivo, defensins are most likely to reach effective microbicidal or cytotoxic concentrations in PMN-rich exudates (pus), in confined environments of the phagolysosomes, or in intercellular clefts between PMN and their targets.

Blood Proteins↗
PubMed1987

Defensins mediate the microbicidal activity of human neutrophil granule extract against Acinetobacter calcoaceticus.

An acid extract of human neutrophil granules was fractionated on a Sephadex G-100 column and tested for microbicidal activity against Acinetobacter calcoaceticus HO-1 as described previously (M.C. Modrzakowski and C. M. Paranavitana, Infect. Immun. 32:668-674, 1981). The low-molecular-weight protein fraction, peak D, accounted for about 30% of the protein and nearly all of the activity of the crude extract against strain HO-1. Peak D protein proved to be a mixture of the three human defensin peptides HNP1, HNP2, and HNP3. Purified defensins reproduced the microbicidal activity of peak D. The data suggest that defensins could play a major role in the killing of A. calcoaceticus by human neutrophils.

Acinetobacter↗
PubMed1987

Defensin-rich dense granules of human neutrophils.

Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.

Blood Proteins↗
PubMed1986

In vitro killing of spores and hyphae of Aspergillus fumigatus and Rhizopus oryzae by rabbit neutrophil cationic peptides and bronchoalveolar macrophages.

The fungicidal activity of six rabbit neutrophil cationic peptides (NP) against resting (dormant) spores, preincubated (swollen) spores, and hyphae of Aspergillus fumigatus and Rhizopus oryzae was examined. Whereas resting spores of both fungi were totally resistant to killing by up to 500 micrograms of NP/ml, preincubated spores and hyphae were sensitive, under optimal conditions, to less than 1 microgram /ml. NP-1 and NP-2, also found in rabbit bronchoalveolar macrophages, were the most active of the peptides. Activity was inhibited with increasing ionic strength, calcium but not magnesium, incubation at 4 C, and the chitin fragments chitobiose and chitotriose. 125I-labeled NP-1 bound to all three forms of A. fumigatus. Finally, killing of A. fumigatus conidia by rabbit bronchoalveolar macrophages was faster and greater if the conidia were first preincubated in RPMI 1640 medium. Thus, although NP did not kill the resting A. fumigatus conidia, a role for cationic peptides in macrophage-mediated killing is not precluded, as fungicidal activity of both isolated cationic peptides and intact bronchoalveolar macrophages appeared to occur only after the conidia became activated.

Animals↗
PubMed1986

Synergistic activity of rabbit granulocyte peptides against Candida albicans.

Rabbit granulocytes contain six antimicrobial peptides that are structurally homologous to the human neutrophil "defensins." NP-5, a rabbit defensin, lacks significant activity against Candida albicans. Nevertheless, its addition to submicromolar concentrations of rabbit NP-1, NP-2, or NP-3a potentiates their candidacidal effect. Thus, granulocyte defensins can act synergistically against potential pathogens.

Animals↗
PubMed1986

In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes.

We examined the activity of defensins, cysteine-rich cationic peptides that are abundant in the cytoplasmic granules of human and rabbit granulocytes, against various tumor targets. The three human defensins, HNP-1, HNP-2, and HNP-3, lysed human and murine targets in chromium release and dye exclusion assays. Defensin-mediated tumor cell lysis was concentration-dependent, inhibited by serum, and dependent on temperature-sensitive events. Lysis was first detected by three hours of incubation and it reached a plateau between eight and 14 hours. In vitro exposure of murine teratocarcinoma cells to HNP 1-3 abrogated their oncogenicity in vivo. Nonmalignant target cells were also susceptible to defensin-mediated lysis. Four rabbit granulocyte defensins exerted marked (NP-1, NP-2) or moderate (NP-3a, NP-3b) cytotoxic activity, whereas defensin NP-5 was not cytotoxic. When tumor cells were incubated with human defensins in combination with hydrogen peroxide, synergistic cytotoxicity was detected. As defensins are released from granulocytes by various stimuli, their release could contribute to extracellular cytotoxicity which is independent of reactive oxygen intermediates.

Animals↗
PubMed1985

Activity of rabbit leukocyte peptides against Candida albicans.

Six related cysteine-rich, low-molecular-weight peptides were purified from rabbit peritoneal granulocytes and tested in vitro for fungicidal activity against Candida albicans. Two peptides (NP-1 and NP-2) were highly effective, one (NP-3a) was moderately active, and three (NP-3b greater than NP-4 much greater than NP-5) had substantially less potency. There was a general, but imperfect, correlation between the candidacidal potency of each peptide and its net cationic charge. Candidacidal activity by NP-1 was concentration and time dependent and occurred rapidly under optimal low-ionic-strength conditions. It was inhibited by increasing either the ionic strength or Ca2+ concentration of the incubation mixtures, but was relatively unaffected by Mg2+. Candidacidal activity was independent of H+ concentrations between pH 5 and 8, but decreased below pH 5. Candidacidal activity was temperature sensitive and was virtually abolished when NP-1 was incubated with C. albicans at 0 degrees C. Cysteine-rich antimicrobial peptides such as NP-1 and NP-2 may equip leukocytes to deal with infections caused by C. albicans and other fungi that are susceptible to their microbicidal effects.

Age Factors↗
PubMed1985

Correlation of binding of rabbit granulocyte peptides to Candida albicans with candidacidal activity.

NP-1, a candidacidal peptide purified from rabbit granulocytes, bound extensively and with biphasic kinetics to Candida albicans. The primary phase of binding was temperature independent and occurred even at 0 degrees C. This primary binding was relatively specific, reversible, saturable, and of high capacity. It was inhibited by increased salt concentrations in the incubation medium, but was relatively unaffected by increasing the calcium ion concentration or by lowering the incubation temperature to 0 degrees C. The secondary phase of binding was only noted under conditions that supported candidacidal activity. Secondary binding was inhibited by millimolar concentrations of calcium, but not magnesium, ions and did not occur at 0 degrees C or when subtoxic concentrations of NP-1 were tested. NP-2 and NP-3a, other potent candidacidal peptides from rabbit granulocytes, also bound directly and extensively to C. albicans and competed for binding with NP-1. NP-4 and NP-5, less candidacidally active homologs of the aforementioned peptides, showed relatively little direct binding activity and competed poorly for binding with NP-1 or NP-2. NP-3b, another less candidacidal homolog, bound extensively to C. albicans, but did not compete effectively with NP-1 or NP-2. By comparing candidacidal and binding activity of the peptides, we conclude that the candidacidal activity of NP-1 involves primary binding to C. albicans followed by postbinding events that are temperature dependent and inhibitable by calcium ions.

Age Factors↗
PubMed1985

Direct inactivation of viruses by MCP-1 and MCP-2, natural peptide antibiotics from rabbit leukocytes.

Six homologous peptides were purified to homogeneity from rabbit granulocytes or alveolar macrophages and tested for their ability to inactivate herpes simplex virus type 1 (HSV-1). Two of the peptides, MCP-1 and MCP-2, showed considerable in vitro neutralizing activity, whereas four structurally homologous peptides (NP-3a, NP-3b, NP-4, and NP-5) were relatively ineffective. Inactivation of HSV-1 by MCP-1 or MCP-2 depended on peptide concentration and on the time, temperature, and pH of the incubation. HSV-2, vesicular stomatitis virus, and influenza virus A/WSN were also susceptible to direct neutralization by MCP-1 or MCP-2, whereas cytomegalovirus, echovirus type 11, and reovirus type 3 were not. We speculate that MCP-1 and MCP-2, peptides that are abundant in rabbit granulocytes and lung macrophages, may contribute to antiviral defenses by mediating the direct inactivation of HSV-1 and selected other viruses.

Animals↗
PubMed1985

Newborn rabbit alveolar macrophages are deficient in two microbicidal cationic peptides, MCP-1 and MCP-2.

Adult rabbit alveolar macrophages (AM) contain 2 cationic peptides with a broad spectrum of antimicrobial activity in vitro against bacteria, fungi, and some enveloped viruses. We determined the amounts of both peptides qualitatively in 1-day-old (1d), 7-day-old (7d), 21-day-old (21d), and adult rabbit AM and found that 1d AM were deficient in both peptides. The levels of MCP-1 extractable from AM were quantitated relative to known standards of purified peptides and were found to increase 6-fold between 1d and 21d AM. Adult AM yielded 9 times as much MCP-1 as did 1d AM despite nearly the same acid-extractable protein content per cell. Using immunoperoxidase techniques we showed that the deficiency of MCP-1 and MCP-2 involves 1d AM uniformly and that all AM 7 days or older have detectable MCP. Seven-day-old AM (and to a lesser extent 1d AM) incorporated 35S-cysteine into intracellular MCP in cell culture, indicating that AM actively synthesize these peptides. The deficiency of these antimicrobial substances may contribute to functional immaturity of newborn rabbit AM.

Aging↗
PubMed1985

Defensins. Natural peptide antibiotics of human neutrophils.

We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.

Amino Acids↗
PubMed1985

Primary structures of three human neutrophil defensins.

The primary structures of three human neutrophil antimicrobial peptides (HNP) were determined. The peptides, HNP-1, HNP-2, and HNP-3, which we have termed defensins, were rich in cystine, arginine, and aromatic residues, but were devoid of free sulfhydryl groups and carbohydrate moieties. They were 29-30 residues in length and identical in sequence in all but their amino terminal residues. The defensins were homologous in sequence to peptides of similar size and biological activity previously purified from rabbit polymorphonuclear leukocytes, but unrelated to other neutrophil proteins of known sequence. 11 amino acid residues of the human defensins, including all six cysteinyl residues, were invariantly conserved in the six rabbit members of this multigene peptide family. That similarly structured antimicrobial peptides are present in both rabbit and human leukocytes supports their purported role as cidal agents in phagocyte-mediated host defense.

Amino Acid Sequence↗
PubMed1983

Molecular cloning of the rat metallothionein 1 (MT-1) mRNA sequence.

Starting with liver mRNA from cadmium chloride-treated rats, we constructed a cDNA library. A mouse metallothionein 1 (MT-1) cDNA restriction fragment (Durnam et al. 1980) was used as a hybridization probe to isolate from this library a cDNA (p2A10) which is a nearly complete copy of the rat MT-1 mRNA sequence. Southern analysis of rat DNA, using p2A10 as a probe, together with the screening of a rat genomic library, reveal that the rat MT-1 gene belongs to a family of related sequences. The rat MT-1 gene is inducible both by glucocorticoids and by metals.

Animals↗
PubMedDate not supplied

Oxygen-independent microbicidal mechanisms of phagocytes.

The principal biological function of phagocytic cells is the destruction of invading microorganisms. Following phagocytosis, microbes are exposed to multiple antimicrobial substances ranging in complexity from simple oxygen radicals to large proteins. These substances disrupt various microbial structures and eventually kill and digest most of the invaders. This review is focused on oxygen-independent microbicidal mechanisms in granulocytes and macrophages.

Allergy and Immunology↗