Biomedical subjects

Terje Espevik

Publications and source records attributed to Terje Espevik.

31 records · Page 2Linked to original sources

PubMed2003

Toll-like receptor-mediated tumor necrosis factor and interleukin-10 production differ during systemic inflammation.

Major trauma is associated with a decreased capacity of patients' leukocytes to produce proinflammatory cytokines on in vitro stimulation. We studied leukocytes from 48 patients with trauma and showed that this hyporeactivity was restricted to gram-negative bacteria, Escherichia coli endotoxin, and unmethylated bacterial DNA, whereas Leptospira interrogans endotoxin-induced tumor necrosis factor production was similar to that observed with healthy donors. As well, tumor necrosis factor and interleukin-6 production in response to gram-positive bacteria was not altered. The expression of toll-like receptor (TLR) 2 was not reduced on patients' monocytes as compared with healthy control subjects, whereas that of TLR4 was reduced. However, the hyporeactivity to gram-negative bacteria and E. coli endotoxin cannot be fully explained by the downregulation of TLR4. Indeed, unlike proinflammatory cytokines, after stimulation with these microbial products the release of antiinflammatory cytokines was increased as compared with healthy control subjects. The increased interleukin-10 production was analyzed in terms of intracellular signaling in peripheral blood mononuclear cells from patients with trauma: our results suggest the involvement of p38 mitogen-activated protein kinase, Sp-1 transcription factor, heterotrimeric Gi protein, and phosphatidylinositol-3'-kinase. In conclusion, the immunodysregulation described for patients with trauma is not a generalized phenomenon but depends on the stimulus and the signaling pathway.

APACHE↗

PubMed2003

Microcapsules made by enzymatically tailored alginate.

Alginate is widely used for encapsulation of cells. Alginate is a linear block copolymer consisting of mannuronic acid (M) and guluronic acid (G). It has been shown that enzymes known as C-5 epimerases convert M to G in the polymer chain, giving rise to novel alginates with tailored properties. One of these enzymes, AlgE4, converts M blocks into blocks of strictly alternating M and G. In this study we investigated how alginate epimerized by AlgE4 affected capsule properties such as stability and permeability. Inhomogeneous calcium-alginate gel beads were made with original and AlgE4-epimerized alginates of different origin. The epimerized alginates formed initially smaller alginate gels that showed increased resistance to osmotic swelling compared with the original nonmodified alginate samples. The permeability, measured as diffusion of immunoglobulin (Ig) G into Ca/Ba-alginate gel beads, was reduced by epimerization and further reduced by addition of poly-L-lysine (PLL). The osmotic stability of alginate-poly-D-lysine(PDL)-alginate capsules was enhanced by the use of epimerized alginate; indeed, stable capsules with low permeability to tumor necrosis factor (TNF) could be made with low PDL exposures. Finally, alginate with an alternating structure interacted more strongly with the alginate-PLL capsule than did alginate with a high content of M blocks or G blocks or than an alginate consisting mainly of M.

Alginates↗

PubMed2003

Cell distributions and functions of Toll-like receptor 4 studied by fluorescent gene constructs.

Bacterial lipopolysaccharide (LPS) is recognized in mammals by a receptor complex composed of CD14, Toll-like receptor 4 (TLR4), and MD-2. The detailed mechanisms of how TLR4 transmits the signal from the outside to the inside of the cell remain to be elucidated. One way of studying TLR4 signaling mechanisms is to construct chimeras of TLR molecules C-terminally fused to fluorescent proteins and stably express these constructs in cells. Such constructs are functional when transfected into HEK293 epithelial cells. Confocal microscopy of TLR4 expression in live cells demonstrated pronounced expression on the plasma membrane as well in the Golgi apparatus. Studies were performed to clarify whether expression of TLR4 in the Golgi was necessary for LPS stimulation. Rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane was a prominent phenomenon. In agreement with other types of plasma membrane receptors, aggregation of TLR4 by immobilized TLR4 antibodies was sufficient to induce signaling. Also, pharmacological disruption of the Golgi did not inhibit LPS induced NF-kappaB activation. Furthermore, LPS stimulation recruited the adapter molecule, MyD88, to the inside of the plasma membrane. Thus, LPS signaling commences on the plasma membrane and is independent of trafficking to the Golgi.

Animals↗

PubMed2003

The LPS receptor generates inflammatory signals from the cell surface.

Bacterial lipopolysaccharides (LPSs) are recognized in mammals by a receptor complex composed of CD14, Toll-like receptor 4 (TLR4), and MD-2. The mechanism of TLR4 function remains to be elucidated. We constructed chimeric TLR molecules C-terminally fused to fluorescent proteins and stably expressed these chimeric constructs in cells. Confocal microscopy revealed TLR4 to be expressed on the plasma membrane and the Golgi apparatus. Time-lapse confocal imaging showed rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane. Membrane TLR4 engagement by antibody was sufficient to induce signaling and pharmacological disruption of the Golgi did not affect cellular responses to LPS. Thus, LPS signaling commences after LPS recognition by surface-expressed TLR4 independent of LPS trafficking to the Golgi.

Cell Line↗

PubMed2002

A novel host-parasite lipid cross-talk. Schistosomal lyso-phosphatidylserine activates toll-like receptor 2 and affects immune polarization.

Schistosome infections are characterized by prominent T cell hyporesponsiveness during the chronic stage of infection. We found that schistosome-specific phosphatidylserine (PS) activated TLR2 and affected dendritic cells such that mature dendritic cells gained the ability to induce the development of IL-10-producing regulatory T cells. Using mass spectrometry, schistosomal lysophosphatidylserine (lyso-PS) was identified as the TLR2-activating molecule. This activity appears to be a unique property of schistosomal lyso-PS, containing specific acyl chains, because neither a synthetic lyso-PS (16:0) nor PS isolated from the mammalian host activates TLR2. Taken together, these findings provide evidence for a novel host-parasite interaction that may be central to long term survival of the parasite and limited host pathology with implications beyond parasitology.

Animals↗

PubMed2002

Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction.

Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2. We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi. Similar distributions were observed in human monocytes. Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane. Fluorescent LPS followed these trafficking pathways in CD14-positive cells. The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling. Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved. We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Involvement of toll-like receptor (TLR) 2 and TLR4 in cell activation by mannuronic acid polymers.

The alginate capsule produced by the human pathogen Pseudomonas aeruginosa is composed mainly of mannuronic acid polymers (poly-M) that have immunostimulating properties. Poly-M shares with lipopolysaccharide the ability to stimulate cytokine production from human monocytes in a CD14-dependent manner. In the present study we examined the role of Toll-like receptor (TLR) 2 and TLR4 in responses to poly-M. Blocking antibodies to TLR2 and TLR4 partly inhibited tumor necrosis factor production induced by poly-M in human monocytes, and further inhibition was obtained by combining the antibodies. By transiently transfecting HEK293 cells, we found that membrane CD14 together with either TLR2 or TLR4/MD-2 could mediate activation by poly-M. Transfection of HEK293 cells with TLR2 and fluorescently labeled TLR4 followed by co-patching of TLR2 with an antibody revealed no association of these molecules on the plasma membrane. However, macrophages from the Tlr4 mutant C3H/HeJ mice and TLR4 knockout mice were completely non-responsive to poly-M, whereas the tumor necrosis factor release from TLR2 knockout macrophages was half of that seen with wild type cells. Taken together the results suggest that both TLR2 and TLR4 are involved in cell activation by poly-M and that TLR4 may be required in primary murine macrophages.

Animals↗

PubMed2002

The proinflammatory CD14+CD16+DR++ monocytes are a major source of TNF.

In human blood two monocyte populations can be distinguished, i.e., the CD14(++)CD16(-)DR(+) classical monocytes and the CD14(+)CD16(+)DR(++) proinflammatory monocytes that account for only 10% of all monocytes. We have studied TNF production in these two types of cells using three-color immunofluorescence and flow cytometry on whole peripheral blood samples stimulated with either LPS or with the bacterial lipopeptide S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH,trihydrochloride (Pam3Cys). After stimulation with LPS the median fluorescence intensity for TNF protein was 3-fold higher in the proinflammatory monocytes when compared with the classical monocytes. After stimulation with Pam3Cys they almost exclusively responded showing 10-fold-higher levels of median fluorescence intensity for TNF protein. The median fluorescence intensity for Toll-like receptor 2 cell surface protein was found 2-fold higher on CD14(+)CD16(+)DR(++) monocytes, which may explain, in part, the higher Pam3Cys-induced TNF production by these cells. When analyzing secretion of TNF protein into the supernatant in PBMCs after depletion of CD16(+) monocytes we found a reduction of LPS-induced TNF by 28% but Pam3Cys-induced TNF was reduced by 64%. This indicates that the minor population of CD14(+)CD16(+) monocytes are major producers of TNF in human blood.

Adjuvants, Immunologic↗

PubMed2002

Inflammatory response after open heart surgery: release of heat-shock protein 70 and signaling through toll-like receptor-4.

BACKGROUND: Coronary artery bypass grafting with the use of cardiopulmonary bypass is known to mediate an inflammatory response. The stress-inducible heat-shock protein (HSP) 70 has been detected in myocardial cells after CABG, and toll-like receptors (TLRs) are suggested as putative signaling receptors for the HSPs, mediating synthesis of inflammatory cytokines. The main aims of our study were to explore the release of HSP70 and the regulation of monocyte TLR-2 and TLR-4 expression after CABG. METHODS AND RESULTS: Twenty patients referred for elective CABG were included in this study. Using immunoassays, we detected HSP70 in plasma after CABG, with peak concentration immediately after surgery. Interleukin-6 in plasma reached peak concentration 5 hours after surgery. Monocyte CD14, TLR-2, and TLR-4 expression, as analyzed by flow cytometry, was initially downregulated. On day 1, CD14 expression normalized, whereas TLR-2 and TLR-4 expression was upregulated. TLR-4 was significantly upregulated even on postoperative day 2. Additional experiments revealed that peritoneal macrophages from control (C3H/HeN) mice responded to HSP70 with release of tumor necrosis factor, whereas macrophages from mutated TLR-4 (C3H/HeJ) mice were unresponsive. In vitro, human adherent monocytes responded to recombinant HSP70 with interleukin-6 and tumor necrosis factor release. CD14 and TLR-4 monoclonal antibodies inhibited the cytokine response. CONCLUSIONS: In this study, we observed an immediate release of HSP70 into the circulation and a modulation of monocyte TLR-2 and TLR-4 expression after CABG. TLR-4 and CD14 appear to be involved in an HSP70-mediated activation of innate immunity.

Aged↗

PubMed2002

Development, characterization and use of monoclonal antibodies against sTRAIL: measurement of sTRAIL by ELISA.

Two monoclonal antibodies against tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL), designated VI10E and III6F, have been generated. These antibodies were useful in flow cytometry analysis, immunohistochemistry, immunoprecipitation and in the development of an immunoassay for the detection of soluble TRAIL (sTRAIL)in biological samples. The immunoassay was based on two monoclonal antibodies against TRAIL. VI10E was used as the capture antibody and bound TRAIL was detected with anti-TRAIL from R&D Systems which was digoxigenin (DIG)-labeled. This enzyme-linked immunosorbent assay (ELISA) was specific for TRAIL since a panel of other cytokines did not affect the signal. The immunoassay was suitable for the detection of sTRAIL in human serum and plasma samples, cell culture supernatants and cell lysates. In a preliminary screening, it was found that serum samples from human immunodeficiency virus (HIV)-infected patients contained sTRAIL, and all these positive samples were found in the AIDS group. Using the immunoassay, it was found that phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) to produce significant amounts of sTRAIL, the levels of which increased with exposure time. Thus, the immunoassay for TRAIL presented here represents a useful tool for measuring sTRAIL in various biological samples. It will also permit studies of release mechanisms as well as possible functions of the soluble form of this molecule.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Human gingival CD14(+) fibroblasts primed with gamma interferon increase production of interleukin-8 in response to lipopolysaccharide through up-regulation of membrane CD14 and MyD88 mRNA expression.

Gamma interferon (IFN-gamma)-primed human gingival fibroblasts (HGF) have been shown to produce higher levels of interleukin-8 (IL-8) upon stimulation with bacterial products and inflammatory cytokines than nonprimed controls. In this study, we examined whether priming of HGF with IFN-gamma up-regulates IL-8 production by the cells in response to purified lipopolysaccharide (LPS). The priming effect of IFN-gamma was clearly observed in the high-CD14-expressing (CD14(high)) HGF but not in the low-CD14-expressing (CD14(low)) HGF. The CD14(high) HGF were most effectively primed with IFN-gamma (1,000 IU/ml) for 72 h. To elucidate the mechanism of the priming effects of IFN-gamma for the LPS response by HGF, we examined whether IFN-gamma regulated expression of CD14, Toll-like receptor 2 (TLR2), TLR4, MD-2, and MyD88, all of which are molecules suggested to be associated with LPS signaling. In CD14(high) HGF, IFN-gamma markedly up-regulated CD14 and MyD88 but not TLR4 protein and MD-2 mRNA expression, while in CD14(low) HGF, IFN-gamma slightly increased MyD88 and scarcely affected CD14, TLR4 protein, and MD-2 mRNA levels. LPS-induced IL-8 production by IFN-gamma-primed CD14(high) HGF was significantly inhibited by monoclonal antibodies (MAbs) against CD14 and TLR4, but not by an anti-TLR2 MAb. These findings suggested that IFN-gamma primed CD14(high) HGF to enhance production of IL-8 in response to LPS through augmentation of the CD14-TLR system, where the presence of membrane CD14 was indispensable for the response of HGF to LPS.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Induction of cytokine production in human T cells and monocytes by highly purified lipoteichoic acid: involvement of Toll-like receptors and CD14.

BACKGROUND: The pro-inflammatory potential of lipoteichoic acid (LTA) from Staphylococcus aureus is controversial. The present study was undertaken to examine the ability of highly purified and characterized S. aureus LTA to stimulate the production of pro-inflammatory cytokines in human leukocytes at both mRNA and protein level, and to study the involvement of Toll-like receptors (TLRs) and CD14 in this response. MATERIAL/METHODS: Purified LTA was administered to whole human blood ex-vivo (or primary adherent monocytes) and the cytokine response assessed in plasma by EIA. Cytokine mRNA was measured by RT-PCR on leukocyte subsets isolated following stimulation. To study the involvement of specific receptors for LTA signaling, CHO cells transfected with CD14 and/or TLR2, TLR4 were used, as well as antibodies directed against these receptors. RESULTS: Addition of highly purified LTA to human whole blood or primary adherent human monocytes elicited a time and concentration dependent release of tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6 and IL-8. Messenger RNA encoding TNF-alpha, IL-1 beta and IL-6 seemed to be accumulated in monocytes and T cells, but not in granulocytes and B cells. Expression of TLR2, but not TLR4, in chinese hamster ovary cells conferred responsiveness to LTA. However, antibodies directed towards TLR2 (clone TL2.1) or TLR4 (clone HTA125) failed to inhibit TNF-a release induced by LTA in both the whole blood model and in adherent monocytes. In contrast, blockade of the CD14 receptor with MAb18D11 strongly attenuated the LTA induced release of TNF-alpha in both models. CONCLUSIONS: We propose that (i) LTA from S. aureus triggers the release of cytokines during staphylococcal infections, and (ii) both monocytes and T cells contribute to cytokine production induced by LTA. TLR2 may mediate cellular activation by LTA, but the functional significance of this receptor during staphylococcal infections remains elusive.

Actins↗

PubMed2002

Microencapsulation of cells producing therapeutic proteins: optimizing cell growth and secretion.

Microencapsulation of genetically engineered cells may have important applications as delivery systems for therapeutic proteins. However, optimization of the microcapsules with regard to mechanical stability, cell growth, and secretion of proteins is necessary in order to evaluate the future use of this delivery technology. We have explored the growth, survival, and secretion of therapeutic proteins from 293-EBNA cells producing endostatin (293 endo cells) and JJN3 myeloma cells producing hepatocyte growth factor (HGF) that have been embedded in various types of alginate capsules. Parameters that affect capsule integrity such as homogenous and inhomogenous gel cores and addition of an outer poly-L-lysine (PLL)-alginate coating were evaluated in relation to cell functions. When cells were encapsulated, the PLL layer was found to be absolutely required for the capsule integrity. The JJN3 and 293 endo cells displayed completely different growth and distribution patterns of live and dead cells within the microcapsules, as shown by 3D pictures reconstructed from images taken with confocal laser scanning microscopy (CLSM). Encapsulated JJN3 cells showed a bell-shaped growth and HGF secretion curve over a time period of 5 months. The 293 endo cells reached a plateau phase in growth after 23 days postencapsulation; however, after around 30 days a fraction of the microcapsules started to disintegrate. Microcapsule disintegration occurred with time irrespective of capsule and cell type, showing that alginate microcapsules possessing relatively high gel strength are not strong enough to keep proliferating cells within the microcapsules for prolonged time periods. Although this study shows that the stability of an alginate-based cell factory can be increased by a PLL-alginate coating, further improvement is necessary with regard to capsule integrity as well as controlling the cell growth before this technology can be used for therapy.

Alginates↗