Influence of lipid diets on the number of metastases and ganglioside content of H59 variant tumors.

We investigated the influence of the fatty acid composition of the diet on the number of hepatic metastases and the ganglioside profile of the primary tumor and metastases. C57BL/6 female mice were fed different diets containing either no fats (TEK) or 8% of fish oil (POL), linseed oil (LIN), safflower oil (SAF) or beef tallow (BT) and were injected subcutaneously in the dorsum with H59 cells, a variant of the Lewis lung carcinoma (3LLc) that metastasizes preferentially to the liver. The omega3 polyunsaturated fatty acid (PUFA)-rich diets (LIN and POL) elicited more metastases than the omega6 PUFA-rich (SAF), fat-free (TEK), or saturated fats (BT) diets.

Preservation of the pattern of tyrosine phosphorylation in human neutrophil lysates.

Activation of various cell types by different agonists is known to stimulate a transient increase in the level of tyrosine phosphorylation of certain cellular proteins. Such phosphorylation is essential for mediating signalling by these agonists. The preservation of the tyrosine phosphorylation of proteins in lysates has proven to be a difficult task in neutrophils because of their large arsenal of proteases and phosphatases.

Epstein-Barr virus modulates 5-lipoxygenase product synthesis in human peripheral blood mononuclear cells.

The effect of short-term coincubations of Epstein-Barr virus (EBV) with mononuclear cells on the synthesis of leukotrienes (LT) by monocytes was investigated. Although treatment of mononuclear cells with EBV alone had no significant effect on LT synthesis by monocytes, the preincubation of mononuclear cells with EBV before the further stimulation of the cells with either the ionophore A23187, the chemoattractant formyl-Met-Leu-Phe, or the phagocytic particles zymosan strikingly enhanced the formation of both LTB4 and LTC4 above the levels of synthesis observed with the stimuli alone. Such priming effect of EBV on LT synthesis was maximal after 15 minutes of preincubation of mononuclear cells with EBV and slowly declined at longer preincubation times; the priming effect of EBV was observed both in Hank's Balanced Salt Solution and plasma.

Granulocyte-macrophage colony-stimulating factor-activated signaling pathways in human neutrophils. I. Tyrosine phosphorylation-dependent stimulation of phosphatidylinositol 3-kinase and inhibition by phorbol esters.

Phosphatidylinositol 3-kinase (PI3-kinase) is a cytosolic enzyme that plays key roles in mediating signaling through many receptors. The heterodimeric form of PI3-kinase is made up of a regulatory subunit, p85, and a catalytic subunit, p110. Although granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to activate PI3-kinase, the mechanisms by which this activation is mediated and regulated are incompletely understood.

Role of protein kinase C alpha, Arf, and cytoplasmic calcium transients in phospholipase D activation by sodium fluoride in osteoblast-like cells.

The effect of fluoride on phospholipase D (PLD) activation was studied using in vitro culture of Saos-2, MG-63 osteosarcoma cells, and normal osteoblast-like cells derived from human bone explants. Millimolar concentrations of NaF induced a significant accumulation of phosphatidylethanol (PEt) in Saos-2 cells but not in MG-63 and normal osteoblast-like cells. PLD activation was evident at 15 mM and concentration-dependent up to 50 mM.

Identification of G-protein binding sites of the human interleukin-8 receptors by functional mapping of the intracellular loops.

Interleukin 8 (IL-8) is considered to be a major mediator of the inflammatory response. Recent evidence indicates that a direct physical association occurs between IL-8 receptors and the alpha subunit of guanine nucleotide regulatory protein (Gi(alpha)2) upon stimulation of human neutrophils by IL-8. In the present study, we identified by site-directed mutagenesis key residues within the three intracellular loops of the IL-8RA receptor involved in the interaction with Gi(alpha)2.

Interleukin-4, transforming growth factor beta 1, and dexamethasone inhibit superantigen-induced prostaglandin E2-dependent collagenase gene expression through their action on cyclooxygenase-2 and cytosolic phospholipase A2.

Signalling via MHC class II in human fibroblast-like synoviocytes selectively induces interstitial collagenase gene expression over its natural inhibitor, the tissue inhibitor of metalloproteinase (TIMP), through a prostaglandin E2 (PGE2)-dependent pathway involving cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2). In the present study, we investigated the effect of three different agents the T-cell-derived cytokine IL-4, transforming growth factor beta 1 (TGF-beta 1), and dexamethasone (DXS) on this response. Our results indicate that treatment of superantigen-stimulated synoviocytes with DXS or IL-4 inhibited collagenase gene expression without affecting TIMP gene expression.

Gangliosides in human uveal melanoma metastatic process.

The inability of current therapy to prevent metastases arising from uveal melanoma often results in patient mortality. With the goal of developing a treatment for metastasis, gangliosides were studied as potential tumor-associated antigens. Our report describes the production of a metastatic liver variant (MH) from a human uveal melanoma cell line (SP6.

Diverging signal transduction pathways activated by interleukin 8 (IL-8) and related chemokines in human neutrophils. IL-8 and Gro-alpha differentially stimulate calcium influx through IL-8 receptors A and B.

Interleukin 8 (IL-8) and Gro-alpha are members of the CXC branch of a family of cytokines recently designated the "chemokine" superfamily. Recent evidence indicates that, contrary to previously held beliefs, IL-8 and Gro-alpha may not be perceived equivalently by neutrophils. In this study, we have evaluated the effects of IL-8 and Gro-alpha on the rate of calcium influx in human neutrophils and in 293 cells transfected with type A or type B IL-8 receptors.

Paradoxical effects of colchicine on the activation of human neutrophilis by chemotactic factors and inflammatory microcrystal.

Neutrophil activation by chemotactic factors and by inflammatory microcrystals is accompanied by increases in protein tyrosine phosphorylation and by the activation of the NADPH oxidase. The addition of colchicine inhibited both responses induced by triclinic monosodium urate or calcium pyrophosphate crystals. On the other hand, colchicine enhanced the tyrosine phosphorylation of specific protein in neutrophils stimulated by chemotactic factor and augmented the production of superoxide anions induced by these same agonists.

Physical association of Gi2alpha with interleukin-8 receptors.

Interleukin-8 (IL-8), one of the major mediators of the inflammatory response, belongs to a family of chemokines that includes NAP-2 (neutrophil-activating peptide-2) and Gro-alpha and whose biological activities are directed to a great extent toward neutrophils. Two distinct receptors have been described with overlapping, but not identical, binding affinities for IL-8, NAP-2, and Gro-alpha. This study was designed to examine the intracellular pathways activated upon the occupation of each of the IL-8 receptors (IL-8R).

Colocalization of cytosolic phospholipase A2, 5-lipoxygenase, and 5-lipoxygenase-activating protein at the nuclear membrane of A23187-stimulated human neutrophils.

The distribution of cytosolic phospholipase A2 (cPLA2), arachidonate 5-lipoxygenase, and 5-lipoxygenase-activating protein (5-LAP) was investigated in subcellular fractions of human neutrophils disrupted by three techniques. As determined by immunoblot analysis, the bulk of cPLA2 and 5-lipoxygenase was detected in cytosolic fractions of unstimulated neutrophils disrupted by sonication or cavitation. After cell stimulation with the calcium ionophore A23187, both proteins accumulated primarily in nuclei-containing fractions; this accumulation was accompanied by a loss of these enzymes from cytosolic fractions.

Priming of human peripheral blood mononuclear cells with lipopolysaccharides for enhanced arachidonic acid release and leukotriene synthesis.

In a previous study, we have shown that the ability of lipopolysaccharides (LPS) to prime isolated neutrophils for enhanced leukotriene B4 (LTB4) synthesis was dependent on the presence of plasma and involved the CD 14 antigen. In the present study, we have investigated the priming of human peripheral blood mononuclear cells (PBMC) with LPS for the subsequent release and metabolism of arachidonic acid. When PBMC were incubated with LPS for up to 2 h or when freshly isolated PBMC were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with LPS alone, little or no synthesis of 5-lipoxygenase products nor arachidonic acid liberation were detected.

Adenosine A2 receptor-induced inhibition of leukotriene B4 synthesis in whole blood ex vivo.

1. Engagement of adenosine A2 receptors suppresses several leukocyte functions. In the present study, we examined the effect of adenosine on the inhibition of leukotriene B4 (LTB4) synthesis in heparinized human whole blood, pretreated with lipopolysaccharide (LPS) and tumour necrosis factor alpha (TNF-alpha) and stimulated with the chemotactic peptide, N-formyl-Met-Leu-Phe (FMLP).

C2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils.

Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C2-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O2-) production. The preincubation of PMN for 15 min with C2-ceramide increased by up to almost 3-fold the amounts of O2- generated in response to 0.

Small GTPase-regulated phospholipase D in granulocytes.

This review examines the functional role of phospholipase D in the neutrophil. Phospholipase D is emerging as an important component in the signal transduction pathways leading to granulocyte activation. Through the second messenger it produces, phosphatidic acid, phospholipase D plays an active role in the regulation of granulocyte NADPH oxidase activation and granular secretion.

ADP-ribosylation factor translocation correlates with potentiation of GTP gamma S-stimulated phospholipase D activity in membrane fractions of HL-60 cells.

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors from both the membrane and the cytosol. The small GTP-binding protein ADP-ribosylation factor (ARF) has been established as one important component of PLD activation. By stimulating HL-60 cells with various agonists and then isolating the membrane fraction and assaying PLD activity in the presence and absence of GTP gamma S, we observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a potentiation of GTP gamma S-stimulated PLD activity in the membrane fractions of these cells.

Leukotriene synthesis in calcium-depleted human neutrophils: arachidonic acid release correlates with calcium influx.

The relationship between intracellular calcium concentration ([Ca2+]i), the release of arachidonic acid and the synthesis of leukotriene B4 (LTB4) was investigated using Ca(2+)-depleted human polymorphonuclear leucocytes (PMNs) in which [Ca2+]i can be manipulated by varying the concentration of exogenous Ca2+ added with agonists. In this model, Ca2+, platelet-activating factor (PAF) and N-formyl-Met-Leu-Phe (FMLP), added alone, were unable to induce arachidonic acid release or LTB4 synthesis, as assessed by measurements of the products by MS and HPLC, respectively. However, the simultaneous addition of Ca2+ and either PAF or FMLP to these Ca(2+)-depleted PMNs resulted in an influx of Ca2+ proportional to the extracellular concentration of Ca2+ and caused a substantial release of arachidonic acid and synthesis of LTB4.

Superantigens initiate cognate CD4+ T cell/B cell interactions leading to early activation and proliferation of B cells.

Dimerization or even multimerization of various receptors is commonly required for signal transduction. We report here that clustering of major histocompatibility complex class II molecules in human B cells by biotinylated staphylococcal enterotoxin A (SEA) cross-linked with avidin induces an increase in the level of intracellular calcium. This response was abolished by prior treatment with protein tyrosine kinase (PTK) inhibitors, suggesting that SEA-triggered calcium mobilization in B cells is probably dependent on the activation of PTK.

Alpha-fetoprotein (AFP) expression in clones of McA-RH 7777 rat hepatoma: correlation with the occurrence of homogeneously staining regions on chromosome 14.

alpha-Fetoprotein (AFP) is a well-established cell differentiation and tumor marker. We showed previously that McA-RH 7777 hepatoma cells are heterogeneous in terms of their AFP cellular expression. In the present study, we developed stable and unstable 7777 hepatoma clones in terms of their AFP phenotype: AFP-producing (AFP+) or AFP-nonproducing (AFP-) clones, and investigated in these clones (a) AFP phenotype related to protein and mRNA levels; (b) cellular morphology; and (c) expression of several liver-specific markers.

A natural mutation of the amino acid residue at position 60 destroys staphylococcal enterotoxin A murine T-cell mitogenicity.

A variety of techniques have been used to identify the amino acid residues of bacterial superantigens involved in their interactions with major histocompatibility complex (MHC) class II and T-cell receptor (TCR). In this study, we isolated a naturally mutated staphylococcal enterotoxin A (SEA) from three different Staphylococcus aureus strains, in which the amino acid at position 60 has been changed from aspartic acid (D) to asparagine (N). We then studied the influence of this change on the immunological activities of SEA.

Low molecular weight GTP-binding proteins in HL-60 granulocytes. Assessment of the role of ARF and of a 50-kDa cytosolic protein in phospholipase D activation.

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions.

Diverging signal transduction pathways activated by interleukin-8 and related chemokines in human neutrophils: interleukin-8, but not NAP-2 or GRO alpha, stimulates phospholipase D activity.

Interleukin-8 (IL-8) and the structurally related cytokines neutrophil-activating peptide-2 (NAP-2) and GRO alpha are powerful chemotactic agents for human neutrophils. Although these three chemokines act by binding to overlapping but not identical receptor subsets, the data available to date have stressed the similarities in their mechanisms of action. The present studies were undertaken to further our understanding of the signal transduction mechanisms associated with these neutrophil agonists.

Induction of chemokine gene expression by major histocompatibility complex class II ligands in human fibroblast-like synoviocytes. Differential regulation by interleukin-4 and dexamethasone.

Chemokines play a key role in recruiting leukocytes into inflamed synovial environment, and the cells of the synovial membrane, which express high levels of major histocompatibility complex (MHC) class II molecules, are a major source of these chemokines. Our data indicated that engagement of MHC class II molecules by staphylococcal enterotoxin A superantigen resulted in the induction of chemokine gene expression as well as protein synthesis. Pretreatment of the cells with cycloheximide potentiated the effect of superantigen on chemokine mRNA induction, suggesting that the expression of these genes may occur independently of prior protein synthesis.

Posttransplant antibodies and fresh venous allograft failure in dogs.

Small diameter arterial reconstruction is usually achieved by use of the autologous long saphenous vein. As an alternative to this blood conduit, the venous allograft has been used with some success in the past, but is likely to be the target of an immune rejection reaction from the host. This study was designed to characterize humoral immune reactions possibly involved in the outcome of venous allografts.