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Biomedical subjects

A J Ulmer

Publications and source records attributed to A J Ulmer.

At least 19 recordsLinked to original sources
PubMed2006

Role of MIF and glutathione, in association with fetal ovine globin chain (Hbgamma) and LPS, in induction of TNFalpha from cells of young and aged mice, and PBL from healthy human populations.

Previous reports from our group have established that the fetal ovine gamma globin chain (Hbgamma) and LPS can synergize in the induction of pro-inflammatory cytokines, especially TNFalpha, from mouse and human leukocytes. A fetal sheep liver extract (FSLE) which was observed to have marked immunoregulatory properties in vivo and in vitro had independently been observed to contain significant amounts of each of these molecules. However, the biological activity of this extract (hereafter FSLE) was not explained solely by its content of Hbgamma and LPS, and independent analysis confirmed also the presence of migration inhibitory factor, MIF, and glutathione in FSLE. We have investigated whether MIF and the cellular anti-oxidant glutathione can further synergize with Hbgamma and LPS in TNFalpha induction from human cells in vitro, and mouse cells activated in vivo/in vitro. Our data show that indeed there is evidence for such a synergy. Treatment or mouse cells with FSLE produced an enhanced TNFalpha production which could be inhibited independently both by anti-Hbgamma and by anti-MIF, and optimally by a combination of these reagents.

Aging↗
PubMed2003

The LPS receptor (CD14) links innate immunity with Alzheimer's disease.

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.

Alzheimer Disease↗
PubMed2002

Inhibition of bladder carcinoma cell adhesion by oligopeptide combinations in vitro and in vivo.

PURPOSE: A presumed reason for the high recurrence rate of superficial bladder cancer after transurethral tumor resection is the reimplantation of tumor cells. Because tumor cell adhesion to the extracellular matrix is mediated by integrin molecules, we tested specific integrin receptor blocking oligopeptides to prevent this mechanism. MATERIALS AND METHODS: An in vitro cell adherence assay with various bladder cancer cell lines and extracellular matrices, including fibronectin, collagen type I, laminin and combinations, was used to analyze the inhibition of tumor cell adhesion by the matrix specific oligopeptides GRGDS, DGEA and EILDV. In therapeutic in vivo experiments the orthotopic murine bladder tumor model MB49 was used. The ability of oligopeptides to interfere with tumor cell adhesion and consecutive tumor outgrowth was evaluated and compared with that of nonspecific peptides, commercially available irrigation fluid and single dose epirubicin chemotherapy. RESULTS: In vitro fibronectin specific oligopeptides showed a concentration dependent inhibition of tumor cell adherence to fibronectin, whereas adhesion to laminin, collagen and combined matrices was not inhibited. In contrast, combinations of integrin receptor blocking oligopeptides were highly active. In vivo local tumor take was not affected by irrigation fluid, nonspecific peptides or monospecific oligopeptides alone, whereas the combination of the 3 oligopeptides effectively inhibited tumor outgrowth. CONCLUSIONS: Combining oligopeptides with various specificities significantly inhibited tumor cell adhesion and tumor outgrowth. Application of this principle in a clinical setting may be an effective method for reducing the recurrence rate of superficial bladder cancer.

Animals↗
PubMed2001

The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent.

Bacterial lipopolysaccharide (LPS, endotoxin) is a ubiquitous component of dust and air pollution and is suspected to contribute after inhalation to an activation of eosinophils in bronchial tissues of asthmatic patients, provoking inflammatory and allergic processes. We were therefore interested in the interaction of eosinophil granulocytes with LPS and have examined the activation of and uptake to human peripheral blood eosinophils by LPS. Eosinophils were stimulated by LPS and the endotoxic component lipid A and the release of tumor necrosis factor alpha (TNF-alpha) and of the eosinophil-specific granule protein eosinophil cationic protein (ECP) was estimated. The results show induction of TNF-alpha and ECP-release by LPS and lipid A in a dose-dependent manner. Anti-CD14 monoclonal antibody (moAb) (clone MEM-18) and the synthetic lipid A partial structure 406 blocked the release of TNF-alpha and ECP by LPS-stimulated eosinophils. Studies with radioactively labeled LPS showed dose-dependent uptake of (3)H-LPS to eosinophils. The (3)H-LPS uptake was found to be specific because preincubation with unlabeled LPS, compound 406 and also anti-CD14 antibodies inhibited uptake of (3)H-LPS to eosinophil granulocytes. By flow cytometry using anti-CD14 moAb and by reverse transcriptase-polymerase chain reaction (RT-PCR) technique, CD14 expression was detectable. Furthermore, messenger RNA (mRNA) expression of Toll-like receptors (TLR) 2 and TLR 4 was detected, indicating the presence of these CD14 coreceptors. The results indicate that eosinophils can take up LPS and can be stimulated by LPS in a CD14-dependent manner. Hence, in addition to allergens, eosinophils interact with endotoxin, a process that possibly exacerbates ongoing inflammatory and allergic processes.

Antibodies, Monoclonal↗
PubMed2001

Lipopolysaccharide and ceramide docking to CD14 provokes ligand-specific receptor clustering in rafts.

The glycosylphosphatidylinositol-anchored receptor CD14 plays a major role in the inflammatory response of monocytes to lipopolysaccharide. Here, we describe that ceramide, a constituent of atherogenic lipoproteins, binds to CD14 and induces clustering of CD14 to co-receptors in rafts. In resting cells, CD14 was associated with CD55, the Fcgamma-receptors CD32 and CD64 and the pentaspan CD47. Ceramide further recruited the complement receptor 3 (CD11b/CD18) and CD36 into proximity of CD14. Lipopolysaccharide, in addition, induced co-clustering with Toll-like receptor 4, Fcgamma-RIIIa (CD16a) and the tetraspanin CD81 while CD47 was dissociated. The different receptor complexes may be linked to ligand-specific cellular responses initiated by CD14.

Antigens, CD↗
PubMed2001

The biology of endotoxin.

Endotoxin (lipopolysaccharide, LPS) is the major component of the outer leaflet of Gram-negative bacteria and has profound immunostimulatory and inflammatory capacity. The septic shock syndrome caused by endotoxin still has an unacceptably high mortality rate and, owing to increasing numbers of resistant strains, remains an ongoing threat throughout the world. However, the past years have provided new insights especially into the receptors of the innate immune system that are involved into the recognition of LPS and the initial signal transduction pathways that are engaged after the primary recognition on the cell surface. The knowledge about the molecular basis for the responses to endotoxin may eventually lead to the development of new drugs to fight the fatal effects of bacterial infections.

Animals↗
PubMed2001

Structural and biological characterisation of a novel tetra-acyl lipid A from Escherichia coli F515 lipopolysaccharide acting as endotoxin antagonist in human monocytes.

We here report on the structural analysis of a novel tetra-acyl lipid A (LA (tetra) ) isolated from Escherichia coli deep rough (Re)-mutant strain F515. In addition to the biologically active hexa-acyl E. coli-type lipid A (compound 506), this incompletely acylated lipid A was found to be also present in the native LPS. Its structure was studied without further derivatisation by chemical analysis, matrix-assisted laser desorption/ionization mass spectrometry, and one- and two-dimensional (1)H- and (13)C-NMR spectroscopy. It was found to be structurally distinct from the tetra-acyl lipid A biosynthetic precursor Ia (compound 406) in lacking the primary (R)-3-hydroxytetradecanoic acid 14:0(3-OH) in position 3' ester-linked to the 'non-reducing' glucosamine (GlcN II). The hydroxyl group at the (R)-3-hydroxytetradecanoic acid attached to position 2' of GlcN II was found to be substituted by dodecanoic acid (12:0), thus forming a dodecanoyloxytetradecanoyl residue 14:0[3-O(12:0)]. The acylation pattern at the 'reducing' GlcN I was identical to that of compound 406 in having two primary (R)-3-hydroxy tetradecanoic acid residues [14:0(3-OH)] attached to positions 3 (ester-linked) and 2 (amide-linked), respectively. In human mononuclear cells (hMNC) the new LA (tetra) antagonized LPS-induced release of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) in a dose-dependent manner with identical antagonistic potency as compared with compound 406. Also like compound 406, it was found to be an agonist in murine macrophage-like J774.1 cells.

Animals↗
PubMed2001

Decay-accelerating factor (DAF/CD55) is a functional active element of the LPS receptor complex.

Previously, we identified an 80 kDa membrane protein (LMP80) that is capable of binding to LPS and lipid A in the presence of LBP and sCD14. LMP80 could also be detected after immuno-coprecipitation of cell membranes with LPS and lipid A, indicating a physical contact of LMP80 and LPS/lipid A. Further analysis and peptide sequencing revealed that LMP80 is identical to CD55 (decay accelerating factor, DAF), a regulatory molecule of the complement cascade. Transfection of LPS-hyporesponsive Chinese hamster ovary (CHO) cells with human CD55 resulted in the translocation of NF-B upon stimulation with LPS or lipid A. Our results demonstrate a new functional role of CD55 as a molecule able to mediate LPS-induced activation of cells that may be part of a multimeric LPS receptor complex.

Animals↗
PubMed2000

Induction of proliferation and cytokine production in human T lymphocytes by lipopolysaccharide (LPS).

Lipopolysaccharide (LPS), also known as endotoxin, is a compound of the cell wall of Gram-negative bacteria, which has been demonstrated to induce inflammatory reactions in vitro as well as in vivo, including lethal shock. A great number of different cells have been documented to be reactive to LPS, e.g. monocytes/macrophages, vascular cells, polymorphonuclear cells, and even B lymphocytes. We have now established that T lymphocytes could also contribute to an inflammatory reaction to LPS. LPS is a potent inducer of human T-lymphocyte proliferation and cytokine production. The activation of T lymphocytes by LPS requires direct cell-to-cell contact with viable accessory monocytes. This interaction was found to be MHC-unrestricted, but strongly dependent on costimulatory signals provided by B7/CD28 interactions. The frequency of responding T lymphocytes is less than 1:1000. A very exciting finding was that not only monocytes, but also CD34+ hematopoietic stem cells, which circulate in peripheral blood in very low frequency, exert essential accessory cell activity during stimulation of T lymphocytes by LPS. In contrast, the response of T lymphocytes to conventional recall antigens is not controlled by blood stem cells. These conclusions are based on the observation that depletion of CD34-positive blood stem cells resulted in a complete loss of LPS-induced T-lymphocyte stimulation. Addition of CD34-enriched blood stem cells led to a recovery of reactivity of T lymphocyte to LPS. The characteristics of T-lymphocyte activation indicate that LPS is neither active as a mitogen, or as a superantigen, or as a classical antigen, but may activate T lymphocyte through a new, so far undescribed, mechanism. Furthermore, the involvement of hematopoietic blood stem cells in the activation of T lymphocytes by LPS demonstrates a role of these cells in inflammatory and immunological events.

Animals↗
PubMed2000

Binding of lipopolysaccharide (LPS) to CHO cells does not correlate with LPS-induced NF-kappaB activation.

Activation of myeloid cells by lipopolysaccharide (LPS) is a key event in the development of gram-negative sepsis. One crucial step within this process is the binding of LPS to CD14. CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein requiring at least one additional membrane-spanning molecule for signal transduction. It is not clear whether the function of CD14 is to merely catalyze LPS binding, followed by the interaction of LPS with the signal transducer, or whether CD14 has a more specific function and may be a part of the signaling complex. To address this question we generated Chinese hamster ovary (CHO) cells expressing a human GPI-anchored form of LPS-binding protein (mLBP) to substitute for CD14 as LPS acceptor molecule. By comparison of CHO / mLBP with CHO / vector and CHO / CD14 cells we found that expression of GPI-linked LBP results in an enhanced binding of LPS but not in an increase in cell activation as determined by translocation of NF-kappaB. Furthermore, excess of recombinant soluble LBP resulted also in increased LPS binding without affecting NF-kappaB translocation. These data show that LPS binding alone is not sufficient to induce signaling. We conclude that CD14 is more than a catalyst for LPS binding: it seems to be directly involved in LPS signaling and thus appears to be an essential part of the signaling complex.

Acute-Phase Proteins↗
PubMed2000

In vitro generation of bacillus Calmette-Guérin-activated killer cells.

Tumor regression induced in cancer patients by local instillation of bacillus Calmette-Guérin (BCG) into the bladder is considered to be mediated by cellular immune and inflammatory reactions. In an attempt to elucidate which of these effects are relevant to tumoricidal activity, an in vitro system was employed in which the immunostimulatory effects of BCG could be studied. This report describes the induction of BCG-activated killer (BAK) cells, which effectively lyse bladder tumor cells. Human peripheral blood mononuclear cells (PBMC) were stimulated with viable and sonicated BCG (v-BCG and s-BCG, respectively) to generate BAK cells. Cytotoxicity of BAK cells was comparable with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interferon (IFN)-gamma but did not reach the level of interleukin-2 (IL-2)-generated LAK cells. Induction of BAK cells was possible only with v-BCG and not with s-BCG. By depletion and enrichment of defined cell populations, the cytotoxic potential of BAK cells could be attributed to a population of CD8(+) and CD56(+) double-positive lymphocytes. Macrophages and CD4(+) cells were required for the induction of killing activity but had no such activity by themselves. Furthermore, the presence of IFN-gamma and IL-2 in the supernatants harvested during the generation of BAK cells was demonstrated. Monoclonal antibodies neutralizing these cytokines abolished BCG-mediated cytotoxicity. From these results, it is concluded that the known beneficial effect of local instillation of BCG on maintenance of the relapse-free state in superficial bladder cancer may be due to local generation of BAK cells.

BCG Vaccine↗
PubMed2000

Reduction of side effects of intravesical therapy with bacille Calmette-Guérin by pentoxifylline?--an in vitro approach.

Immunotherapy with intravesical bacille Calmette-Guérin (BCG) is the treatment of choice against superficial bladder cancer recurrences. However, this therapy is associated with side effects that are considered to be the result of inflammatory cytokines. Since pentoxifylline is known to interfere with the production of cytokines, this drug was tested in vitro with regard to a later clinical application in BCG-treated patients. The cytokine release and the cytotoxicity of interleukin-2 or BCG-stimulated mononuclear cells were analyzed, and the growth of BCG under the influence of pentoxifylline was assayed. The results showed an inhibition of cytokine release of stimulated mononuclear cells. The cytotoxicity of BCG-stimulated mononuclear cells but not of lymphokine-stimulated mononuclear cells against bladder carcinoma cells was significantly inhibited. Restimulation with fresh BCG restored cytotoxicity. Direct coincubation of BCG and pentoxifylline resulted in a reduction of mycobacterial metabolism. From these data, we conclude that the use of pentoxifylline to reduce BCG-related side effects should be tested further in a clinical study.

Administration, Intravesical↗
PubMed1999

Differential impact of substitution of amino acids 9-13 and 91-101 of human CD14 on soluble CD14-dependent activation of cells by lipopolysaccharide.

The soluble form of the endotoxin receptor CD14 is required for the LPS-induced activation of cells lacking membrane-bound CD14. It has been shown that a deletion mutant of human CD14 consisting of the N-terminal 152 amino acids has the capacity to mediate the stimulation of different cell types by LPS. To identify the structural domains of the molecule related to this functional property, we screened a set of alanine substitution mutants using CD14-negative U373 astrocytoma cells. We show that 3 of 18 soluble mutants of human CD14 failed to mediate the LPS-induced IL-6 production in U373 cells. These mutants were located in two regions of the molecule (aa 9-13 and 91-101) that are not essential for LPS binding. In addition, the mutants had a reduced capacity to mediate LPS-stimulated IL-6 production in human vascular endothelial and SMC. In contrast, the potential of sCD14(91-94,96)A, and sCD14(97-101)A to signal LPS-induced activation of human PBMC was not significantly reduced. These results show that the regions 9-13 and 91-101 are involved in the sCD14-dependent stimulation of cells by LPS but that the mechanisms by which different cell types are activated may not be identical.

Alanine↗
PubMed1999

CD34(+) hematopoietic stem cells exert accessory function in lipopolysaccharide-induced T cell stimulation and CD80 expression on monocytes.

CD34(+) hematopoietic stem cells, which circulate in peripheral blood with very low frequency, exert essential accessory function during lipopolysaccharide (LPS)-induced human T lymphocyte activation, resulting in interferon gamma production and proliferation. In contrast, stimulation of T cells by "conventional" recall antigens is not controlled by blood stem cells. These conclusions are based on the observation that depletion of CD34(+) blood stem cells results in a loss of LPS-induced T cell stimulation as well as reduced expression of CD80 antigen on monocytes. The addition of CD34-enriched blood stem cells resulted in a recovery of reactivity of T cells and monocytes to LPS. Blood stem cells could be replaced by the hematopoietic stem cell line KG-1a. These findings may be of relevance for high risk patients treated with stem cells or stem cell recruiting compounds and for patients suffering from endotoxin-mediated diseases.

Adult↗
PubMed1999

Bacillus-Calmette-Guérin (BCG) and 3D tumors: an in vitro model for the study of adhesion and invasion.

PURPOSE: To study adhesion, penetration and internalization of BCG and effector-cells to and into three-dimensional in vitro cell aggregates from benign and malignant urothelial origin mimicking small in vitro tumors. MATERIALS AND METHODS: Multicellular spheroids (MCS) were generated by "liquid-overlay" technique. Adhesion and penetration of viable FITC-labelled BCG into MCS from urothelial cancer cell lines and normal urothelial cells was studied by electron microscopy (TEM) and fluorescence microscopy. Spheroid growth during BCG-co-incubation was determined by light microscopy. Peripheral blood mononuclear cells (PBMC) were stimulated with BCG to generate BCG-activated-killer (BAK) cells. The infiltration of these effectors and of lymphokine-activated killer (LAK) cells into MCS was examined at different intervals by means of immunohistochemistry. The resulting cytotoxicity was judged in a 3H-l-methionine release assay. RESULTS: BCG adhered to MCS from tumor cells but not to benign cell MCS. Intracellular internalization of the bacteria was detectable in superficial tumor cell-layers (1-5) whereas BCG was not found in deeper layers. Proliferation of malignant MCS was reduced in the presence of BCG. Benign MCS showed contact inhibition growth arrest, which was not altered by BCG. BAK and LAK effector cells both infiltrated tumor cell MCS as opposed to unstimulated PBMC. In contrast to LAK cells, BAK cells did not infiltrate into benign cell MCS and were not cytotoxic towards them. CONCLUSION: With regard to the clinical situation the selective adhesion and internalization of BCG to malignant cells might explain why BCG has been rarely found in follow-up biopsies in tumor free patients. More interestingly, the selective adhesion of BCG to and infiltration of BAK effector cells into malignant cell spheroids suggests a selective mode of action of BCG.

Adjuvants, Immunologic↗
PubMed1999

Identification of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55).

The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it forms complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated.

Animals↗