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Biomedical subjects

Z A Cohn

Publications and source records attributed to Z A Cohn.

At least 73 records · Page 4Linked to original sources
PubMed1986

Local and systemic effects of intradermal recombinant interferon-gamma in patients with lepromatous leprosy.

Evidence that interferon-gamma may be a physiologic macrophage-activating factor, and that macrophage activation may be defective in lepromatous leprosy, led us to test the effects of intradermal injection of low doses of recombinant interferon-gamma in six patients with this disease. Interferon-gamma, 1 or 10 micrograms, was administered daily by jet gun for three days into a single cutaneous lesion. A biopsy specimen was taken from the injection site on the sixth study day and compared with specimens obtained previously from a site where no injection had been made or where excipient alone had been injected in the same way as the interferon. Interferon-gamma elicited local effects similar to certain features of delayed-type hypersensitivity reactions or tuberculoid leprosy, including induration, T-cell and monocyte infiltration, keratinocyte proliferation, diminution of epidermal Langerhans cells, and dermal and epidermal cell HLA-DR (Ia) antigen expression. At some of the sites of interferon-gamma injection, there was also an apparent decrease in acid-fast bacilli. Before treatment, monocytes from patients with lepromatous leprosy released 48 percent as much hydrogen peroxide as did monocytes from controls in response to phorbol myristate acetate, and 36 percent as much as those from controls in response to Mycobacterium leprae. When recombinant interferon-gamma was injected, these responses became normal. No toxic effects were observed. These observations suggest that interferon-gamma can mediate certain manifestations of delayed-type hypersensitivity or cell-mediated immunity in vivo, and that recombinant interferon-gamma should be tested for possible therapeutic effects in certain nonviral infectious diseases.

Adult↗
PubMed1986

Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules.

Histocompatibility-restricted cytotoxic T lymphocytes produce circular lesions on target cell membranes. The pore-forming protein (PFP or perforin 1) that forms these membrane lesions has been purified from lymphocytes. At 37 degrees C, in the presence of Ca2+, this protein polymerizes into a supramolecular tubular complex of Mr greater than 10(6) that partially resists dissociation by SDS and reducing agents. It incorporates spontaneously into planar lipid bilayers during polymerization to form nonselective ion channels, showing heterogeneous size distribution, the smallest conductance per unit being identified as 400 pS in 0.1 M NaCl. PFP/P1 that had been assembled in lipid vesicles before incorporation into planar bilayer show much larger single channel conductance, ranging from 1 to 6 nS in 0.1 M NaCl, suggesting that PFP/P1 may assume multiple functional sizes in proportion to its state of polymerization. The reconstituted channels are relatively voltage-insensitive, with most channels persisting in the open state for seconds to minutes. Nucleated cells are rapidly depolarized by this protein. The purified protein lyses a variety of tumor cells. Polymerization and functional channel activity are absolutely Ca2+-dependent. The activity of this protein may play a direct role in T lymphocyte-mediated cytolysis.

Animals↗
PubMed1986

Bacterial lipopolysaccharides prime macrophages for enhanced release of arachidonic acid metabolites.

Preincubation of resident peritoneal macrophages with 10-100 ng/ml LPS for 60 min resulted in the cells becoming primed for enhanced (three-to eightfold higher) arachidonic acid (20:4) secretion in response to a variety of triggers. The half-maximal concentration of LPS required for priming was 10 ng/ml irrespective of whether the trigger was particulate (examples: zymosan or immune complexes) or soluble (such as PMA or A23187). Similarly, the time required for half-maximal priming of macrophages was 20 min irrespective of which trigger was used. The primed state persisted for at least 30 h. LPS-priming of macrophages also affected the kinetics of 20:4 metabolite secretion. The lag phase characteristically observed when 20:4 secretion is triggered was reduced in LPS-primed cells. Furthermore, LPS-primed cells secreted 20:4 metabolites when challenged with latex beads, while unprimed cells did not. These data suggest that stimuli such as zymosan, which elicit 20:4 secretion in macrophages, promote two signals, a priming signal and a triggering signal. LPS is capable of establishing the priming signal but not the triggering signal, while latex promotes the triggering signal but is unable to prime the cells for 20:4 release. LPS did not effect the profile of 20:4 metabolites secreted in response to any of the triggers, nor did it effect the profile of products synthesized from exogenously added 20:4, suggesting that it did not regulate the 20:4 cascade at the level of either the cyclooxygenase or lipoxygenase pathways. Macrophages respond to LPS without the intervention of T lymphocytes, since the macrophages from nude mice could be primed for enhanced 20:4 secretion.

Animals↗
PubMed1986

A semiautomated hemolysis microassay for membrane lytic proteins.

A simple, rapid, semiautomated microassay for hemolysis using a microtiter plate spectrophotometric system is described. The assay relies on the differences in light scattering (turbidity) properties of intact and of lysed erythrocytes. Lysis of erythrocyte suspensions in 96-well plates is determined by absorbance at 690 nm. A linear correlation between the percentage of hemolysis and the turbidity decrease is observed, indicating that this assay may be used for both rapid screening and quantitation of the hemolytic activity. This assay allows screening of 300 samples in less than 6 min. Small samples derived from protein fractionation columns (HPLC, for example) can be rapidly screened. This assay has been used in the successful isolation of a cytolytic membrane-lytic protein from the granules of cloned cytotoxic T lymphocytes and NK cells.

Animals↗
PubMed1986

Purification and characterization of a cytolytic pore-forming protein from granules of cloned lymphocytes with natural killer activity.

A cytolytic pore-forming protein (PFP, perforin) was purified from isolated granules of cloned NK-like cytolytic cells, which showed an apparent Mr of 70-75 kd (reduced) and 62-66 kd (nonreduced). Cytolysis produced by this protein occurred only in the presence of Ca2+ and was accompanied by the formation of membrane lesions of 160 A diameter. The purified protein depolarized cells and made lipid vesicles leaky to monovalent and divalent ions. This protein formed large, voltage insensitive and nonselective ion channels in planar bilayers that remained preferentially in the open state. The channels were heterogeneous in size distribution averaging 400 pS/U in 0.1 M NaCl. The membrane lesions formed by PFP were morphologically and functionally similar to those formed by intact NK-like cells and their granules. This PFP could be released from granules during cell killing, followed by its polymerization on target membranes to form large transmembrane pores.

Calcium↗
PubMed1986

Evidence for sequential signals in the induction of the arachidonic acid cascade in macrophages.

We have examined the requirement for Na+, Ca2+, and protein synthesis in the induction of the arachidonic acid (20:4) cascade in cultured murine peritoneal macrophages. Replacement of extracellular Na+ with choline or with K+ inhibited receptor-mediated 20:4 release by 60-90%, but did not inhibit release stimulated by the soluble triggers PMA and A23187. Cells that had preingested zymosan particles in a K+ medium could be induced to secrete 20:4 metabolites merely by changing the medium to one containing Na+. The Ca2+ ionophore A23187 caused cells in Na+-free medium to release and metabolize 20:4 to prostacyclin, PGE2, leukotriene C, and hydroxyeicosatetraenoic acids, suggesting that the phospholipase(s), cyclooxygenase, and lipoxygenase enzymes do not have a requirement for extracellular Na+. These data suggest that receptor-mediated 20:4 secretion has a requirement for extracellular Na+, while 20:4 release triggered by soluble stimuli do not. Immune complex- and A23187-induced 20:4 release was absolutely dependent on extracellular Ca2+. PMA-triggered 20:4 secretion was inhibited 50% in Ca2+-free medium, but could be inhibited completely by preloading the cells with the Ca2+ antagonist quinine. Protein and RNA synthesis was required for 20:4 release induced by zymosan, immune complex, and PMA, but not by A23187. Cycloheximide and emetine were effective within 15 min of addition, while actinomycin D was an effective inhibitor within 45 min. We suggest that receptor-mediated signal response coupling in the 20:4 cascade in macrophages comprises a sequential series of signals that includes an Na+ influx, synthesis of a rapid turnover-protein, and finally an increase in intracellular Ca2+.

Animals↗
PubMed1986

Bacterial lipopolysaccharides modify signal transduction in the arachidonic acid cascade in macrophages.

Macrophages are a potent source of arachidonic acid (20:4) metabolites. When macrophages interact with an appropriate stimulus, phospholipase activity is induced, resulting in the liberation of 20:4 from the membrane phospholipid and its quantitative oxygenation via either the lipoxygenase or cyclooxygenase pathways. We have attempted to dissect the molecular events coupling the initial membrane-perturbing signal to the phospholipase activity. Using a variety of stimuli and uncoupling agents we have found that receptor-mediated 20:4 release is triggered by a series of sequential signals, including ligand-receptor binding, receptor clustering, Na+-dependent events, the synthesis of a rapidly turning over protein and finally an influx of Ca2+ into the cell. Bacterial lipopolysaccharides (LPS) are poor triggers of the 20:4 cascade. However, pretreatment of cells with LPS leads to the establishment of a 'primed' or 'intermediate' state which can act synergistically with subsequent signals. Hence, the amount of 20:4 metabolites secreted in response to a variety of triggers is increased 3-10-fold in LPS-primed cells, and the lag phase usually observed in 20:4 secretion disappears. The observations presented suggest a two-stage mode of signalling in the receptor-mediated induction of the 20:4 cascade.

Animals↗
PubMed1986

Analysis of enzymatic activities of subcellular and chromatographic fractions by an automated colorimetric microassay system.

A simple, automated colorimetric microassay system has been designed to quantitate enzyme activities commonly used as markers for subcellular compartments. This system relies on the spectrophotometric reading of microtiter wells containing the chromophore products. The microassay allows rapid, economical, and quantitative analysis of enzyme activities associated with sucrose or Percoll gradient fractions used for subcellular fractionation studies as well as the screening of a large number of fractions derived from HPLC and other separation columns used for enzyme purification. We describe its use for the quantitation of activities associated with acid and alkaline phosphatases, alkaline phosphodiesterase, beta-glucuronidase, alpha-N-acetylglucosaminidase, alpha-mannosidase, alpha-L-fucosidase, glycosidases, serine esterases, and succinate dehydrogenase, and give the range of their sensitivities. This microassay system has been applied to the isolation of granules of cytolytic lymphocytes and to the identification and purification of a serine esterase from the isolated granules of these cells.

Chromatography↗
PubMed1986

Functional channel formation associated with cytotoxic T-cell granules.

Lymphocyte granules from cytotoxic T-lymphocyte lines A2, A11, and R8 were enriched by subcellular fractionation using a Percoll gradient. Granule-enriched fractions showed potent hemolytic activity in the presence of Ca2+. Isolated granules induced rapid Ca2+-dependent membrane depolarization of J774 macrophage-like cells. When tested in planar bilayers, granules induced the formation of Ca2+-dependent functional ion channels of large conductance steps of 1-6 nS in 0.1 M NaCl. Granule-induced channels were resistant to closing by an increase in transmembrane potential, with few channels shifting to the closed state only at voltages of greater than 70 mV, following a Poisson process. These channels showed poor ion selectivity and were permeable to all monovalent and divalent ions (K+, Na+, Li+, Cl-, Ca2+, Mg2+, Zn2+, Ba2+). Ultrastructural examination of soluble granule proteins incubated for 48 hr at 37 degrees C in the presence of Ca2+ revealed ring-like structures of 150-200 A. Structural and functional channel formation may be involved in cytolysis induced by cytotoxic T lymphocytes.

Animals↗
PubMed1986

Influence of delayed immune reactions on human epidermal keratinocytes.

The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from Ia- to Ia+. Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis.

Antigens, Bacterial↗
PubMed1986

Extracellular release of lymphocyte cytolytic pore-forming protein (perforin) after ionophore stimulation.

The cytolytic pore-forming protein (PFP, perforin) of lymphocyte granules has recently been isolated and characterized. The lytic activities expressed by both the isolated granules and the purified PFP require the presence of Ca2+. Here, we report on the extracellular release of PFP after stimulation of lymphocytes with the Ca2+ ionophore A23187, which degranulates the cells. The secreted protein associates with lipid to form structural and functional channels and supramolecular complexes that partially resist dissociation by sodium dodecyl sulfate and reducing agents. Immunoblots of the released material reveal positive identification with antibodies specific for mouse PFP and human complement component C9, indicating cross-reactivity between these two molecules. By using these specific antibodies as immunoadsorbents, the lymphocyte PFP has been affinity purified from the supernatant of stimulated cells. The extracellular release of PFP is associated with simultaneous formation of functional ion-nonselective channels with conductances of 550-600 pS in 0.15 M NaCl, as measured in planar model bilayers. In the absence of extracellular Ca2+, 15% of the maximal release activity is observed. Ca2+ appears to be required to elicit both secretion by lymphocytes and the assembly of the released PFP into tubular polymers. Similar secretion of PFP may occur during cell killing by lymphocytes, resulting in its assembly on target membranes to form tubular transmembrane lesions.

Animals↗
PubMed1986

Bacterial lipopolysaccharides, phorbol myristate acetate, and zymosan induce the myristoylation of specific macrophage proteins.

We demonstrate stimulus-dependent incorporation of exogenously added [3H]myristic acid into specific macrophage proteins. In control unstimulated cells an 18-kDa protein is the major acylated species. In cells incubated with bacterial lipopolysaccharide (LPS), or its monoacyl glucosamine phosphate derivative, fatty acid is incorporated into proteins with molecular mass of 68 kDa and a doublet of approximately 42-45 kDa. Phorbol 12-myristate 13-acetate (PMA) or a phagocytic stimulus (zymosan) promotes the acylation of a similar array of proteins. However, PMA and zymosan also promote the myristoylation of unique proteins of 92 and 50 kDa. The fatty acid associated with each of the acylated proteins is myristic acid. The myristate is probably linked to the proteins through amide bonds, since it is not released by treatment with hydroxylamine. Palmitate and arachidonate are not incorporated into proteins in the same manner. Temporal analysis revealed that LPS-induced proteins are myristoylated by 30 min, while the 50-kDa protein myristoylated in response to PMA is labeled later. Most myristoylated proteins appear to be associated with the membrane fraction. Macrophages from C3H/HeJ mice, which do not respond to LPS, do not show any LPS-dependent protein acylation. Interestingly, zymosan and PMA induce the myristoylation of the 50-kDa protein in C3H/HeJ macrophages, but not the acylation of the 68-kDa and 42-kDa doublet species. We suggest that myristoylation of specific proteins is an intermediary in the capacity of LPS, PMA, and zymosan to alter macrophage functions such as arachidonic acid metabolism.

Animals↗
PubMed1986

Effects of reagent and cell-generated hydrogen peroxide on the properties of low density lipoprotein.

Low density lipoprotein (LDL) isolated from human plasma anticoagulated with EDTA (EDTA/LDL) was 4-fold more resistant to oxidation by reagent H2O2, as assayed by the thiobarbituric acid (TBA) assay, than LDL prepared from plasma anticoagulated with citrate (CDP/LDL). The LDLs required 1-3 mM H2O2 for maximal oxidation by this assay, and ED50S were 1.7 X 10(-3) M for EDTA/LDL and 4.5 X 10(-4) M for CDP/LDL. Oxidation was enhanced 2.3-fold by Cu2+ ions. Rabbit endothelial cell line monolayers released two orders of magnitude less H2O2 than was required to oxidize LDL and failed to induce TBA reactivity in either EDTA/LDL or CDP/LDL after a 24-hr coincubation. However, this LDL was subsequently degraded by mouse macrophages more rapidly than untreated LDL. Freshly isolated human monocytes (2 X 10(6) cells per ml), with or without phorbol myristate acetate (100 ng/ml) to trigger the respiratory burst, did not oxidize LDL in the TBA assay, despite producing large amounts of reactive oxygen intermediates. EDTA/LDL, CDP/LDL, and acetoacetylated LDL failed to trigger H2O2 release from human monocytes or macrophages. These results separate oxidation of LDL as measured by TBA assay from the modification of LDL by rabbit aortic endothelial cell line that leads to its subsequent enhanced degradation by macrophages.

Animals↗
PubMed1986

The alveolar macrophage.

The alveolar macrophage is one of the few tissue macrophage populations readily accessible to study both in the human and in animals. Since harvesting of these cells by bronchoalveolar lavage was first described in 1961, alveolar macrophages have been extensively investigated. This population is the predominant cell type within the alveolus, and undoubtedly serves as the first line of host defense against inhaled organisms and soluble and particulate molecules. Early studies focussed on this endocytic role and delineated the cells' phagocytic and microbicidal capacities. More recent investigations demonstrated an extensive synthetic and secretory repertoire including lysozyme, neutral proteases, acid hydrolases and O2 metabolites. In addition, the complex immunoregulatory role of the macrophage has also been appreciated. These cells have been shown to produce a wide variety of pro- and anti-inflammatory agents including arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways, cytokines which modulate lymphocyte function and factors which promote fibroblast migration and replication.

Animals↗