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Biomedical subjects

K Bendtzen

Publications and source records attributed to K Bendtzen.

At least 271 records · Page 15Linked to original sources
PubMed1988

Detection of tumour necrosis factor from lipopolysaccharide-stimulated human mononuclear cells by enzyme-linked immunosorbent assay and cytotoxicity bioassay.

Tumour necrosis factor (TNF) or cachectin is an important mediator of endotoxic activity. To investigate the production of TNF from human mononuclear cells (MNC) in response to lipopolysaccharide (LPS), we developed a sensitive and specific enzyme immunoassay (ELISA) and a cytotoxicity bioassay for TNF. The ELISA utilizes the biotin/avidin system and includes four incubation steps. The detection limit was 25 pg recombinant TNF (rTNF)/100 microliter. There was no interference of medium, serum, plasma, spinal fluid, or urine and no cross-reaction with natural or recombinant IL-1-alpha, IL-1-beta, IL-2, IFN-gamma, or lymphotoxin (TNF-beta). Recovery of TNF added to the media was 85-123% (n = 22). The relative standard deviations within and between assays were 7% and 8%, respectively. TNF-induced cytotoxicity was measured on actinomycin-D-treated L-M mouse fibroblasts. The detection limit in this bioassay was 0.5 U/30 microliter or 12.5 pg/30 microliter of rTNF. IL-1-alpha and IL-1-beta slightly inhibited the cytotoxic activity of rTNF. In this bioassay, cytotoxic activity (50-300 U/ml) was detected only when MNC were stimulated with high concentrations of LPS (100-1000 ng/ml). In contrast, using 0.01-100 ng/ml of LPS, the ELISA detected TNF in a dose-dependent manner (0.25 ng/ml to 40 ng/ml). It is concluded that TNF is liberated from human blood MNC if stimulated with minute amounts of LPS. It is suggested that human TNF may be secreted in a relatively inactive form or that inhibitors of TNF are generated along with the monokine. Because of this, and because commonly used bioassays for TNF fail to distinguish between TNF and lymphotoxin, specific ELISA are recommended to supplement TNF bioassays.

Cytotoxicity Tests, Immunologic↗
PubMed1988

Inhibitor of interleukin 1 in normal human urine. Different effects on mouse thymocytes and on a murine T-cell line.

We investigated whether urine from normal individuals contains inhibitors of interleukin 1 (IL-1). Diafiltered urine from normal afebrile donors suppressed IL-1-induced interleukin 2 (IL-2) activity of mouse thymocyte supernatants. These supernatants, however, strongly suppressed IL-2-induced [3H]thymidine incorporation into the IL-2-sensitive cell line CTLL-2, whereas the urine preparations did not. This phenomenon was caused by an increased amount of thymidine secreted by the urine-treated thymocytes. Therefore, in order to prevent interference, experiments were carried out with excess [3H]thymidine. Under these circumstances, suppression of IL-1- and to a lesser extent IL-2-induced DNA synthesis was still observed, whereas the synergistic effect of IL-1 on IL-2-induced DNA synthesis was only marginally reduced. We conclude that suppression of IL-1-induced IL-2 production by mouse thymocytes is a major effect of the IL-1-inhibitory factor(s) in normal urine. When the murine EL4 cell line was used, the diafiltered urine failed to inhibit IL-1-induced IL-2 production. The detection of an IL-1 inhibitor in urine is therefore dependent on the target cells as well as the effects of IL-1 on these cells.

Animals↗
PubMed1988

Modulation of natural killer cell activity in peripheral blood by physical exercise.

The present study was designed to examine the effect of physical exercise on human natural killer (NK) cells. Six healthy volunteers underwent two different acute physical exercise tests with an interval of at least 1 week: (1) 60 min bicycle exercise at 80% of maximal oxygen uptake (VO2max) and (2) 60 min back-muscle training at up to 29% of VO2max; blood samples were collected before and during the last few minutes of exercise, as well as 2 h and 24 h afterwards. The NK cell activity (lysis/fixed number of mononuclear cells) increased during bicycle exercise, dropped to a minimum 2 h later and returned to pre-exercise levels within 24 h. Back-muscle exercise did not significantly influence NK cell activity. Plasma levels of adrenaline, noradrenaline, and cortisol were elevated during bicycling, but not during back-muscle exercise, indicating that exercise intensity is a determinant of NK cell activity. During bicycle exercise the NK cell subset (CD16- cells) of mononuclear cells increased significantly. Furthermore an improved interleukin 2 (IL-2) boosting of the NK cell activity was found during work as compared to IFN-alpha and indomethacin-enhanced NK cell activity. These results indicate that NK cells with a high IL-2 response capacity are recruited to the peripheral blood during exercise. The decreased NK cell activity demonstrated 2 h after work was probably not due to fluctuations in size of the NK cell pool, since the proportion of CD16+ cells was normal. The finding that indomethacin fully restored the suppressed NK cell activity in vitro and the demonstration of a twofold increase in monocyte (CD20+ cells) proportions 2 h after work, strongly indicate that prostaglandins released by monocytes during the heavy physical exercise are responsible for the down-regulation of the NK cells.

Adult↗
PubMed1988

Interleukin 1-induced down-regulation of antibody binding to CD4 molecules on human lymphocytes.

Interleukin 1 (IL-1) is involved in the early activation of T lymphocytes. The CD4 antigen, described as a phenotypic marker of helper T cells, is also important in early T-cell activation by its ability to bind to MHC class II molecules on antigen-presenting cells, and to transmit positive (and negative) signals to the cells. We observed that purified human monocyte IL-1 as well as recombinant IL-1 alpha and IL-1 beta selectively decreased the binding of monoclonal antibodies to CD4 on the surface of otherwise unstimulated blood T cells, in contrast to prestimulated and continuously grown CD4+ cells. Under optimal growth conditions, the initial reduction in antibody binding to CD4 was followed by an apparent re-expression of the CD4 antigen even in the presence of high concentrations of IL-1. This re-expression did not occur if the cells were cultured at 4 degrees C, or after treatment with actinomycin D or cytochalasin B, indicating that protein synthesis and intact microfilament function were essential for re-expression of CD4 binding. The mechanism by which CD4 molecules are physically and/or functionally modulated by IL-1 is unclear.

Antibodies, Monoclonal↗
PubMed1988

Interleukin-1 affects the function of cultured human thyroid cells.

Cytokines are peptide hormones essential for cellular communication in the immune response. The purpose of this study was to investigate the influence of cytokines, especially recombinant interleukin 1 beta (rIL-1 beta), on human thyroid cells. Thyroglobulin (Tg) was measured by a double antibody radioimmunoassay, and cyclic AMP (cAMP) by a competitive protein binding assay. Supernatants from unstimulated and phytohaemagglutinin-stimulated blood mononuclear cells were added to human thyroid cells cultured in monolayers. A dose-dependent inhibition of the secretion of Tg and cAMP was demonstrated. Both subcultured and primary cultured cells incubated with rIL-1 beta at pharmacological levels (10(-1)-10(2) U/ml) exhibited an inhibition of Tg and cAMP secretion, while at physiological levels (10(-5)-10(-3) U/ml), the secretion of Tg was enhanced. The similar stimulation of cAMP was demonstrated in subcultures. These in vitro studies suggest that IL-1 beta may play a role in the pathogenesis of autoimmune thyroid diseases. Further, the stimulations at low concentrations indicate that IL-1 beta may regulate the function of the thyroid gland under physiological conditions.

Autoimmune Diseases↗
PubMed1988

Inhibition of human lymphocyte proliferation and cleavage of interleukin-2 by Pseudomonas aeruginosa proteases.

This study was undertaken to determine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (ELA) on human lymphocyte function. AP at 50 micrograms/ml and ELA at 12 micrograms/ml caused a 50% inhibition of phytohemagglutinin-induced proliferation. There was no difference in the effect of proteases on CD4- and CD8-positive cells. To determine the effect of proteases on interleukin-2 (IL-2)-induced cell proliferation, the proteases and IL-2 were added to the IL-2-dependent CTLL-2 cell line. AP and ELA inhibited the proliferation of these cells. When IL-2 was added in excess, the inhibition was partly reversed. ELA at 10 micrograms/ml cleaved IL-2, as judged by size chromatography of a reaction mixture containing 125I-labeled IL-2 and the proteases. The ELA-digested IL-2 exhibited a reduced binding capacity to IL-2 receptors on the lymphocytes. Furthermore, treatment of phytohemagglutinin-stimulated lymphocytes with AP and ELA resulted in inhibition of binding of intact IL-2 to IL-2 receptors on the stimulated lymphocytes. These results indicated that P. aeruginosa-derived enzymes are able to interfere with human lymphocyte function in vitro and that this effect might be due to cleavage of IL-2.

Binding, Competitive↗
PubMed1988

Functional characteristics of rat pancreatic islets maintained in culture after exposure to human interleukin 1.

Recent observations suggest a role for interleukin 1 beta (IL-1) in the autoimmune beta-cell destruction observed in type I (insulin-dependent) diabetes mellitus. We investigated the acute and long-term effects of IL-1 on pancreatic beta-cell function in vitro. Rat pancreatic islets were isolated and kept in tissue culture for 5 days. The islets were subsequently transferred to media containing RPMI-1640 plus 1% human serum with or without human recombinant IL-1 beta (300 pM) and cultured for another 48 h. The islets were examined either immediately after IL-1 exposure (day 0) or after an additional 6-day culture period without IL-1. On day 0, IL-1 was found to totally inhibit glucose-stimulated insulin release, partially inhibit glucose oxidation, and induce a decrease in islet DNA content. However, these islets were able to release insulin after stimulation with glucose plus theophylline, although the absolute rate of insulin secretion was lower than that of the control group. After 6 days in culture, the insulin-secretory response to glucose and the glucose oxidation rates of the IL-1-pretreated islets were completely restored, but there remained a reduced islet DNA content. We conclude that IL-1 is cytotoxic to islet beta-cells. However, surviving beta-cells are able to recover their functional capacity after a period of inhibited function.

Animals↗
PubMed1988

Effects of natural and recombinant interleukin-1 alpha and -beta on cytosolic free calcium in human and murine fibroblasts.

Changes in the concentration of cytosolic free calcium ((Ca2+)i) in response to purified blood monocyte IL-1 and human rIL-1 alpha and rIL-1 beta (17.5 kDa) were measured in murine L-M fibroblasts and in human foreskin fibroblasts using the fluorescent Ca2+ indicator, fura-2. In L-M fibroblasts, each of these IL-1 species, but not a recombinant 24-kDa precursor of the predominant IL-1 beta, produced a prompt, dose-related, and transient increase in (Ca2+)i. The effect was smaller but not eliminated when the cells were stimulated in EGTA-containing calcium, suggesting that the rise in (Ca2+)i was due to influx from both intracellular and extracellular Ca2+ pools. In human fibroblasts, however, the (Ca2+)i increased gradually, reaching a maximum after 1 hr of incubation with IL-1 and returning slowly to near basal levels in the following 2 hr. In contrast to the L-M cells, this accumulation of Ca2+ was abolished by EGTA, suggesting that in human fibroblasts, Ca2+ is mobilized solely from the extracellular space. Addition of the Ca2+ channel blockers verapamil and nifedipine was ineffective. IL-1 alpha and IL-1 beta both induced a dose-related increase in prostaglandin E2, but only in the human fibroblasts. These findings indicate that an increase in (Ca2+)i may be an important second mediator by which IL-1 initiates cell activation, but the signal may differ between cells.

Animals↗
PubMed1987

Human tumor necrosis factor potentiates human interleukin 1-mediated rat pancreatic beta-cell cytotoxicity.

Previous studies have established that the cytokine interleukin 1 (IL-1) is selectively cytotoxic for isolated human and rat pancreatic beta-cells. This observation raises the possibility that insulin-dependent diabetes mellitus is in part due to immunologically mediated mechanisms involving IL-1. However, other cytokines are produced during immunologic responses. To study possible modulatory effects of other cytokines on IL-1-mediated beta-cell cytotoxicity, we added human recombinant IL-1 alpha and beta (rIL-1 alpha, rIL-1 beta), tumor necrosis factor (rTNF), lymphotoxin (rLT), and interferon-gamma (rIFN-gamma) separately or in combinations to the culture medium of isolated rat islets of Langerhans. A half-maximal inhibition of glucose-stimulated insulin release after 7 days of culture was obtained with 100 pg/ml of rIL-1 beta, whereas 1000 pg/ml of rIL-1 alpha were necessary to obtain an equivalent effect. While ineffective in causing inhibition of beta-cell function or morphologic damage to islets alone 2.5 to 25 ng/ml of rTNF, but not 40 ng/ml of rLT, or 25 ng/ml of rIFN-gamma markedly potentiated the inhibition of beta-cell secretory response and dissolution of islet integrity caused by rIL-1 alpha and beta. The potentiating effect of rTNF was more pronounced if the rTNF was added after 60 min of preincubation of the islets with rIL-1 beta, than if rIL-1 beta was added after 60 min of preincubation with rTNF. rTNF did not interfere with the activity of rIL-1 alpha or beta on lymphocytes. Combinations of rIFN-gamma and rTNF or rLT did not affect beta-cell function. In conclusion, rTNF strongly potentiates the functional inhibition of beta-cells and the morphologic disintegration of islets caused by rIL-1 in vitro. These data, seen in context with previous observations of rIL-1-mediated beta-cell cytotoxicity, suggest that macrophages present in the intra-islet mononuclear cell infiltrate in insulin-dependent diabetes mellitus may secrete monokines that could be important effector molecules in beta-cell destruction.

Animals↗
PubMed1987

Effect of antimalarial drugs on stimulation and interleukin 2 production of human lymphocytes.

Effect of pyrimethamine, an antimalarial antifolate, and of mefloquine, chloroquine, and quinine, which belong to the quinoline group of antimalarials, on proliferation and interleukin 2 (IL-2) production of human lymphocytes was studied in vitro. Pyrimethamine at concentrations above therapeutic levels suppressed the lymphocytes' proliferation, but not their IL-2 production. All three quinolines suppressed the proliferation of lymphocytes, but not equally, with mefloquine having the strongest effect. Quinine suppressed the growth at therapeutic concentrations. The IL-2 production was suppressed at concentrations twice as high as those required to suppress lymphocyte proliferation. Addition of exogenous IL-2 only partially reversed the suppressive effect on lymphocyte proliferation. Delayed addition of the quinolines decreased their suppressive effect, but not completely. The mechanisms of action on human mononuclear cells of the various antimalarial drugs and the potential adverse effects of antimalarial chemotherapy are discussed.

Antimalarials↗
PubMed1987

Selective modulation of the CD4 molecular complex by Pseudomonas aeruginosa alkaline protease and elastase.

The binding of monoclonal antibodies against CD4 was specifically inhibited by treatment of human CD4+ cells with either alkaline protease (AP) or elastase (Ela), purified from Pseudomonas aeruginosa. Binding of antibodies against CD3 (pan T), CD5 (pan T), CD8 (T suppressor/cytotoxic), HLA-ABC, HLA-DR, HLA-DQ, HLA-DP/DR, and beta 2 microglobulin was not inhibited by AP or Ela. Heat-inactivation of the proteases at 65 degrees C for 20 min or treatment with the metal chelator EDTA abolished the inhibitory activity of both proteases. These findings may serve to develop novel immunological methods for the isolation and study of the lymphocyte CD4 structure, which plays an important part in the immune response.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Ultrastructural studies of time-course and cellular specificity of interleukin-1 mediated islet cytotoxicity.

Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed to interleukin-1 for short time periods, isolated rat or human islets were incubated with or without 25 U/ml highly purified human interleukin-1 for 24 h. Samples of rat islets were taken after 5 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and samples of human islets after 5 min, 30 min and 24 h of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h of incubation with interleukin-1, more than 80% of rat beta cells showed signs of degeneration. Beta cell specific changes similar to those observed in rat islets exposed to IL-1 for 30 min were seen in human islets exposed to IL-1 for 24 h. The described changes were not observed in alpha cells in interleukin-1-treated rat or human islets, or in alpha and beta cells in control islets. Passing interleukin-1 over columns containing Sepharose-coupled anti-interleukin-1 antibody completely removed the beta cell cytotoxic action on rat islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Islet cytotoxicity of interleukin 1. Influence of culture conditions and islet donor characteristics.

We recently demonstrated that the macrophage product interleukin 1 (IL-1) is cytotoxic to isolated pancreatic islets and hypothesized that IL-1 is responsible for beta-cell destruction in insulin-dependent diabetes mellitus (IDDM). We studied whether the variation in IDDM preponderance with age, sex, and genetic background in vivo is reflected in different susceptibility to IL-1 toxicity of islets in vitro. In addition, we studied the effect of preculture conditions that support endocrine islet cell function and decrease nonendocrine passenger-cell survival on the susceptibility of beta-cells to IL-1 because it is unknown whether IL-1 acts directly on beta-cells or via passenger cells. No differences in susceptibility to various doses of IL-1-containing mononuclear cell supernatants were found between islets isolated from newborn or adult rats, male or female rats, or rats of four inbred strains, indicating that age, sex, and genetic background do not influence the susceptibility of the beta-cell to IL-1. Preculture of islets for 1-7 days in normal atmosphere and preculture of islet clusters in 95% O2 to delete passenger cells did not affect IL-1-mediated cytotoxicity, suggesting that IL-1 acts directly on beta-cells. Increasing the glucose concentration (22 mM) in the culture medium, which is known to protect beta-cells against alloxan toxicity, reduced IL-1 toxicity. Five or 25% normal human serum as well as 5% normal rat serum, but not equivalent concentrations of human serum albumin, inhibited IL-1 toxicity, indicating the presence of IL-1 inhibitors, IL-1 antagonists, or beta-cell-protecting factors in normal serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1986

Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans.

Activated mononuclear cells appear to be important effector cells in autoimmune beta cell destruction leading to insulin-dependent (type 1) diabetes mellitus. Conditioned medium from activated mononuclear cells (from human blood) is cytotoxic to isolated rat and human islets of Langerhans. This cytotoxic activity was eliminated from crude cytokine preparations by adsorption with immobilized, purified antibody to interleukin-1 (IL-1). The islet-inhibitory activity and the IL-1 activity (determined by its comitogenic effect on thymocytes) were recovered by acid wash. Purified natural IL-1 and recombinant IL-1 derived from the predominant pI 7 form of human IL-1, consistently inhibited the insulin response. The pI 6 and pI 5 forms of natural IL-1 were ineffective. Natural and recombinant IL-1 exhibited similar dose responses in their islet-inhibitory effect and their thymocyte-stimulatory activity. Concentrations of IL-1 that inhibited islet activity were in the picomolar range. Hence, monocyte-derived pI 7 IL-1 may contribute to islet cell damage and therefore to the development of insulin-dependent diabetes mellitus.

Animals↗
PubMed1986

Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans.

Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets. These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play an important role in the molecular mechanisms underlying autoimmune B-cell destruction leading to Type 1 (insulin-dependent) diabetes mellitus.

Animals↗
PubMed1986

Mechanisms of pancreatic islet cell destruction. Dose-dependent cytotoxic effect of soluble blood mononuclear cell mediators on isolated islets of Langerhans.

Supernatants of peripheral blood mononuclear cells from healthy human donors stimulated with recall antigen (purified protein derivative of tuberculin) or lectin (phytohaemagglutinin) markedly inhibited the insulin release from isolated human and rat islets of Langerhans, and decreased rat islet contents of insulin and glucagon in a dose-dependent manner. A maximal effect on islet function was obtained with supernatant concentrations down to 5%. Supernatants of mononuclear cells stimulated with tuberculin were more potent than supernatants produced by lectin stimulation. Culture medium reconstituted with tuberculin or phytohaemagglutinin did not impair islet function. Electron microscopy demonstrated that supernatants were cytotoxic to islet cells. The cytotoxic mononuclear cell mediator(s) was non-dialysable, sensitive to heating to 56 degrees C, labile even when stored at -70 degrees C, but stable when lyophilised.

Animals↗