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T Ganz

Publications and source records attributed to T Ganz.

At least 37 records · Page 2Linked to original sources
PubMed1993

The effect of biotinylation on the antigenic specificity of anti-defensin monoclonal antibodies.

We studied the effects of biotinylation on three monoclonal antibodies (Mabs) that were raised against carrier protein conjugates of human defensin HNP-1, and of rabbit defensins NP-2 and NP-5 respectively. Before biotinylation, each Mab specifically bound to its peptide hapten. Biotinylation of these Mabs by the N-hydroxysuccinimide-biotin (NHS-biotin) resulted in crossreactivity of each Mab with the two irrelevant defensin peptides. In contrast, Mab specificity was preserved after biotinylation with biotin hydrazide, which links biotin to the glycan moiety of antibodies. The effects of NHS-biotinylation were in part mimicked by 2,4-dinitrofluorobenzene, another agent that modified primary amine groups of proteins, suggesting that this modification contributed to the change in antibody specificity. When a high degree of antigenic specificity against peptide immunogens is required, biotinylation on the glycan moiety of Mabs may be preferable.

Amino Acid Sequence↗
PubMed1993

Defensins: antimicrobial and cytotoxic peptides of mammalian cells.

Defensins are antimicrobial and cytotoxic peptides that contain 29-35 amino acid residues, including six invariant cysteines whose intramolecular disulfide bonds cyclize and stabilize them in a complexly folded, triple-stranded beta-sheet configuration. Generated by the proteolytic processing of 93-95 amino acid precursor peptides, the constitute > 5% of the total cellular protein in human and rabbit neutrophils (polymorphonucleated neutrophils--PMN) and are also produced by rabbit lung macrophages and by mouse and rabbit small intestinal Paneth cells. Despite their prominence in rat PMN, defensins are not found in murine PMN. The antimicrobial spectrum of defensins includes gram positive and gram negative bacteria, mycobacteria, T. pallidum, many fungi, and some enveloped viruses. Defensins exert nonspecific cytotoxic activity against a wide range of normal and malignant targets, including cells resistant to TNF-alpha and NK-cytolytic factor. They appear to kill mammalian target cells and microorganisms by a common mechanism, which involves initial electrostatic interactions with negatively charged target cell surface molecules (likely the head groups of polar membrane lipids), followed by insertion into the cell membranes which they permeabilize, forming voltage-regulated channels. In addition to their antimicrobial and cytotoxic properties, some defensins act as opsonins, while others inhibit protein kinase C, bind specifically to the ACTH receptor and block steroidogenesis or act as selective chemoattractants for monocytes. Defensins are a newly delineated family of effector molecules whose contribution to host defense, inflammation, and cytotoxicity may be considerable for humans, even though it is unlikely to be revealed by experimentation with mice.

Amino Acid Sequence↗
PubMed1993

Defensins reduce the barrier integrity of a cultured epithelial monolayer without cytotoxicity.

Polymorphonuclear leukocytes (PMN) contribute to epithelial injury at sites of inflammation, but their mechanisms of action are incompletely understood. PMN can injure target tissues by oxidative and nonoxidative mechanisms. Included in the nonoxidative mechanisms are defensins (DEF), small (3.5 to 4.0 kD), arginine- and cysteine-rich polypeptides. DEF are bactericidal, fungicidal, viricidal, and tumoricidal, but their ability to contribute to inflammatory injury has not been extensively evaluated. One marker of inflammatory injury is disrupted epithelial barrier integrity. Using Madin-Darby canine kidney (MDCK) epithelial monolayers, we measured the effect of both human and rabbit DEF on barrier integrity using mannitol permeability (Pmann) and transepithelial electrical resistance (Rt). Human DEF (HNP1-3, 2:2:1 molar ratio) increased Pmann in a time- and concentration-dependent manner and Rt fell progressively over a 48-h period after exposure of monolayers to HNP1-3. Rabbit DEF peptide 1 (NP-1) also increased Pmann, but rabbit peptide 5 (NP-5) had no effect on Pmann. To investigate the role of charge, HNP1-3 was added to the monolayers with the polyanions heparin or sulfated dextran. Heparin and sulfated dextran only partially inhibited the increase in Pmann. Fetal bovine serum (FBS), however, completely inhibited the effect of HNP1-3, but this protection was only partially explained by the anionic protein, albumin. The FBS protection was time dependent and was present when FBS was added up to 16 h after exposure to HNP1-3. While both HNP1-3 and NP-1 increased epithelial permeability, neither were cytolytic to MDCK cells as measured by lactate dehydrogenase release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Macrophage function.

Macrophages and their secretory products play a central role in host defense against pathogenic microbes in the development of protective immunity, in pathogenesis of septic shock and of acute and chronic infections, and in the pathophysiology of wound healing, tissue remodeling, and fibrosis. This review provides a bird's-eye view of the vast literature of the macrophage.

Animals↗
PubMed1993

Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis.

We measured concentrations of defensins (human neutrophil peptides) in the plasma of healthy volunteers and patients with sepsis and meningitis. When a sensitive enzyme immunoassay was used, defensins were detected in plasma samples from 13 of 24 healthy blood donors, with a mean +/- SD of 42 +/- 53 ng/ml. Defensin levels in plasma samples from seven patients with sepsis at the onset of disease ranged from 900 ng/ml to 170,000 ng/ml. In 10 patients with meningitis in the initial phase of disease, plasma defensin concentrations ranged from 120 ng/ml to 910 ng/ml. Defensin concentrations in the plasma of both patient groups were significantly higher than those in healthy blood donors (p << 0.01), and patients with sepsis had higher defensin levels than patients with meningitis (p < 0.01). Defensin levels were significantly (p < 0.01) lower after the beginning of specific antibiotic therapy. Defensin concentrations in the plasma of patients with sepsis and meningitis correlated only weakly (r = 0.38) with blood neutrophil count. In vitro studies of defensin added to plasma indicated that all defensin was bound to plasma proteins. At high concentrations (1000 micrograms/ml), defensins caused precipitation of plasma proteins. Because plasma defensin levels may reflect neutrophil activation at sites of infection and inflammation, studies of the clinical utility of defensin ELISA are indicated.

Bacteremia↗
PubMed1992

Posttranslational processing of defensins in immature human myeloid cells.

Human neutrophil promyelocytes synthesize, process, and package several microbicidal proteins, including the highly abundant defensins, into their azurophil granules. As deduced from their cDNA sequences, defensins are initially synthesized as 94 amino acid (aa) precursors that must undergo extensive processing. We performed metabolic labeling studies of defensin synthesis in the human promyelocytic cell line HL-60 and in chronic myeloid leukemia cells, and showed that preprodefensins are processed to mature 29 to 30 aa defensins over 4 to 24 hours via two major intermediates: a 75 aa prodefensin generated by the cleavage of the signal sequence, and a 56 aa prodefensin that results from a subsequent preaspartate proteolytic cleavage. Almost all of the 75 aa form was found in the cytoplasmic/microsomal fraction, whereas the 56 aa prodefensin and mature defensins predominated in the granule-enriched fraction. The 75 aa prodefensin was also selectively released into the culture supernatant. Treatment of HL-60 cells with monensin, chloroquine, or ammonium chloride, substances that neutralize acidic subcellular compartments, partially blocked conversion of the 75 aa prodefensin into 56 aa prodefensin, but did not increase the extracellular release of the 75 aa form. Further studies will be required to determine the role of this processing pathway in subcellular targeting to azurophil granules and avoidance of autocytotoxicity.

Amino Acid Sequence↗
PubMed1992

Defensins: endogenous antibiotic peptides from human leukocytes.

A variety of endogenous antimicrobial peptides equip mammals, amphibians, insects and plants to defend themselves against microbial pathogens. Defensins are small peptides of mammalian cells that contain 29-35 amino acid residues, including six invariant cysteines that form three intramolecular disulphide bonds. They are produced by the sequential proteolysis of precursors that contain approximately 95 amino acids and are synthesized by several types of cells, especially the bone marrow precursors of blood neutrophils. Defensins constitute 5-7% of the total protein in human neutrophils and are present in high concentration in the azurophil granules and phagocytic vacuoles. The production of defensins by human neutrophils amounts to approximately 10 mg/kg body weight per day. In certain mammalian species lung macrophages and specialized epithelial (Paneth) cells in the small intestine also produce defensins. Defensins are complexly folded, amphipathic, rich in antiparallel beta-sheet but devoid of alpha-helical domains. Their unusually broad antimicrobial spectrum encompasses Gram-positive and Gram-negative bacteria, many fungi, mycobacteria, spirochetes and several enveloped viruses. The antimicrobial properties of defensins result from their insertion into target cell membranes and the formation of voltage-sensitive channels. Given their abundance and broad spectrum of antimicrobial activity, defensins may contribute substantially to innate resistance to infection.

Amino Acid Sequence↗
PubMed1992

Cationic defensins arise from charge-neutralized propeptides: a mechanism for avoiding leukocyte autocytotoxicity?

Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the principal constituents of cytoplasmic granules of mammalian neutrophils and certain macrophages. To identify conserved structural features of defensin precursors that may be important for their targeting to cytoplasmic granules or for prevention of autocytotoxicity, we isolated and sequenced three neutrophil-specific rabbit defensin cDNAs that code for preproprotein precursors to the mature defensins NP-3a, NP-4, and NP-5. The preprodefensins NP-3a, NP-4, and NP-5, like the previously characterized preprodefensins, lack consensus sequences for N-linked glycosylation, suggesting that defensins are targeted to lysosome-like granules by a mechanism not dependent on the mannose-6-phosphate receptor. Analysis of all seven known myeloid prodefensins revealed a structure wherein an anionic propiece neutralizes the cationicity of the mature peptide. Because defensins apparently require cationic epitopes for cell membrane permeabilization and cytotoxicity, charge neutralization of mature peptides by their anionic propieces may prevent autocytotoxicity during defensin synthesis and processing.

Amino Acid Sequence↗
PubMed1992

Activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare.

We have examined the activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare. M. avium-M. intracellulare at 2.5 x 10(6)/ml or 2.5 x 10(8)/ml was cultured in the presence of defensins at 37 degrees C from 4 to 48 h. After incubation, CFU were enumerated. Human neutrophil peptide 1 (HNP-1) at 5 micrograms/ml had the ability to kill M. avium-M. intracellulare. Treatment with HNP-1 resulted in significant (96.3 to 97.7%) killing of M. avium-M. intracellulare, even after taking clumping into consideration. This activity was not affected by the presence of calcium (0.5 and 1.0 mM), magnesium (0.5 and 1.0 mM), or sodium chloride (25, 50, and 100 mM). The optimal pH for bactericidal activity was higher than 5. We tested numerous M. avium-M. intracellulare strains, and HNP-1 was successful in killing every strain, although the degree of killing varied among them (34.2 to 87.2%). Additionally, this activity was independent of colonial morphology. We also examined the activity of HNP-2 and HNP-3 against M. avium-M. intracellulare and found that they were as effective in killing M. avium-M. intracellulare as HNP-1 was. These observations suggest that defensins may play an important role in the host defense against M. avium-M. intracellulare.

Blood Bactericidal Activity↗
PubMed1991

An enzyme immunoassay for human defensins.

We developed and optimized an enzyme immunoassay for human neutrophil defensins, cationic cysteine-rich peptides that participate in host defense and inflammation. The assay utilizes a sandwich design with a monoclonal capture antibody and a biotinylated monoclonal detecting antibody. Cetrimonium bromide is employed to obviate non-specific binding of defensins to surfaces. The assay has a sensitivity of 0.04-0.05 ng/ml and a working range of 0.05-10 ng/ml.

Antibodies, Monoclonal↗
PubMed1991

Contribution of rabbit leukocyte defensins to the host response in experimental syphilis.

In the companion paper (L. A. Borenstein, M. E. Selsted, R. I. Lehrer, and J. N. Miller, Infect. Immun. 59:1359-1367, 1991), we report that rabbit alveolar macrophage and neutrophil derived defensins possess antimicrobial activity against Treponema pallidum subsp. pallidum, the etiologic agent of syphilis. In this study, antisera specific for NP-1 and NP-2 (defensins present in certain macrophages and polymorphonuclear leukocytes) and NP-5 (a defensin produced only in neutrophils) were used to detect these peptides by immunoperoxidase staining in testicular lesions from infected rabbits. Profound amounts of cell-free and cell-associated defensins were detected in the tunica albuginea and interstitial spaces during the first 24 h of infection. The presence of defensins was transient and almost undetectable by day 4. Interstitial defensins were detected again at day 10 and increased through day 16, at which time lesion healing was evident by hematoxylin and eosin staining. The appearance and increase in detectable defensins between days 10 and 16 of infection correlated with a reduction in numbers and disappearance of T. pallidum, as demonstrated by using silver staining. The extent and pattern of immunostaining for NP-1 and NP-2 corresponded with immunostaining for NP-5 and identified neutrophils as the cellular source of the defensins. These findings indicate that defensins may contribute to the control of local T. pallidum infection and suggest a role for acute inflammatory processes in the resolution of early experimental syphilis.

Animals↗
PubMed1991

Activated alpha 2-macroglobulin is a principal defensin-binding protein.

Defensins are highly abundant and variably cationic peptides that possess antimicrobial, cytotoxic, and chemoattractant properties and equip mammalian phagocytes for participation in host defense and inflammatory processes. We studied the binding of the human defensin HNP-1 by proteins in plasma and serum and identified activated (F-form) alpha 2-macroglobulin (alpha 2M) as a principal binding protein for HNP-1. In contrast, native (S-form) alpha 2M bound little HNP-1. The binding of HNP-1 by F-form alpha 2M was resistant to salt and boiling in 2% sodium dodecyl sulfate but was ablated by dithiothreitol. Pretreatment of methylamine-activated serum or plasma by iodoacetamide substantially decreased the binding of HNP-1 to alpha 2M, suggesting that thiol groups in activated alpha 2M play a role in defensin binding, possibly by covalently trapping defensins via thiol-disulfide exchange. Western blots of conventionally collected sera showed endogenous defensins complexed with the F-form of alpha 2M, indicating that the generation of defensin-alpha 2M complexes was not limited to the in vitro model of methylamine-activated serum or plasma and radiolabeled HNP-1. Previous studies indicated that native alpha 2M can be converted to its F-form by many proteases, including those released by neutrophils and platelets, and that the F-form is recognized and internalized by specific receptors on macrophages and hepatocytes. Our findings suggest that the alpha 2M system may function as a scavenger of defensins and other peptide mediators in inflamed tissues and may constitute an important mechanism for the regulation and containment of inflammation.

Blood Bactericidal Activity↗
PubMed1990

Purification and characterization of a major phosphatidylserine-binding phosphoprotein from human platelets.

We describe the isolation, lipid-binding properties and partial amino acid sequence of PS-p68, a novel 68 kDa phosphatidylserine-binding protein from human platelets. PS-p68 is an abundant constituent of platelets, accounting for 0.5-0.75% of total cell protein. It was purified from platelet cytosol by affinity chromatography. Amino acid sequence analysis yielded no similarity to identified proteins. In contrast with most known phospholipid-binding proteins, PS-p68 does not bind Ca2+ and does not require Ca2+ for its binding of phosphatidylserine. Phosphatidylserine binding to PS-p68 was inhibited by phosphatidic acid and by alkylphospholipids. PS-p68 was isolated as a major phosphoprotein from 32P-labelled platelets and was found to function as a protein kinase C substrate in vitro. However, treatment of intact platelets with phorbol 12-myristate 13-acetate, thrombin or carbacyclin did not increase PS-p68 phosphorylation. Platelets appear to be the only blood cells containing PS-p68, which was not detected in neutrophils, monocytes and lymphocytes.

Amino Acid Sequence↗
PubMed1990

A distinct subset of antineutrophil cytoplasmic antibodies is associated with inflammatory bowel disease.

Antineutrophil cytoplasmic antibodies (ANCAs) have recently been demonstrated to be of importance in Wegener's granulomatosis and certain other forms of vasculitis associated with glomerulonephritis. With a fixed-cell ELISA, we demonstrated that ANCAs occur in the serum of patients with inflammatory bowel disease (IBD) involving the colon. In a blinded study, sera from 21 of 25 patients with ulcerative colitis (UC) and five of 25 patients with Crohn's disease had binding in the fixed-cell ELISA. The five reactive sera from patients with Crohn's disease were associated with the presence of clear colonic involvement. The presence of ANCA in patients with UC was not influenced by disease distribution or activity. Indeed, such antibodies were present in four subjects with UC more than 5 years after colectomy. The IBD-associated ANCAs were distinct from ANCAs reported in patients with Wegener's granulomatosis since the pattern of staining on indirect immunofluorescence exhibited a nongranular perinuclear distribution (P-ANCA). The P-ANCA observed in IBD did not react with myeloperoxidase and thus was distinct from the P-ANCA observed in vasculitis with cresentric glomerulonephritis. IBD and, in particular, UC, is associated with a distinct subset of P-ANCA, which may have important diagnostic and potential pathophysiologic implications.

Adult↗