Biomedical subjects

D Heumann

Publications and source records attributed to D Heumann.

At least 55 records · Page 3Linked to original sources

PubMed1993

Short time exposure to lipopolysaccharide is sufficient to activate human monocytes.

Very little is known about early events in LPS binding and about the duration of LPS exposure required to activate monocytes. In the present study, we have investigated the kinetics of LPS binding to human monocytes and the time of exposure required to trigger the synthesis of TNF-alpha. We directly followed the binding of FITC-labeled LPS to monocytes by flow cytometry or confocal laser microscopy. LPS was able to bind to the cell surface within 1 min of exposure, and was internalized within 5 min. Equilibrium was reached within 15 min at all but the lowest LPS concentration tested (10 ng/ml). Binding was dependent on the presence of LPS-binding protein, supplied either as a plasma component or in purified form, and inhibited by an anti-CD14 mAb (MY4). Polymyxin B, an inhibitor of LPS-mediated activation, essentially abrogated the LPS-binding protein- and CD14-dependent binding of LPS to monocytes. Using either the anti-CD14 mAb or polymyxin B to block further LPS binding, we found that 5 to 10 min of exposure was sufficient to trigger maximal TNF-alpha release. Similarly, monocytes washed after 5 to 15 min exposure to eliminate LPS also produced high levels of TNF-alpha transcripts without further presence of LPS in the medium. We conclude that a few minutes of exposure to physiologically relevant concentrations of LPS are sufficient to trigger both maximal binding and activation of monocytes.

Acute-Phase Proteins↗

PubMed1993

[Cytokines and severe sepsis].

During severe sepsis syndromes, almost every gene coding for cytokines may be activated. The primary purpose of this activation is to defend the organism against infection, but sometimes these inflammatory mediators go out of control. The reasons why this may occur is unclear because the regulation of cytokines production is still poorly understood. Metabolic effects, production of endothelial adhesion molecules and triggering of neutrophils are some important consequences of cytokine overstimulation which may lead to the clinical picture of septic shock. The major cytokines involved in septic shock are tumor necrosis factor alpha (TNF) and interleukin-1 (IL-1). Both may induce lethal shock in experimental models. The effects of these 2 cytokines are difficult to differentiate from one another because they share many similar biological effects, one can induce the synthesis of the other, and they are strikingly synergistic with each other. gamma-interferon may amplify the inflammatory response by stimulating the cells of monocytic lineage and by increasing TNF-receptor expression, thus participating in the pathogenesis of the septic syndrome. The role of other cytokines is still poorly known. Clinical studies with anti-TNF monoclonal antibodies or with an IL-1 receptor antagonist are under way.

Animals↗

PubMed1993

Competition between bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein for lipopolysaccharide binding to monocytes.

The bactericidal/permeability-increasing protein (BPI) inhibits the lipopolysaccharide (LPS)-mediated activation of monocytes. Due to its inhibitory activity for various LPS, BPI has therapeutic potential in endotoxic shock. To be efficient in vivo, BPI should overcome the action of LPS-binding protein (LBP), a serum molecule that increases the expression of LPS-inducible genes via CD14 of monocytes, rBPI23, a recombinant fragment of BPI, prevented in a dose-dependent manner the binding and the internalization of LPS mediated by LBP. Consequently, rBPI23 also inhibited LPS-induced tumor necrosis factor (TNF alpha) synthesis from monocytes. LPS- and LBP-mediated activation of monocytes was totally inhibited when LPS was preincubated with rBPI23. Adding rBPI23 at the same time as LBP resulted in an important but partial inhibition of TNF alpha release, but this inhibition vanished with delaying the time of addition of rBPI23. These studies suggest that the inhibitory activity of BPI is related to its ability to compete with LBP for LPS.

Acute-Phase Proteins↗

PubMed1993

Purification and characterization of murine lipopolysaccharide-binding protein.

The serum protein lipopolysaccharide (LPS)-binding protein (LBP) seems to play an important role in regulating host responses to LPS. Complexes of LPS and LBP form in serum and stimulate monocytes, macrophages, or polymorphonuclear leukocytes after binding to CD14. Previous reports have described the structure and properties of LBP from human and rabbit sera. Since mice are used in some experimental models of endotoxemia or gram-negative bacterial infections, information is needed about the properties of murine LBP. Murine LBP was purified by ion-exchange chromatography and high-pressure liquid chromatography; its NH2-terminal sequence (TNPGLVTRIT) was very similar to those of human and rabbit LBPs (80 to 90% amino acid identity). Murine LBP resembled LBPs from other species in that it promoted the binding of LPS to monocytes and enhanced the sensitivity of monocytes to LPS at least 100-fold. Mouse LBP, like rabbit and human LBPs, was found to be an acute-phase protein. Further in vivo studies with mice and anti-CD14 or anti-LBP reagents should help determine the role of LBP in response to LPS challenges.

Acute-Phase Proteins↗

PubMed1992

Control of lipopolysaccharide (LPS) binding and LPS-induced tumor necrosis factor secretion in human peripheral blood monocytes.

We used flow cytometry to determine how LPS-binding protein (LBP) effects the binding of fluorescein-labeled LPS to human monocytes via receptor-dependent mechanisms. The addition of human, rabbit, mouse, or FCS strikingly increased the binding of LPS to monocytes compared with controls incubated in serum-free medium. This binding was totally prevented by preincubation of monocytes with MY4, an anti-CD14 mAb, or by enzymatic removal of CD14 from monocytes. Depletion of LBP from rabbit serum with anti-LBP antibodies also produced a similar suppression. Solutions of albumin did not support the enhanced binding observed in serum but the addition of purified rabbit LBP to albumin solutions resulted in binding similar to that observed in serum-containing medium. When type-specific anti-LPS mAb was added to human serum, LPS binding to monocytes occurred but was only partly inhibited by anti-CD14 mAb, suggesting that receptors other than CD14 (presumably Fc or complement receptors) were involved. Serum increased by 100- to 1000-fold the sensitivity of monocytes to the triggering by LPS resulting in TNF secretion. TNF secretion was inhibited by anti-CD14 mAb up to 100 ng/ml of LPS and by anti-LPS mAb up to 1 to 10 ng/ml. The inhibition of TNF secretion by anti-LPS mAb appeared to be the result of directing LPS to monocyte receptors other than CD14. In contrast, in medium containing normal as well as acute serum and in the absence of anti-LPS antibodies, the binding of LPS to monocytes and the triggering of TNF secretion appeared to be mediated mainly by interactions between CD14 and LBP-LPS complexes.

Acute-Phase Proteins↗

PubMed1992

Cytokine production after intravenous or peritoneal gram-negative bacterial challenge in mice. Comparative protective efficacy of antibodies to tumor necrosis factor-alpha and to lipopolysaccharide.

The production of TNF-alpha, IL-1, and IL-6 was measured in mice after bolus i.v. Escherichia coli O111 LPS injections and during bacteremia induced either by bolus i.v. or by i.p. challenges of live E. coli O111. High but transient TNF-alpha peaks were observed after bolus i.v. LPS or bacterial challenges. In contrast, the levels during lethal peritonitis increased progressively to values 50- to 100-fold lower than the peak values observed after i.v. injections, and remained sustained until death. Whereas after i.v. challenge with 1000 LD50 of LPS, anti-TNF-alpha antibody fully protected mice from death and reduced serum IL-1 and IL-6 levels, anti-TNF-alpha antibody did not improve the survival of mice nor reduced serum IL-1 and IL-6 levels after i.p. bacterial challenge. In contrast to anti-TNF-alpha antibodies, anti-LPS antibodies were protective in the peritonitis model. Protection was accompanied by a striking reduction of bacterial numbers and of TNF-alpha, IL-1, and IL-6 levels in the serum, but the levels of these cytokines were only marginally affected in the peritoneal lavage fluid. This latter observation demonstrates that the local peritoneal cytokines did not diffuse readily into the circulation, thus suggesting that at least part of the circulating cytokines are produced systemically. In conclusion, the striking differences between cytokine profiles as well as the divergent efficacy of anti-TNF-alpha antibody after i.v. bolus and after i.p. challenges suggest that TNF-alpha may not be as important in the pathogenesis of lethal peritonitis than after lethal acute bacteremia.

Animals↗

PubMed1992

Enhancement of murine macrophage binding of and response to bacterial lipopolysaccharide (LPS) by LPS-binding protein.

We have studied the effects of highly purified rabbit lipopolysaccharide (LPS)-binding protein (LBP) on the ability of murine bone marrow-derived macrophages to respond to bacterial LPS. Macrophage responses studied include the secretion of tumor necrosis factor alpha, production of arginine-derived nitrite (NO2-), and killing of an intracellular pathogen, Leishmania enriettii. Macrophages from either CBA or LPS-hyporesponsive C3H/HeJ mice exhibited significantly greater sensitivity to LPS in the presence of LBP. Furthermore, both CBA and C3H/HeJ macrophages demonstrated an LBP-dependent enhancement of LPS binding. These results suggest that C3H/HeJ macrophages are capable of binding LPS-LBP complexes and support the hypothesis that hyporesponsiveness in this strain involves a step subsequent to LPS binding. Furthermore, these findings provide additional evidence of the important role played by the acute-phase plasma protein LBP in modifying host response to LPS.

Acute-Phase Proteins↗

PubMed1992

Production of tumor necrosis factor-alpha by naive or memory T lymphocytes activated via CD28.

While it is well established that activated T cells can produce tumor necrosis factor alpha (TNF-alpha), it is less clear whether this function is confined to a given subset, e.g., memory cells. To approach this question, we investigated the production of TNF-alpha by human peripheral blood T lymphocytes activated with anti-CD28 mAb since this activation pathway is known to potentiate cytokine production. Under the culture conditions used, the amount of TNF-alpha produced was markedly enhanced compared to that obtained after activation with immobilized anti-CD3 mAb. The enhancement of TNF-alpha production was already apparent after incubation of T cells for 6 hr. Up to 5 ng/ml of TNF-alpha was measured on Day 2 in supernatants of cultures of 10(4) T lymphocytes. To determine the source of the cells producing high amounts of TNF-alpha, T lymphocytes were separated into two subpopulations, namely naive cells (expressing the CD45RA isoform) and memory cells (expressing the CD45RO isoform). While both subpopulations proliferated equally well after stimulation with anti-CD28 mAb, up to 90% of the TNF-alpha produced under these conditions originated from memory T cells. These results thus document that T cell activation via CD28 results in a marked increase in TNF-alpha production without affecting the functional disparity that exists between naive and memory T cells.

Antibodies, Monoclonal↗

PubMed1991

High circulating levels of interleukin-6 in patients with septic shock: evolution during sepsis, prognostic value, and interplay with other cytokines. The Swiss-Dutch J5 Immunoglobulin Study Group.

PURPOSE AND PATIENTS: We measured the serum concentrations of interleukin-6 (IL-6) in 70 patients with established septic shock caused predominantly by gram-negative bacteria. The aims of the study were to determine whether and for how long IL-6 was detectable in the circulation of these patients, to assess whether IL-6 levels were associated with patients' outcomes, and, finally, to examine the interplay between IL-6, tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma). RESULTS: IL-6 was detected in 64% of the patients at study entry but in only 18% on Day 1 and 2% on Day 10. Serum levels of IL-6 were higher (median: 3.5 ng/mL, range: less than 0.1 to 305 ng/mL) in patients dying of fulminant septic shock than in those surviving (median: 0.5 ng/mL, range: less than 0.1 to 135 ng/mL; p = 0.003) or in those with a transient reversal of shock but who ultimately died of a relapse of shock (median: less than 0.1 ng/mL, range: less than 0.1 to 12.5 ng/mL; p = 0.005). However, no cutoff values of IL-6 confidently predicted the outcome of an individual patient. The serum concentrations of IL-6 measured at study entry correlated with the duration of survival (r = -0.51, p = 0.004) and with the levels of TNF-alpha (r = 0.53; p less than 0.0001) but not with the levels of either IL-1 beta (r = 0.01, p = 0.90) or IFN-gamma (r = 0.06, p = 0.60). CONCLUSIONS: These results indicate that circulating levels of IL-6 are detectable in a majority of patients with gram-negative septic shock. Concentrations of IL-6 peaked near the onset of shock and rapidly decreased to undetectable levels within approximately 24 hours in most patients. Levels of IL-6 measured at study entry correlated with levels of TNF and with patients' outcomes. Yet, IL-6 does not appear to be a clinically useful laboratory test for predicting the outcome of an individual patient.

Adolescent↗

PubMed1991

Antibodies to core lipopolysaccharide determinants: absence of cross-reactivity with heterologous lipopolysaccharides.

Using monoclonal antibodies directed against defined epitopes of endotoxin core, this study demonstrated that the presentation of lipopolysaccharide (LPS) to antibodies is critical for measuring the specific binding of antibodies to LPS structures. False cross-reactive reactions apparently were observed when free core LPS or lipid A were used as antigens in ELISA, whereas coating with complexes of high-density lipoproteins with core LPS increased both the sensitivity and the specificity of the test compared with coating with free core LPS, so that nonspecific binding of antibodies was largely avoided. Using this technique, it was not possible to find broadly cross-reactive core LPS antibodies after immunization of rabbits and humans with rough mutants of gram-negative bacteria. These observations underscore the need for careful evaluation of the potential for cross-reactivity of antisera and of monoclonal antibodies directed against endotoxin core.

Animals↗

PubMed1991

Antibodies to lipopolysaccharides after immunization of humans with the rough mutant Escherichia coli J5.

To investigate whether immunization with Escherichia coli J5 boiled cells induces antibodies directed at deep core structures, antibodies against J5 lipopolysaccharide (LPS), Re LPS, and lipid A were measured in the serum of 70 volunteers before and 2 weeks after immunization. To improve the sensitivity and the specificity of ELISA, complexes of core LPS with high-density lipoproteins were used instead of free core LPS as antigens. A median three-fold increase in antibodies directed against J5 LPS was observed, but no significant increase in the antibodies against Re LPS or lipid A was found. Since J5 antiserum did not react with several smooth LPS or with Re LPS and lipid A, cross-reactivity could not be demonstrated. Thus, immunization of volunteers with E. coli J5 produced a modest specific antibody response against J5 LPS. The mechanism of protection previously observed with J5 antiserum remains unclear.

Antibodies, Bacterial↗

PubMed1990

Association between protective efficacy of anti-lipopolysaccharide (LPS) antibodies and suppression of LPS-induced tumor necrosis factor alpha and interleukin 6. Comparison of O side chain-specific antibodies with core LPS antibodies.

Two-core LPS antibodies, the rabbit J5 polyclonal antiserum and the human anti-lipid A IgM mAb HA-1A, did not improve the survival of mice challenged with E. coli O111 or P. aeruginosa 3, or with the LPS extracted from them, and did not decrease the incidence of Shwartzman reactions in rabbits challenged with O111 LPS. In contrast, O side chain-specific rabbit antisera were protective in these models. The protection afforded by O side chain-specific antisera against endotoxin lethality was associated with decreased LPS-induced serum TNF and IL-6 levels, whereas core LPS antibodies had no effect on TNF or IL-6 levels. The absence of reduction of LPS-induced cytokines levels by core LPS antibodies suggests that these antibodies are not able to prevent the interactions between LPS and target cells.

Animals↗

PubMed1989

Human alpha 2-macroglobulin as an inhibitor of insoluble trypsin.

alpha 2-Macroglobulin binds to insoluble trypsin bound on agarose beads inducing a reduction of proteolytic activity of the enzyme towards large substrates such as azocasein. When trypsin was bound on other matrices like sheep red blood cells or latex beads, the inhibition of proteolytic activity by alpha 2-macroglobulin was complete. These results show that alpha 2-macroglobulin inhibits similarly both soluble and insoluble proteinases.

Enzymes, Immobilized↗

PubMed1989

Molecular interactions of Leishmania promastigote surface protease with human alpha 2-macroglobulin.

The interaction of Leishmania promastigote surface protease (PSP) with the plasmatic protease inhibitor alpha 2-macroglobulin (alpha 2M) was investigated. In plasma, solubilized PSP forms covalent complexes only with alpha 2M, at the exclusion of other protease inhibitors. The formation of complexes is accompanied by the proteolytic cleavage of the alpha 2M subunit and by the transition from the 'slow' to the 'fast' form of alpha 2M. The proteolytic activity of solubilized PSP on azocasein is inhibited by alpha 2M. In contrast, we found no evidence for a specific interaction of alpha 2M with the surface of promastigotes and PSP proteolytic activity on intact cells was not inhibited by alpha 2M.

Animals↗

PubMed1988

Immunomodulation by alpha 2-macroglobulin and alpha 2-macroglobulin-proteinase complexes: the effect on the human T lymphocyte response.

Alpha 2-macroglobulin (alpha 2M), various alpha 2M-proteinase complexes and methylamine-treated alpha 2M were added to human lymphocyte cultures stimulated with the specific antigen purified protein derivative of tuberculin (PPD), pokeweed mitogen (PWM) and anti-CD3. alpha 2M-trypsin diminished all reactions in a dose-dependent way. In the PPD-induced stimulation of peripheral blood mononuclear cells, both change of configuration and remaining proteinase activity contributed to the suppressive activity. Separate exposure of adherent cells or the highly purified T lymphocytes to alpha 2M-trypsin complexes indicated that the effect was mediated through adherent cells. Addition of indomethacin did not modify the results. In the interleukin 2 (IL2)-dependent stimulation of purified T lymphocytes by anti-CD3 the effect of alpha 2M-proteinase complexes was probably due to the digestion of IL 2 through remaining proteinase activity. As alpha 2M-proteinase complexes are formed at sites of inflammation, the multiple immunomodulatory effects of alpha 2M-proteinase complexes might contribute to the dysregulation of the immune system in inflammatory diseases.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1987

Activation of resting T lymphocytes by cross-linked anti-CD3 (T3).

In this study we have examined the role of small numbers of adherent cells in the stimulation of highly purified resting T lymphocytes by cross-linked monoclonal anti-CD3 antibodies (T3). T cells were obtained by 3 cycles of purification, using adherence to plastic surface, nylon wool column separation and rosetting with 2-aminoethyl isothiouronium bromide-treated sheep red blood cells. They were stimulated either with T3 antibodies or with solid-phase rabbit anti-mouse IgG-T3 complexes. In standard high density cultures (1 X 10(5)-2 X 10(5) cells), soluble T3, interleukin (IL) 1 and IL 2, used separately, did not induce proliferation of T cells. Soluble T3 together with IL 2 slightly activated T cells to proliferate. Rabbit anti-mouse IgG-T3 complexes attached to the plastic wells induced stimulation in some cultures. The response was markedly increased after addition of IL 2, but not IL 1. The irregular response of these cultures suggested the presence of another variable signal. When cell numbers were reduced (12 X 10(3) cells), neither cross-linking of the CD3-Ti complex nor addition of exogenous IL 1 or IL 2, alone or in combination, stimulated the T cells to increase DNA synthesis. A positive response, comparable to complete peripheral blood mononuclear cells, was restored with 0.5% supplemented adherent cells, provided that IL 2 was present.

Antibodies, Monoclonal↗

PubMed1986

Determination of ectoenzyme activities in leukemic cells and in established hematopoietic cell lines.

Ectoenzyme activities were determined in peripheral blood cells from patients with acute leukemias, from normal controls, and in cells of hematopoietic cell lines. In common acute lymphoblastic leukemia, cell membrane-associated 5'-nucleotidase (5'-N) activity was significantly higher than in acute T and unclassified lymphoblastic leukemias. In acute myeloblastic and myelomonocytic leukemias, cells contained significantly higher gamma-glutamyl transpeptidase (gamma-GT) activity than in lymphoblastic leukemias. Normal B lymphocytes differed from T cells and monocytes mainly in their 5'-N activity, whereas in monocytes, gamma-GT activity was more pronounced than in other normal blood cells. Hematopoietic cell lines showed some distinct patterns of ectoenzyme activity. Most B cell lines had high 5'-N and (Na-K-Mg) adenosine triphosphatase activities. In lines of myeloid origin, elevated gamma-GT values were found. In lymphoid stem cells and in T lymphoblast lines, most ectoenzyme activities were lower than in the other cell lines. In some cell lines, characteristic high-activity marker enzymes were detected.

5'-Nucleotidase↗

PubMed1986

Characterization of membrane vesicles circulating in the serum of patients with common acute lymphoblastic leukemia.

Common acute lymphoblastic leukemia antigen detected by radioimmunoassay in the serum of patients with common acute lymphoblastic leukemia was found to be exclusively associated with the pellet of the serum samples obtained by ultracentrifugation at 100,000 X g. The pellets were shown to contain membrane vesicles or fragments which were characterized by electron microscopy and determination of enzymatic activity. The pelleted fragments had an apparent diameter ranging between 60 and 260 nm and showed a trilaminar membrane structure. On freeze-fracture preparations, the fragments with concave profile, corresponding to the external fracture face of plasma membrane, displayed an intramembrane particle density (ranging from 0 to 750 particles per micron2) which is similar to that recorded on the corresponding fracture face of intact cells from the common lymphoblastic leukemia antigen positive leukemic cell line (Nalm-1) or of vesicles shed in the culture medium by Nalm-1 cells. Furthermore, analysis of the membrane enzyme marker 5'-nucleotidase in the pellet of patient's sera, showed that the presence of this enzyme correlated with that of common lymphoblastic leukemia antigen, but the quantitative relationship between the two surface constituents was not linear. The results suggest that the two markers are located on the same membrane fragments, but that their individual distribution on the shed fragments is heterogeneous.

5'-Nucleotidase↗