Search PubMed⌕ Search

Biomedical subjects

A J Ulmer

Publications and source records attributed to A J Ulmer.

At least 37 records · Page 2Linked to original sources
PubMed1999

The internalization time course of a given lipopolysaccharide chemotype does not correspond to its activation kinetics in monocytes.

The prerequisites for the initiation of pathophysiological effects of endotoxin (lipopolysaccharide [LPS]) include binding to and possibly internalization by target cells. Monocytes/macrophages are prominent target cells which are activated by LPS to release various pro- and anti-inflammatory mediators. The aim of the present study was to establish a new method to determine the binding and internalization rate of different LPS chemotypes by human monocytes and to correlate these phenomena with biological activity. It was found that membrane-bound LPS disappears within hours from the surface being internalized into the cell. Further, a correlation between the kinetics of internalization and the length of the sugar chain as well as an inverse correlation between the time course of internalization and LPS hydrophobicity was revealed. Comparison of the internalization kinetics of different LPS chemotypes with kinetics of tumor necrosis factor alpha release and kinetics of oxidative burst did not reveal any correlation of these parameters. These findings suggest that cellular internalization of and activation by LPS are mechanisms which are independently regulated.

Biological Transport, Active↗
PubMed1999

Tissue factor expression of human monocytes is suppressed by lysophosphatidylcholine.

The expression of tissue factor (TF), the principal initiator of coagulation, is increased during inflammation and atherosclerosis. Both conditions are promoted by lysophosphatidylcholine (lysoPC). We observed in the present study that lysoPC (1 to 10 micromol/L) dose-dependently reduced TF activity in human monocytes, as elicited by lipopolysaccharide (LPS). Lysophosphatidylethanolamine (lysoPE) and other lysophospholipids did not affect LPS-induced TF activity of human monocytes. TF antigen expression as elicited by LPS was also lowered by lysoPC. Phospholipid analyses indicated a selective increase in the lysoPC content of the monocytes after preincubation with the lysophospholipid. LysoPC inhibited the TF activity of Mono Mac-6 cells to a similar extent as in the monocytes. LPS binding to plasma membrane receptors and internalization of LPS into monocytes were not affected by lysoPC. In contrast, LPS-mediated nuclear binding of nuclear factor-kappaB/Rel to a TF-specific kappaB site was inhibited by lysoPC. Induction of TF mRNA expression by LPS tended to be partially reduced by the lysophospholipid. Preincubation with lysoPC increased monocytic cAMP levels. Inhibition of adenylyl cyclase by pretreatment with 2'-deoxy-3'-adenosine monophosphate partially reversed the inhibition of TF activity promoted by lysoPC. In conclusion, lysoPC markedly decreases LPS-mediated TF expression of human monocytes, the effect probably being mediated by both transcriptional and posttranscriptional mechanisms. LysoPC may thus attenuate activation of coagulation during inflammation and atherosclerosis.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Components of gut bacteria as immunomodulators.

In 1885 Louis Pasteur was the first to propose that the human immune system may be influenced by microorganisms. A large body of data has since been accumulated proving this assumption to be correct. Bacteria constitute the main constituents of the microbial flora of the human digestive tract and compounds of the bacterial cell wall have been shown to play an important role in the interaction of microbes with higher organisms. These components include peptidoglycan (PG) and lipopolysaccharide (LPS) of gram-negative bacteria. Both types of molecules are potent activators of the human immune system and exert their activity through the induction of endogenous mediators which are endowed with biological activity. This review focuses on the structure and activity of LPS and PG and illustrates how these bacterial factors stimulate the immune cells resulting in desired physiological or dramatic pathophysiological responses of the host organism.

Adjuvants, Immunologic↗
PubMed1998

Stimulation of human T lymphocytes by LPS is MHC unrestricted, but strongly dependent on B7 interactions.

Recently, we have shown that LPS is a potent inducer of human T cell proliferation and lymphokine production. However, the activation of T cells by LPS has been demonstrated to be monocyte dependent and to require direct cell-to-cell contact. Here, we investigated the role of monocytes as accessory cells and the requirement for costimulatory signals in more detail. We found that the accessory cell activity of monocytes during LPS-induced T cell proliferation is characterized by the following features: LPS-primed monocytes are competent stimulators of T cell proliferation; interaction of LPS with monocytes during the priming step is dependent on CD14 and is sensitive to ammonia; monocyte/T cell interactions are not MHC restricted but are strongly dependent on interactions of CD28 and/or CTLA-4 on T cells and their ligands CD80 and/or CD86 on monocytes. CD80 seems to be crucial for the activation of T cells by monocytes, since monocytes expressing CD86 but not CD80 after LPS stimulation were unable to stimulate T cells; IL-12, at least as a costimulatory factor, but not IL-15, is important in LPS-induced T cell proliferation. Taken together, our results indicate that LPS acts neither as a mitogen, nor as a superantigen, nor as an Ag. The activation of human T cells by LPS requires the help of accessory functions by primed monocytes and is MHC unrestricted but needs costimulatory signals via CD28 and/or CTLA-4.

Ammonium Chloride↗
PubMed1998

Structural elucidation and monokine-inducing activity of two biologically active zwitterionic glycosphingolipids derived from the porcine parasitic nematode Ascaris suum.

The isolated neutral glycosphingolipid fraction from the pig parasitic nematode, Ascaris suum, was fractionated by silica gel chromatography to yield a neutral and a zwitterionic glycosphingolipid fraction, the latter of which mainly contained two zwitterionic glycosphingolipids termed components A and C. Preliminary chemical characterization with hydrofluoric acid treatment and immunochemical characterization with a phosphocholine-specific monoclonal antibody indicated that both components contained phosphodiester substitutions: phosphocholine for component A, and phosphocholine and phosphoethanolamine for component C. Both components were biologically active in inducing human peripheral blood mononuclear cells to release the inflammatory monokines tumor necrosis factor alpha, interleukin 1, and interleukin 6. Component A was the more bioactive molecule, and its biological activity was abolished on removal of the phosphocholine substituent by hydrofluoric acid. The glycosphingolipid components were structurally analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, liquid secondary ion mass spectrometry, methylation analysis, 1H NMR spectroscopy, exoglycosidase cleavage, and ceramide analysis. Their chemical structures were elucidated to be (see Structure I below), [structure: see text] The carbohydrate moiety oligosaccharide core was characterized as belonging to the arthro series of protostomial glycosphingolipids. The ceramide moiety was distinguished by (R)-2-hydroxytetracosanoic acid as the dominant fatty acid species and by the C17 iso-branched sphingosine and sphinganine bases, 15-methylhexadecasphing-4-enine and 15-methylhexadecasphinganine, respectively.

Animals↗
PubMed1998

Molecular mechanisms of sepsis.

Today a great number of problems in the field of bacterial sepsis remain to be solved. Understanding the molecular mechanisms of one of the most important bacterial products in the pathogenesis of sepsis - endotoxin may contribute to innovative and more effective therapies. Therefore, this review focuses on the structural and functional elements of endotoxin, its interaction with immune cells, and its biological activity. Finally, other bacterial components and their impact on sepsis are discussed.

Endotoxins↗
PubMed1998

CD26 (dipeptidyl peptidase i.v.) on human T lymphocytes does not mediate adhesion of these cells to endothelial cells or fibroblasts.

We have examined the role of CD26 (dipeptidyl peptidase IV) in the adhesion of resting and activated T lymphocytes to endothelial cells and fibroblasts. For this purpose, we ran a short-time adhesion assay under different strategies: Adhesion of T lymphocytes was determined in the presence of different anti-CD26 monoclonal antibodies, or in the presence of synthetic inhibitors of the enzymatic function of CD26. In addition, the expression of CD26 on T lymphocytes, which were adherent to endothelial cells or fibroblasts, was performed by flow cytometric analysis. We found that the anti-CD26 monoclonal antibodies tested here were not able to inhibit T cell adhesion to monolayers of endothelial cells or fibroblasts. Secondly, synthetic inhibitors of the enzymatic function of CD26 had no effect on the adhesion of T lymphocytes to endothelial cells or fibroblasts. Furthermore, CD26-positive T cells were not accumulated in the adherent population. These results suggest that CD26 on T lymphocytes plays no role in T cell adhesion to endothelial cells or fibroblasts.

Dipeptidyl Peptidase 4↗
PubMed1998

Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation.

Surface structures of bacteria contribute to the microbial pathogenic potential and are capable of causing local and generalized inflammatory reactions. Among these factors, endotoxin and peptidoglycan are of particular medical importance. Both toxic bacterial polymers are now recognized to interact with the same cellular receptor, the CD14 molecule, which is expressed on different types of immune cells, in particular, monocytes/macrophages. The interaction between these bacterial activators and CD14 leads to the production of endogenous mediators such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, which are ultimately responsible for phlogistic responses. The fact that CD14 recognizes not only endotoxin and peptidoglycan but also other glycosyl-based microbial polymers suggests that this cellular surface molecule represents a lectin.

Humans↗
PubMed1998

ADP-ribosylation: role in LPS-induced phosphorylation of two cytosolic proteins (p36/38) in monocytes.

Human monocytes respond to LPS by releasing proinflammatory cytokines such as TNF-alpha, IL-1 and IL-6. Here we show that inhibitors of ADP-ribosylation namely nicotinamide and meta-iodobenzylguanidine prevent production of TNF-alpha and IL-6 at the protein and mRNA level. The inhibitors also influence the LPS-induced phosphorylation pattern of cytosolic proteins. They consistently lead to changes of the phosphorylation state of two proteins with an apparent molecular mass of 36 kDa and 38 kDa. The changes are both time and dose dependent. The data suggest that the conditions leading to altered phosphorylation of p36/38 may correlate with conditions initiating and regulating TNF-alpha and IL-6 production.

3-Iodobenzylguanidine↗
PubMed1997

Actin: a target of lipopolysaccharide-induced phosphorylation in human monocytes.

We have previously reported that lipopolysaccharide (LPS) causes altered phosphate labelling of cytosolic proteins of 36 kDa and 38 kDa (p36/38) and that inhibition of phosphorylation is accompanied by a loss of cytokine production. Here we have purified the two phosphorylated proteins via two-dimensional polyacrylamide gel electrophoresis. P 36 was found to consist of two proteins p36a and p36b. The proteins were analysed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and p36b was identified as gamma-actin, p36a as beta/gamma-actin. The ability of LPS to cause altered phosphate labelling of beta/gamma-actin suggests a participation of the microfilament network in LPS-induced monocyte activation.

Actins↗
PubMed1997

Detection of lipopolysaccharide (LPS)-binding membrane proteins by immuno-coprecipitation with LPS and anti-LPS antibodies.

In this study we describe a general method for the detection and characterization of endotoxin-(lipopolysaccharide, LPS)-binding membrane proteins. In the past, experimental procedures to detect LPS-binding sites on cells were generally performed with chemically modified LPS derivates. Since any modification of a ligand may lead to a modification of its binding characteristics, the results of those studies are controversial. In our assay, cell membrane preparations are treated with free lipid A, the endotoxic center of LPS, in the presence of normal human serum. After binding of lipid A, membrane proteins are solubilized by mild detergent treatment without disruption of the lipid A-protein complexes. Addition of anti-(lipid A) mAbs and subsequent adding of protein A agarose lead to the precipitation of complexes of lipid A and its binding proteins. By SDS/PAGE and western blot, these precipitates can be screened for the presence of LPS/lipid A-binding proteins. We describe the use of this method for the immuno-coprecipitation of lipid A (or LPS) with an 80-kDa LPS-binding membrane protein (LMP80), which we have previously identified on several human cells. In addition, CD14, the well-known functional LPS receptor on monocytes and macrophages, can be detected. By means of this immuno-coprecipitation approach we could demonstrate binding of either purified LPS preparations or synthetic lipid A to these LPS/lipid A-binding membrane proteins at physiological pH under conditions in which the proteins are in their natural membranous environment.

Acute-Phase Proteins↗
PubMed1997

Effects of colony-stimulating factors on cellular cytotoxicity.

Colony-stimulating factors (CSF) are used clinically in the treatment of chemotherapy-induced myelosuppression and in support of bone marrow transplantation. As CSF are known to have pleiotropic functions, their effects on cellular cytotoxicity were analysed in vitro against bladder carcinoma cell lines. By means of an L-[3H]methionine-release assay, the cytotoxicity of peripheral blood mononuclear cells against the natural-killer(NK)-cell-resistant bladder carcinoma cell lines BT-A and SBC-7 was measured using different effector/target-cell ratios. Costimulatory effects of granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and stem cell factor (SCF) on the generation of lymphokine-activated killer (LAK), bacillus Calmette-Guérin-activated killer (BAK) and natural killer (NK) cell cytotoxicity were investigated in this assay. Furthermore, the effect of CSF on proliferation of urothelial tumor cells in vitro was determined by a [3H]thymidine DNA-labelling technique. GM-CSF, but not G-CSF, IL-3 or SCF, was able to increase NK, BAK and LAK cytotoxicity in a dose-dependent manner. No acceleration of carcinoma cell proliferation was evident under the conditions of our assay. These data indicate the costimulatory effect of GM-CSF on cellular cytotoxicity, which might be used for immunotherapeutic purposes.

Cell Division↗
PubMed1997

Specific binding of soluble peptidoglycan and muramyldipeptide to CD14 on human monocytes.

Previously, we were able to show that soluble peptidoglycan (sPG)-induced monokine production in human peripheral monocytes is inhibited by anti-CD14 monoclonal antibodies and by lipid A partial structures. This suggested but did not prove that monocytic surface protein CD14 is involved in the activation of human monocytes not only by cell wall components of gram-negative bacteria such as lipopolysaccharide (LPS) but also by cell wall components of gram-positive bacteria such as sPG. In the present study, we provide experimental evidence that CD14 indeed constitutes a binding site for sPG recognition and activation of human monocytes. The results show that fluorescein isothiocyanate-sPG (FITC-sPG) binds to human monocytes in a saturable, dose-dependent, and specific manner. For maximal binding, 2 to 3 microg of FITC-sPG per ml was sufficient, and this binding is completed within 90 min; about 40% of the binding is completed within the first 3 min. The FITC-sPG binding is considered specific because unlabeled sPG and also muramyldipeptide (MDP), the minimal bioactive structure of sPG, inhibit the binding of sPG to monocytes in a dose-dependent manner. This specific binding was also inhibited by an anti-CD14 monoclonal antibody, LPS, and lipid A partial structure compound 406. Direct evidence for an interaction of sPG with CD14 is provided by experiments involving native polyacrylamide gel electrophoresis that showed a shift of the electrophoretic mobility of CD14 by LPS as well as by sPG. These results allow the conclusion that sPG binds directly to CD14, that MDP represents the active substructure of sPG, and that CD14 may be a lectin-like receptor which plays a key role in cellular stimulation by bioactive components of not only gram-negative but also gram-positive bacteria.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1996

Alterations in structure and cellular localization of molecular forms of DP IV/CD26 during T cell activation.

Dipeptidyl peptidase IV (DP IV, CD26), known as an activation marker of T lymphocytes, is a proline-specific protease thought to be involved in the regulation of the immune response. The physiological role of dipeptidyl peptidase IV in the immune system and the molecular events of lymphocyte activation mediated by this enzyme are only partly established. Former results suggested the occurrence of different molecular forms of DP IV in distinct human sources. As yet it has been unknown whether DP IV from human hematopoietic cells also appears in different forms and whether similar structural modifications are involved in functional processes of the regulation of the immune response. Here we describe that lymphocytic DP IV/CD26 occurs in various molecular forms and that some of them are associated with the activation process. In cell lysates of mitogen-activated lymphocytes at least 5 enzymatically active DP IV forms and up to 11 immunoreactive molecular forms of this enzyme with isoelectric points between pH 3.5 and 5.9 were discernible. Corresponding analyses of soluble and membrane cell fractions of human lymphocytes showed significant differences in the staining pattern of molecular DP IV structures. After mitogenic stimulation a special molecular form of DP IV arises in the membrane, which was originated either from the soluble part of the cell (translocation) or represents a new synthesized form. Particularly, changes of molecular DP IV forms after mitogenic stimulation strongly suggest that special forms/epitopes of this enzyme are directly involved in the process of lymphocyte activation and growth. Importantly, different monoclonal DP IV antibodies partly define different molecular forms of DP IV. Moreover, the pattern of immunostaining and enzymatic staining (Gly-Pro-beta-methoxynaphthylamide) also reveals drastic differences. These data strongly suggest a direct relationship between the expression/recognition of special DP IV epitopes and the contradictory functional effects of monoclonal DP IV antibodies found by us and other groups.

Cells, Cultured↗