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Biomedical subjects

S Fiore

Publications and source records attributed to S Fiore.

At least 37 records · Page 2Linked to original sources
PubMed1995

Lipoxin A4 receptor activation is distinct from that of the formyl peptide receptor in myeloid cells: inhibition of CD11/18 expression by lipoxin A4-lipoxin A4 receptor interaction.

Lipoxin A4 (LXA4) interacts with high-affinity receptors in human neutrophils and differentiated HL-60 cells. Recently, we characterized a myeloid-derived cDNA that encodes a LXA4 high-affinity receptor (LXA4R) [Fiore, S., Maddox, J. F., Perez, H. D., and Serhan, C. N. (1994) J. Exp. Med. 180, 253-260] denoted earlier as a related N-formyl peptide receptor (RFP). To examine the selectivity of this receptor we tested its preference for specific binding of 3H-LXA4 versus 3H-N-formylmethionyl-leucyl-phenylalanine (3H-FMLP). When receptor-transfected Chinese hamster ovary cells were exposed to either 3H-LXA4 or 3H-FMLP, the receptor affinity for LXA4 exceeded by 1000-fold that of FMLP (6.1 nM vs 5 microM). Upon differentiation, HL-60 cells acquire high-affinity binding sites and respond to both LXA4 and FMLP. Northern blot analysis of differentiated HL-60 cells using an RFP probe showed a characteristic band at 2.1 kb. Differentiated HL-60 cells exposed to an RFP antisense oligonucleotide selectively lost 3H-LXA4 binding as well as LXA4-stimulated lipid remodeling that paralleled the loss of mRNA for LXA4R. In contrast, the specific mRNA for the FMLP receptor, 3H-FMLP specific binding, and FMLP-induced phospholipase D activity were still observed. Treatment of human neutrophils with antisera raised against a peptide in the LXA4R third extracellular domain also resulted in selective abrogation of 3H-LXA4 specific binding with polymorphonuclear leukocytes (PMN) without blocking 3H-FMLP binding. FMLP-stimulated CD11b upregulation as well as homotypic aggregation of PMN was inhibited by LXA4 (which at 10(-9) M gave approximately 1 log unit shift to the right in the FMLP dose-response curve). The addition of LXA4R antisera did not alter FMLP-induced responses in PMN but completely blocked LXA4 actions. These results indicate that altering the expression of the LXA4R protein by blockage of transcriptional mechanisms or hindrance of the LXA4R extracellular domains leads to loss of LXA4 specific binding and blockage of LXA4 signaling. Moreover, they indicate that in myeloid cells LXA4-LXA4R interactions are dissociable from those of FMLP and that LXA4 regulates CD11/18 on the PMN surface.

Animals↗
PubMed1995

The incidence of complications after caesarean section in 156 HIV-positive women.

OBJECTIVE: To investigate the risks of post-operative complications in HIV-positive mothers who undergo a caesarean section (CS) because the delivery cannot be safely accomplished by the vaginal route or to protect the infant from viral infection. DESIGN: In a multicentre study, we reviewed the incidence and type of post-operative complications in 156 HIV-positive women who underwent a CS. These results were compared with those observed in an equal number of HIV-uninfected women who matched for the indication requiring a caesarean delivery, the stage of labour, the integrity or rupture of membranes, and the use of antibiotic prophylaxis. SETTING: Seven teaching hospitals providing obstetrical care for mothers infected with HIV. RESULTS: We found that six HIV-infected mothers suffered a major complication (two cases of pneumonia, one pleural effusion, two severe anaemia and one sepsis) compared with only one HIV-negative woman who required blood transfusion after surgery. Minor complications like post-operative fever, endometritis, wound and urinary tract infections were significantly more frequent in HIV-positive women than controls. Multivariate analysis revealed that in HIV-infected women the only factor associated with a significant increase in the rate of complications was a CD4 lymphocyte count < 200 x 10(6)/l. CONCLUSIONS: The results of our study indicate that HIV-positive mothers are at an increased risk of post-operative complications when delivered by CS. The risk of post-operative complications is higher in HIV-infected women who are severely immunodepressed.

CD4 Lymphocyte Count↗
PubMed1994

Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor.

Lipoxin A4 (LXA4) triggers selective responses with human neutrophils that are pertussis toxin sensitive and binds to high affinity receptors (Kd = 0.5 +/- 0.3 nM) that are modulated by stable analogues of guanosine 5'-triphosphate (GTP). Here, we characterized [11,12-(3)]LXA4 specific binding with neutrophil granule and plasma membranes, which each display high affinity binding sites (Kd = 0.7 +/- 0.1 nM) that were regulated by GTP gamma S. Since functional LXA4 receptors are inducible in HL-60 cells, we tested orphan cDNAs encoding 7-transmembrane region receptors cloned from these cells for their ability to bind and signal with LXA4. Chinese hamster ovary (CHO) cells transfected with the orphan receptor cDNA (pINF114) displayed specific 3H-LXA4 high affinity binding (1.7 nM). When displacement of LXA4 binding with pINF114-transfected CHO cells was tested with other eicosanoids, including LXB4, leukotriene D4 (LTD4), LTB4, or prostaglandin E2, only LTD4 competed with LXA4, giving a Ki of 80 nM. In transfected CHO cells, LXA4 also stimulated GTPase activity and provoked the release of esterified arachidonate, which proved to be pertussis toxin sensitive. These results indicate that pINF114 cDNA encodes a 7-transmembrane region-containing protein that displays high affinity for 3H-LXA4 and transmits LXA4-induced signals. Together, they suggest that the encoded protein is a candidate for a LXA4 receptor in myeloid cells.

Amino Acid Sequence↗
PubMed1994

Treatment with interferon for genital HPV in HIV-positive and HIV-negative women.

The administration of interferons can be resorted to, either on its own or in combination with physical destruction methods, when the extent of genital HPV is widespread. Extensive genital HPV involvement is often seen in HIV-positive patients as a consequence of their immunodeficiency. The extension of these lesions may invalidate treatment by physical destruction, while an underlying immunodeficiency renders interferon therapy less efficacious. We studied HIV-positive and HIV-negative patients with a similar HPV involvement of their genital tract and compared the effectiveness of systemically administered alpha 2b and beta interferons in clearing HPV. Our results confirm that interferon therapy will cure most patients with extensive genital HPV when they are HIV-negative. HIV-positive patients with CD4 counts over 400 lymphocytes/mm3 may expect a similar cure rate, but this halves when this critical threshold is crossed. In these severely immunodeficient patients repeated courses of interferon therapy alone or in combination with physical destruction methods may be required to cure HPV infection.

CD4-Positive T-Lymphocytes↗
PubMed1994

Carrier-mediated transport of lipoxin A4 in human neutrophils.

Lipoxins and other eicosanoids display potent and selective biological effects on leukocytes. In this study, we utilized radiolabeled lipoxin A4 ([3H]LXA4) to investigate whether carrier-mediated transport of LXA4 might occur in human neutrophils. At a concentration of 5 nM, uptake of [3H]LXA4, above that due to specific binding to receptors, amounted to approximately 0.6 fmol.10(6) cells-1.min-1. This influx was sensitive to a number of anionic inhibitors, including 3,5-diiodosalicylic acid (K0.5 12 microM), pentachlorophenol (K0.5 25 microM), alpha-cyano-beta-(1-phenylindol-3-yl) acrylic acid, and the organomercurial agents mersalyl (K0.5 110 microM) and p-hydroxy-mercuribenzoate. Influx, which was Na+ and membrane voltage independent, exhibited a striking dependence on pH (negative log of dissociation 5.9), results compatible with an H+ + LXA4 anion cotransport system. The LXA4 carrier did not appear to interact with arachidonic acid, prostaglandin E2, 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, or the leukotrienes B4, C4, and D4. Moreover, transport activity was not observed in human erythrocytes, lymphocytes, or platelets, but it was inducible in HL-60 cells on differentiation by exposure to retinoic acid. These findings represent the identification and initial characterization of a novel carrier-mediated pathway in human neutrophils that facilitates transport of LXA4 into cells.

Biological Transport↗
PubMed1993

Lipoxin A4 metabolism by differentiated HL-60 cells and human monocytes: conversion to novel 15-oxo and dihydro products.

Lipoxins are tetraene-containing eicosanoids that possess biological activity in several organ systems. To determine their route of further metabolism, [11,12-3H]lipoxin A4 was prepared and incubated with human neutrophils, promyelocytic leukemia (HL-60) cells, and adherent monocytes. Intact neutrophils and undifferentiated HL-60 cells did not significantly metabolize [11,12-3H]LXA4, while HL-60 cells differentiated with PMA to monocyte/macrophage lineage rapidly (< 15 s) transformed this eicosanoid. The major radiolabeled LXA4-derived metabolites were characterized by physical methods and were shown to be 15-oxo-LXA4, 13,14-dihydro-15-oxo-LXA4, and 13,14-dihydro-LXA4. Substrate competition with cell-free supernatants from differentiated HL-60 cells suggests that lipoxins compete for 15-hydroxyprostaglandin dehydrogenase activity or an equivalent enzyme system. In addition, adherent monocytes exposed to [11,12-3H]LXA4 rapidly metabolized (> 60% within 30 s) the label to its oxo and dihydro derivatives. These results indicate that, unlike leukotrienes, LXA4 is subject to dehydrogenation and reduction of its conjugated tetraene to form triene-containing products. Moreover, they suggest that monocytes participate in lipoxin metabolism in their local milieu.

Alprostadil↗
PubMed1993

Induction of functional lipoxin A4 receptors in HL-60 cells.

The appearance of [11,12-3H]lipoxin A4 (LXA4) specific binding sites was examined with human acute promyelocytic leukemic cell line 60 (HL-60) cells exposed to either retinoic acid, phorbol 12-myristate 13-acetate (PMA), or dimethyl sulfoxide (DMSO). All three agents induced a threefold to fivefold increase in the expression of specific [11,12-3H]LXA4 binding. Similar results were obtained in parallel with [14,15-3H]leukotriene (LT) B4. For both 3H-ligands, homologous displacement curves were similar and independent of the agent used to induce differentiation. Specific binding of [11,12-3H]LXA4 to differentiated HL-60 cells gave a kd = 0.6 +/- 0.3 nmol/L. The appearance of both [11,12-3H]LXA4 and [14,15-3H]LTB4-specific binding sites was inhibited by actinomycin D, and LXA4 binding was sensitive to protease treatment. Specific binding of [11,12-3H]LXA4 was not evident with human platelets, red blood cells (RBCs) or the cultured B-cell (Raji), T-cell (Jurkat) lines save human endothelial cells (kd = 11.0 +/- 0.3 nmol/L). The structural specificity of induced [11,12-3H]-LXA4 recognition sites was assessed with LXB4, LTC4, LTB4, and trihydroxyhepatanoic methyl ester. Only LTC4, at 3-log molar excess, competed for 3H-LXA4-specific binding with HL-60 cells and gave a 30% reduction. The leukotriene D4 receptor antagonist SKF 104353 was ineffective in blocking [11,12-3H]LXA4-specific binding with HL-60 cells while it competed for specific [11,12-3H]LXA4 binding with endothelial cells where LTD4 binding appears to be virtually identical to that of LXA4 binding. In addition, the LTB4 receptor antagonist ONO 4057 was ineffective at competing for [11,12-3H]LXA4 binding. When phospholipase D activation was monitored in human polymorphonuclear leukocytes (PMN) and HL-60 cells, a correlation was shown between activation and specific 3H-LXA4 binding. LXA4-induced phospholipase D (PLD) activation gave a biphasic concentration-dependent response comprised of at least two components: one phase being islet-activating protein (IAP)-sensitive (LXA4 10(-9) mol/L peak activity) and the other was staurosporine-sensitive (LXA4 10(-7) mol/L peak activity). Results indicate that HL-60 cells exposed to differentiating agents express [11,12-3H]LXA4 recognition sites also present in PMN. In addition, specific LXA4 recognition sites of myeloid cells can be distinguished by competition binding with SKF 104353 and 3H-LXA4 cross-reactivity with putative LTD4 receptors present on human endothelial cells. Moreover, they provide evidence indicating that binding of LXA4 to its recognition sites confers functional responses.

Binding, Competitive↗
PubMed1993

Inhibition of granuloma formation induced by potassium permanganate in the mouse by a specific human recombinant receptor antagonist for interleukin-1 (hrIL-1ra).

Interleukin-1 (IL-1) is a polypeptide which mediates several systemic changes associated with infection, inflammation and injury, such as fever, neutrophilia, increased acute phase protein synthesis, and arachidonic acid metabolites. Recently, a natural inhibitor of IL-1 has been cloned, called IL-1 receptor antagonist (IL-1ra), which prevents Escherichia coli-induced shock in rabbits and blocks PGE2 induced by IL-1. In this report we study the effect of human recombinant (hr) IL-1ra on chronic inflammation induced by dorsal injections of 200 microliters of a 1:40 saturated crystal solution of potassium permanganate (KMnO4) in mice. After 7 days, all mice developed a subcutaneous granulomatous tissue indicative of a chronic inflammatory response, at the site of injection. KMnO4-treated mice, injected intraperitoneally twice with hrIL-1ra, 20 micrograms/dose (the first at the same time of induction of the granuloma and the second 24 hr later), show significant decreases in size and weight of the granuloma when compared to mice not treated with hrIL-1ra (controls); the inhibitory effect was approximately 32-46 and 25-51%, respectively. In addition, in all mice treated with hrIL-1ra, there was a strong inhibition of PGE2 and LTB4 on assay of freshly minced granuloma tissue. Moreover, when hrIL-1 beta (1.0 ng/ml) or LPS (100 ng/ml) were added overnight to the minced granuloma tissue cultures, these compounds enhanced the production of LTB4 and PGE2 from the untreated mice, whereas in IL-1ra-treated mice they failed. In the histological studies of the granuloma, animals treated with hrIL-1ra show a lesser degree of mononuclear cell (MC) accumulation. The inhibitory effect of hrIL-1ra on PGE2 production was also confirmed on peritoneal macrophages from untreated mice, stimulated overnight with hrIL-1 beta or LPS in vitro. The resulting inhibition was dose-dependent. In these studies we show, for the first time, the anti-inflammatory effect of hrIL-1ra on chronic inflammation as assessed by the inhibition of granuloma formation, PGE2, LTB4, and white cell accumulation in inflamed tissue.

Animals↗
PubMed1992

Lipoxin recognition sites. Specific binding of labeled lipoxin A4 with human neutrophils.

Lipoxin A4 stimulates rapid lipid remodeling and a pertussis toxin-sensitive release of arachidonic acid in polymorphonuclear leukocytes (PMN) (Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Physiol. 143, 512-523) and has been shown to inhibit leukocyte responses in several systems. To examine the basis underlying these actions, we have prepared [11,12-3H]lipoxin A4 (LXA4) and characterized its interactions with human PMN. Time course studies (0-90 min) with intact PMN demonstrated cell association of 3H label which was specific and reversible. PMN bound [3H]LXA4 with a Kd of 0.5 +/- 0.3 nM, representing approximately 1,830 sites/PMN, and the Hill plot value of 1.9 suggests cooperative binding. [3H]LXA4 binding was stereoselective since neither leukotriene B4 (LTB4), lipoxin B4 (LXB4), (6S)-LXA4, 11-trans-LXA4, nor SKF 104353 competed for [3H]LXA4-specific binding while LTD4 and LTC4 partially competed. Subcellular fractionation revealed that specific binding with [3H]LXA4 was associated with membrane (42.1%)-, granule (34.5%)-, and nuclear (23.3%)-enriched fractions, a distribution distinct from that of [14,15-3H] LTB4 binding. [11,12-3H]LXA4-specific binding was modulated by guanosine analogs, suggesting the involvement of G proteins. A fluorescent LXA4 derivative (methyl-7-methoxycoumarin-LXA4) competed with [3H]LXA4 binding to intact PMN and showed specific and reversible binding as monitored by flow cytometric analysis. These results indicate that PMN possess specific recognition sites for LXA4 which may mediate its actions.

Binding Sites↗
PubMed1992

Blocking the interleukin-1 receptor inhibits leukotriene B4 and prostaglandin E2 generation in human monocyte cultures.

Interleukin-1 is a potent stimulator of arachidonic acid (AA) metabolism and this activity could be attributed to the activation of the prostaglandin-forming enzyme cyclooxygenase or of the arachidonic-releasing enzyme phospholipase A2 or both. Prostaglandin E2 (PGE2), a cyclooxygenase product, and LTB4 (5-(S),12-(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid), a lipoxygenase product, are potent mediators of inflammation. Recently a new cytokine produced by macrophages and named interleukin-1 receptor antagonist (IL-1ra) (MW 22,000 Da) which specifically binds and blocks IL-1 receptors, has proven to be a potent inflammatory inhibitor. In our studies we found that monocyte suspensions, pretreated with hrIL-1ra at increasing concentrations (0.25-250 ng/ml) for 10 min and then treated with LPS in an overnight incubation inhibits, in a dose-dependent manner, the generation of LTB4 as measured by the highly sensitive radioimmunoassay method. In monocytes pretreated with hrIL-1ra (250 ng/ml) for 10 min and treated with arachidonic acid (10(-5)-10(-9) M) and LPS overnight, the release of LTB4 was partially inhibited when compared to hrIL-1ra-untreated cells. Moreover, hrIL-1ra (250 ng/ml) caused a partial inhibition of monocyte LTB4 production when the cells were activated with AA (10(-7) M) and then treated with IL-1 beta (5 ng/ml) overnight or 24 hr incubation. In addition, human monocytes pretreated for 10 min with increasing doses of hrIL-1ra (0.25-250 ng/ml) and then treated with hrIL-1 alpha (5 ng/ml) or beta (5 ng/ml) for 18 hr, also resulted in the inhibition of PGE2 generation as measured by RIA when compared with hrIL-1ra-untreated cells. When the cells were treated with hrIL-1ra (250 ng/ml) and activated for 18 and 48 hr with increasing doses of hrIL-1 beta a strong inhibitory effect was found on PGE2 production. HrIL-1ra used at 15 ng/ml gave a partial inhibition of LTB4 generation, after LPS (1-100 ng/ml) treatment, while NDGA totally blocked the production of LTB4. Moreover, PGE2 released by macrophages activated with LPS (100 ng/ml) or hrIL-1 beta (5 ng/ml) at 18 hr incubation time was strongly inhibited when hrIL-1ra (250 ng/ml) was used. These data suggest that the inhibition of LTB4 and PGE2 by this new macrophage-derived monokine IL-1ra occurs through the block of the IL-1 receptor, rather than phospholipase A2, and thus IL-1ra may offer a potential therapeutic approach to inflammatory states.

Arachidonic Acid↗
PubMed1992

Granulocyte-macrophage colony stimulating factor potentiates human polymorphonuclear leukocyte aggregation responses to formyl-methionyl-leucyl-phenylalanine.

Polymorphonuclear leukocytes (PMN) are known to be activated by several lymphokines and can be induced to release lysosomal enzymes, prostaglandins (PG), thromboxanes (TX) and lipoxygenase products that may be involved in PMN aggregation responses during inflammatory reactions. Granulocyte-macrophage colony stimulating factor (GM-CSF), a glycoprotein cytokine released by immunocompetent cells, has been found to prime neutrophil responses, such as increased cell aggregation after exposure to various biological stimulants. In this study, we examined the effects of the cytokine GM-CSF on human neutrophilic aggregation stimulated by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and its influence on the production of various arachidonic acid metabolites. Neutrophil aggregation of purified PMNs was measured by the percent change in light transmission in a standard aggregometer, and the arachidonic acid products leukotriene B4 (LTB4) and thromboxane A2 (TXA2) were quantified by radioimmunoassay. We found that GM-CSF and other cytokines, used alone, did not cause any significant increase in aggregation of the PMN. However, prior exposure of PMN to GM-CSF markedly increased the aggregation induced by FMLP as opposed to that detected with PMN stimulated with only FMLP. This priming effect was not observed with PMN preincubated with interleukin-1 (IL-1), tumor necrosis factor (TNF) or interleukin-6 (IL-6). In addition, GM-CSF and IL-6 both failed to stimulate the production of LTB4 and TXA2, products which are known to induce PMN aggregation. These findings provide new evidence suggesting that GM-CSF facilitates the action of FMLP on the adhesion dependent cellular functions of the inflammatory response, serving as an important co-factor in neutrophil aggregation.

Calcimycin↗
PubMed1990

Formation of lipoxins and leukotrienes during receptor-mediated interactions of human platelets and recombinant human granulocyte/macrophage colony-stimulating factor-primed neutrophils.

The generation of lipoxygenase products of arachidonic acid is considered an important event in inflammation. This study demonstrates the levels of both lipoxins and leukotrienes (LTC4, LTD4, LTB4, and omega-oxidized LTB4) generated from endogenous sources of arachidonate by PMN primed with recombinant human granulocyte/macrophage colony-stimulating factor and in coincubations with platelets (1:1 to 1:100 ratio). Upon exposure to receptor-mediated stimuli (FMLP and thrombin), the levels of lipoxins generated were within the range of both LTB4 and LTC4. Co-incubation of [1-14C]arachidonate-labeled platelets with primed polymorphonuclear leukocytes (PMN) followed by addition of thrombin and FMLP led to the formation of both 5- and 15-LO products that carried 14C label. Thus, in addition to the transcellular conversion of LTA4 to platelet-derived lipoxins and LTC4, PMN can use platelet-derived arachidonate to generate lipoxygenase products. These results are the first to document the relationship between the levels of lipoxins and leukotrienes generated by receptor-mediated activation of cytokineprimed PMN interacting with platelets. Moreover, they indicate that PMN-platelet interactions utilize bidirectional transcellular routes to contribute to lipoxin formation.

Arachidonic Acids↗