Biomedical subjects

F E Maly

Publications and source records attributed to F E Maly.

At least 55 records · Page 3Linked to original sources

PubMed1989

Low-affinity interaction of fibrinogen carboxy-gamma terminus with human monocytes induces an oxidative burst and modulates effector functions.

The interaction of highly purified, monomeric fibrinogen (Fg) with human monocytes (MO) was investigated. In contrast to commercial Fg, no high-affinity binding of monomeric Fg to MO or mononuclear cells could be demonstrated. MO preincubated with Fg in the presence or absence of Ca++ elicited an oxidative burst when triggered with anti-Fg antibodies. Divalency of the antibody and specificity were required, but an intact Fc portion was not. Surface-adsorbed monomeric Fg also promoted an oxidative burst. Evidence is presented that Fg-MO interaction is mediated by the carboxy-gamma terminus of Fg. MO treated with monomeric Fg or exposed to Fg-coated surfaces show a reduced oxidative burst upon triggering with unrelated stimuli. Thus, MO function may be modulated upon interaction with surface-adsorbed Fg or with fibrin.

Fibrinogen↗

PubMed1989

A dual microtiter plate (192 sample) luminometer employing computer-aided single-photon imaging applicable to cellular luminescence and luminescence immunoassay.

In spite of the increasing applications of luminescence measurements, no instrumentation is yet commercially available that permits sensitive luminescence measurements to be performed simultaneously on a large number of samples. Based on the principle of single-photon imaging, we describe here the performance of a computer-aided image luminometer with a capacity for two microtiter plates. Applications to the study of chemiluminescence by a B lymphocyte cell line, and to an IgE luminescence immunoassay are used to exemplify the capabilities of the system, which we have termed 'chemiluminescence multiwell analyser'.

B-Lymphocytes↗

PubMed1989

Properties of the superoxide-generating oxidase of B-lymphocyte cell lines. Determination of Michaelis parameters.

The capacity of three B-lymphocyte cell lines to generate superoxide (O2.-) was examined. The Burkitt lymphoma lines P.3HR-1 and Jijoye gave no response to phorbol 12-myristate 13-acetate (PMA) at 100 ng/ml but produced up to 0.35 nmol of O2.-/min per mg of protein when stimulated with 5 micrograms of PMA/ml; the cell line RPMI 1788 produced Nitro Blue Tetrazolium-positive responses to low PMA concentrations and approx. 0.4 nmol of O2.-/min per mg of protein at 5 micrograms of PMA/ml. Each cell line contained approx. 10 pmol of low-potential cytochrome b (cytochrome b-245)/mg of protein. Homogenates of PMA-activated cells gave 10-20-fold greater rates of O2.- produced per mg of protein. The Km for NADPH varied between approx. 250 microM for P3.HR-1 and RPMI 1788 cell lines and 30.5 +/- 6.5 microM for the Jijoye cell line; the Km values for NADH were higher. Determination of intracellular NADPH concentration showed that this might limit the rate of O2.- production since in each cell line it was at or below the Km concentration.

B-Lymphocytes↗

PubMed1989

Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines.

EBV-transformed B lymphocyte cell lines can generate superoxide, using an electron transport chain homologous, or even identical, to phagocytic NADPH-oxidase. We searched for normal, not virally transformed, B lymphocytes with analogous properties, using tonsils as the source of B cells. Unseparated tonsillar leukocytes contained cells capable of PMA-triggered superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT+ cells) well in excess of phagocytes (18.9 +/- 6.4% NBT+ cells with 1.3 +/- 0.9% granulocytes and 1.9 +/- 2.3% monocytes/macrophages, n = 8). NBT reduction was also inhibited by diphenylene iodonium, a selective inhibitor of phagocytic NADPH-oxidase. Cross-linking of surface Ig was equally effective as PMA in inducing NBT reduction among tonsillar leukocytes. NBT+ cells co-distributed with B cells on Percoll density gradients and were enriched among purified B cells obtained by SRBC rosetting twice and Sephadex G10 adherence (47.8 +/- 15.2% NBT+ cells among 90.5 +/- 5.5% B cells, 4.8 +/- 5.1% T cells, 1.2 +/- 0.77% monocytes/macrophages, and 0.73 +/- 0.6% granulocytes, n = 10). Further, mAb 7D5, directed against an extracellularly located epitope of the small subunit of cytochrome b-245 of phagocytes, stained the majority of tonsillar B cells (85 +/- 9.2% 7D5+ cells and 91.6 +/- 4.04% B cells, n = 3). Superoxide production, staining with 7D5 antibody, and expression of mRNA for the beta chain of cytochrome b-245 were further analyzed in cell lines. The EBV-BLCL F1 and the Burkitt lymphoma P3HR-1 both carried 7D5-detectable cytochrome b-245 Ag and expressed mRNA for the beta chain of the cytochrome b, both in similar amounts. However, only F1, not P3HR-1, was capable of PMA-triggered superoxide production. These data indicate that also normal nontransformed B lymphocytes possess the capacity to generate superoxide by a system apparently similar to phagocytic NADPH-oxidase, provisionally termed "B cell oxidase." Discrepancies observed in certain B cells and lines between expression of cytochrome b components and stimulus-induced superoxide production may be related to an absence or low level of other oxidase components or of the signal transduction mechanism. Conceivably, production of superoxide and derived reactive oxygen species by B cells may have cytotoxic, immunomodulatory, or mutagenic effects on the B cells themselves or on cells in their immediate vicinity.

Adult↗

PubMed1988

Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals.

Neutrophils (PMN) preincubated with recombinant human granulocyte/macrophage colony-stimulating factor (rhGM-CSF) for 2 h and then stimulated with the chemotactic factors, C5a or FMLP, produce substantial amounts of the lipoxygenase products 5-Hete, LTB4, and omega-oxidised LTB4 metabolites (4.36 +/- 0.95 (SEM) pM (n = 21) LTB4 and LTB4 metabolites/10(6) PMN). No lipoxygenase metabolites are detected by HPLC and RIA if purified PMN are stimulated by either GM-CSF or chemotactic factors in the absence of exogenous arachidonate. The priming effect of GM-CSF upon chemotactic factor induced generation of lipid mediators is a relatively slow process, clearly evident after 1 h and optimal after 2 h. Leukotriene generation is measurable with 0.8 U GM-CSF/10(6) PMN and is maximal with 80 U (10(-11)-10(-9) M). Upon activation of primed PMN with chemotactic factors, leukotriene synthesis is induced very rapidly. Already 2.5 min after activation the major lipoxygenase metabolites present are 20-OH LTB4 and 20-COOH LTB4. Our study shows that the synthesis of lipoxygenase metabolites from endogeneous AA can be initiated in PMN through receptor mediated processes by the appropriately timed combination of biological soluble inflammatory mediator peptides. Furthermore, these results indicate that GM-CSF not only enhances effector cell functions but can qualitatively change the mediator profile formed after activation with a second triggering signal. Such a mechanism might be important in amplifying inflammatory responses. Alternatively, lipid mediators formed might also have an intracellular or autocoid role and be responsible for the enhancement of other PMN functions like oxygen radical release.

Adult↗

PubMed1988

The superoxide generating system of B cell lines. Structural homology with the phagocytic oxidase and triggering via surface Ig.

EBV-transformed B lymphocyte cell lines (EBV-BLCL) produce superoxide after stimulation with phorbol ester, a capacity unique among nonmyeloid cells. The superoxide producing system of EBV-BLCL (B cell oxidase) was compared with the phagocytic NADPH-oxidase and the relationship of the capacity to produce superoxide to the presence of the EBV-genome was analyzed. The two EBV-transformed B cell lines F1 and HELL generated superoxide in response to PMA (2.3 nmol/10(6) F1 cells x 1 h and 6.27 nmol/10(6) HELL cells x 1 h with 1 microgram/ml of PMA), whereas no superoxide release was detected with the EBV-positive Burkitt lymphoma line WIL-2 and the EBV-negative plasmocytoma line U-266. Also, F1 and HELL showed lucigenin-dependent chemiluminescence (CL) after PMA-treatment, whereas no CL responses were detected from WIL-2 or U-266. Further, F1 and HELL cells contained a low potential cytochrome b-245 (10.9 and 61.0 pmol/mg protein, respectively) and also a 45 kDa diphenylene-iodonium (DPI)-binding peptide, both components of the phagocytic NADPH-oxidase. In contrast, neither the cytochrome b-245 nor the 45 kDa DPI-binding peptide were detected in WIL-2 and U-266. In addition, DPI inhibited O2- production by PMA-stimulated EBV-BLCL and polymorphonuclear granulocytes. Further, F1 line cells showed superoxide dismutase-inhibitable lucigenin-dependent CL when triggered by protein A-bearing staphylococci (Cowan strain I) or by a mAb directed against human IgG in the presence of solid-phase goat anti-mouse-Ig antibody. From a panel of eight EBV-BLCL, only five responded with CL when exposed to protein A-bearing staphylococci, whereas all showed CL when treated with phorbol ester. Inasmuch as all eight EBV-BLCL possessed surface Ig and a "functional" oxidase, their differential response to cross-linking of surface Ig may be determined by differences in signal transduction. Superoxide production by EBV-BLCL appears thus related to expression of an electron transport chain structurally homologous, if not identical, with the "phagocytic" NADPH-oxidase. Apparently, the presence of EBV-genome in B cell lines does not per se lead to expression of this oxidase. This suggests that nontransformed B cells may, at a certain differentiation stage, also express a superoxide-generating chain. From the finding of stimulation of superoxide production of EBV-BLCL via surface Ig it appears possible that also Ag may be able to trigger such B cells to production of superoxide which might have an important role in the physiology of B cells.

B-Lymphocytes↗

PubMed1988

A single-photon imaging system for the simultaneous quantitation of luminescent emissions from multiple samples.

A combination of a two-dimensional photon detector (double-microchannel plate) with single-photon sensitivity and an optical projection system that allows space-resolved quantitation of luminescent emissions from spatially extended objects is described. A "luminescent image" of the object focused onto the detector is accumulated over a preset time and stored in a digital frame memory from which photon counts over areas of interest can be read. In this study, the object consisted of a microtiter plate containing luminescent samples which was placed below a projecting lens (2.0/21 mm, 36 X 24-mm format camera lens) at a distance of 38.5 cm. Although geometry substantially limited photon collection, the sensitivity achieved was only 10X less than that obtained with a dedicated photon-counting luminometer. A slightly diminished photon collection from peripheral wells was apparently caused by the projection system and could be corrected arithmetically. Both chemically generated luminescence (ATP bioluminescence) and cell-derived, superoxide-dependent luminescence (with lucigenin as chemilumigenic probe) were detected with excellent spatial resolution and linearity of response over a wide range.

Adenosine Triphosphate↗

PubMed1987

A novel interleukin stimulating free radical production by granulocytes.

Polymorphonuclear granulocytes (PMN) are potent producers of free oxygen-derived radicals. Since other granulocyte functions are affected by interleukins, we investigated whether free-radical production can be initiated by a similar mediator. For estimation of free radical production, SOD-inhibitable lucigenin-dependent chemiluminescence and SOD-inhibitable cytochrome C reduction were used. As a source of interleukins, serum-free 24 h culture supernatants of human mononuclear cells (MNC) stimulated with bacterial lipopolysaccharide were prepared. Addition of such supernatants to PMN caused stimulation of sod-inhibitable chemiluminescence and superoxide production. Studies with separated MNC showed that monocytes were the cellular source of the activity. Biochemically, this activity of the supernatants was due to a heat-labile glycoprotein with a MW of approx. 60 KDa. This mediator, termed granulocyte chemiluminescence inducer (GCI), appears to be distinct from interleukin 1 (alpha and beta) and interferon (alpha and gamma). In conclusion we describe a novel monokine, granulocyte chemiluminescence inducer (GCI), which initiates granulocyte free radical production. This interaction of monocytes and granulocytes may also in vivo constitute a new and potent pathway leading to stimulation of free oxygen production by granulocytes.

Antibodies, Monoclonal↗

PubMed1986

Granulocyte-activating mediators (GRAM): I. Generation by lipopolysaccharide-stimulated mononuclear cells.

We undertook a study to determine whether cytokines exist which are responsible for the activation of polymorphonuclear neutrophilic granulocytes (PMN) besides the already well-known stimuli. Lucigenin-dependent chemiluminescence was used to measure human PMN activation. Addition of supernatants from mononuclear cells stimulated with bacterial lipopolysaccharide produced a long-lasting activation of granulocytes. Induction of chemiluminescence was dose-dependent and inhibitable by superoxide dismutase. Fractionation of mononuclear cells by adherence to plastic dishes or counterflow elutriation proved that monocytes were able to generate granulocyte-activating mediators (GRAM). Production of GRAM was dependent on the dose of the stimulus and appeared to be maximal after 24 h of incubation. Addition of cycloheximide resulted in significantly decreased release of GRAM. Partial characterization of the activity showed GRAM to be heat-labile and sensitive to trypsin, indicating a protein nature of GRAM. The activity fractionated into 2 distinct peaks, one corresponding to 60 kD and another below 10 kD. The interleukin 1 activity did not appear to co-fractionate with GRAM. Evidence presented suggests that the activity corresponds to factors unlikely to have been described previously.

Chemotactic Factors↗

PubMed1986

Induction of granulocyte chemiluminescence by a mediator derived from human monocytes.

Neutrophil granulocytes are most active producers of potentially toxic free oxygen radicals. Since other functions of granulocytes can be affected by lymphokines or monokines, we investigated whether granulocyte oxygenation activity can also be influenced by such cellular mediators. Human granulocytes emitted strong chemiluminescence after addition of culture supernatants from human mononuclear cells stimulated with bacterial lipopolysaccharide (LPS). Response of the granulocytes was dose-dependent and was inhibited up to 90% or more by superoxide dismutase. This granulocyte chemiluminescence inducer-activity (GCI-activity) in the LPS-induced supernatants was heat-labile and sensitive to trypsin treatment. Addition of cycloheximide to the cultures inhibited the generation of GCI-activity by 80%. On HPLC gel filtration GCI-activity eluted with two distinct peaks corresponding to molecular weights of 60 +/- 10 KDa and between 1 and 5 KDa. Murine interleukin 1, human recombinant interferon-alpha and -gamma were devoid of GCI-activity. When mononuclear cells were fractionated by plastic adherence or counterflow elutriation, monocytes appeared to be the source of GCI-activity. Therefore, it appears that granulocyte oxygenation activity can be enhanced strongly by a cellular mediator derived from monocytes. This interaction of monocytes and granulocytes may constitute a new and potent pathway of phagocyte-dependent production of highly reactive and potentially toxic oxygen radicals.

Cell Adhesion↗

PubMed1985

Detection of complement activation in human serum using C5a-induced chemiluminescence of human granulocytes.

The complement system can be activated by many factors, including immune complexes, leading to the generation of biologically active split products like C5a anaphylatoxin. This study presents a technique which may be used for measuring C5a activity in human serum. The tetanus-anti-tetanus immune complex and aggregated human IgG were used as model activators of the complement cascade. The C5a activity was measured by C5a-induced chemiluminescence of granulocytes; furthermore, a radioimmunoassay was used to detect the C5a peptide. There was a strongly positive correlation between the two assay systems. The described method should be useful as an alternative means of detecting complement activation in the serum of patients with inflammatory diseases.

Antigen-Antibody Complex↗

PubMed1985

Dissociation of natural killing and luminol-dependent chemiluminescence.

A long lasting luminol-dependent chemiluminescence was seen when mouse NK cell preparations or human NK cell preparations from Percoll gradients were mixed with NK susceptible targets. This CL could be abolished by extensive removal of adherent cells though normal NK cell activity remained. In addition, the NK cell line HY 3-Ag3 did not show any CL; although it expressed a very strong cytolytic activity. Thus, we conclude that there are two cells reacting with NK susceptible targets. First, the NK cell whose cytolytic activity does not necessarily depend on the formation of oxygen metabolites detectable by CL, and second, a more adherent cell population, found in NK cell preparations obtained after Percoll gradients, that reacts with NK-sensitive targets and leads to luminol-dependent CL. The observed CL paralleled the cytotoxicity measured by 51Cr release. The time course of the CL signal was similar in human and mouse. The maximal CL-signal obtained was about 10(5) cpm at 37 degrees C when 10(6) human effector cells were used at a ratio of 5:1 effector to target cells. The CL was shown to be highly temperature dependent.

Animals↗

PubMed1984

Differential generation of chemiluminescence--detectable oxygen radicals by normal polymorphonuclear leukocytes challenged with sera from systemic lupus erythematosus and rheumatoid arthritis patients.

The generation of chemiluminescence (CL)-detectable oxygen radicals by normal human polymorphonuclear leukocytes (PMN) after challenging with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) sera is described. CL was measured in a luminol-dependent assay and referred to a standard obtained when performed immune complexes (Ic) (human tetanus toxoid-antitoxoid Ic resuspended in normal pooled serum) were tested on PMN. Normal sera gave rise to CL activity by PMN between 0% and 50% of the standard Ic (mean +/- standard error of the mean (SEM): 20.7 +/- 4.8). Sera from SLE and RA patients induced strikingly different biological effects on PMN. SLE sera generally induced a high CL-detectable generation of oxygen metabolites which may be causally related to the intense tissue damage (vasculitis) frequently observed in this disease. In contrast to SLE, RA sera induced a CL-detectable respiratory burst by PMN that was included in the normal range. Thus, the biological effects of these sera in terms of stimulation of toxic oxygen radical generation by phagocytes are quite different. This generation of oxygen radicals might reflect a different clearance of circulating Ic by PMN in SLE and RA disease.

Antigen-Antibody Complex↗

PubMed1983

C5a-induced chemiluminescence of human granulocytes and its amplification by a serum factor.

Purified human C5a elicits a fast chemiluminescence (CL) response from isolated human granulocytes in the presence of Lucigenin (bis-N-methylacridinium nitrate). The reaction is inhibitable to more than 90% by superoxide dismutase (SOD) - final concentration 200 micrograms/ml -, to about 60% by catalase - final concentration 10 mg/ml - and to 30% by the hydroxyl radical scavenger D-mannit - final concentration 100 mM. Therefore O2- seems to be the oxygen radical responsible for most of the CL, while OH and H2O2 are also involved. Addition of normal pool serum to the cells for 1-2 min before stimulation with C5a strongly enhances the effect in a dose and time-dependent manner. Therefore the existence of a "helper activity" in serum amplifying the C5a-induced CL of granulocytes is postulated. This "helper activity" is, however, no specific for C5a, since CL responses elicited with the chemotactic peptide f-met-phe or by phorbol-myristate-acetate (PMA) are also enhanced by preincubation with serum. In contrast, ConA-induced CL is not enhanced but decreased. Therefore, though not unique to C5a-induced CL, the "helper activity" seems not to represent a general "adjuvans" effect of serum on the granulocytes, but to be restricted to certain stimuli.

Chemotaxis, Leukocyte↗

PubMed1983

Quantitation of immunoadsorbed flavoprotein oxidases by luminol-mediated chemiluminescence.

The detection of the flavoenzymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase at the sub-femtomol level was achieved by coupling the reaction of the immunoadsorbed proteins to the peroxidase-catalysed oxidation of luminol. The H2O2-producing oxidases retained their full activity when bound to the respective immobilized antibodies. This fact allowed the concentration of the enzymes from very dilute solutions and the quantitative assay of their activities in the microU range. Due to strict stereoselectivity and the absence of immunological cross-reactivity, the two flavoproteins could be determined in the same solution. This method was used to measure the 6-hydroxy-D-nicotine oxidase and 6-hydroxy-L-nicotine oxidase activities in Escherichia coli RR1 and different Arthrobacter strains cultured under non-inducing conditions. The same activity ratio of 6-hydroxy-L-nicotine oxidase/6-hydroxy-D-nicotine oxidase as in D L-nicotine-induced cells of A. oxidans was observed in non-induced wild type and in riboflavin-requiring (rf-) mutant cells of this aerob.

Antigen-Antibody Complex↗

PubMedDate not supplied

Chemiluminescence of mononuclear cells is enhanced during antigen recognition.

Stimulation of phagocytes by several cytokines causes superoxide generation and consequently chemiluminescence. Since antigen-activated lymphocytes generate cytokines, we investigated whether antigen recognition by mononuclear cells, which contain both lymphocytes and monocytes, is accompanied by changes in lucigenin-dependent chemiluminescence. Mononuclear cells which underwent antigen-induced proliferation showed a delayed rise in lucigenin-dependent chemiluminescence in the absence of other stimuli. The common recall antigen Candida albicans increased spontaneous chemiluminescence of mononuclear cells from unselected donors up to 20-fold over control values after 48-72 h of culture. With Rabies virus vaccine as specific antigenic stimulus, only mononuclear cells from rabies immunized individuals responded with enhanced delayed chemiluminescence. In contrast to opsonized zymosan and phorbol myristate acetate, antigens induced no oxidative burst within one hour after addition. Delayed mononuclear cell chemiluminescence was inhibited by the superoxide scavenger superoxide dismutase and by di-phenylene iodonium, a selective inhibitor of the phagocyte NADPH oxidase. A neutralizing monoclonal antibody against interferon-gamma completely abrogated antigen-induced chemiluminescence. Recombinant interferon-gamma by itself induced delayed mononuclear cell chemiluminescence. Thus, antigen-induced delayed mononuclear cell chemiluminescence represents activation of phagocyte NADPH oxidase by interferon-gamma generated by activated lymphocytes.

Antibodies↗

PubMedDate not supplied

[Liberation of the oxygen radical from peripheral human phagocytes (granulocytes and monocytes) in patients with chronic polyarthritis].

Monocytes from patients with definite or classical rheumatoid arthritis show a significantly higher release of O2-radicals in the chemiluminescence assay (2925 X 10(3) cpm) than those of healthy volunteers (267 X 10(3) cpm) (p = 0.01). This difference does not appear in a similar test with granulocytes, which in both groups tested show a mean chemiluminescence of 2739 X 10(3) and 2547 X 10(3) cpm. The chemiluminescence of monocytes is clearly time-course dependent: in the morning we saw the least response, at noon the highest. Following our results, corticosteroids lower chemiluminescence significantly (p = 0.05), while non steroidal antirheumatic drugs and long-term therapy with gold compounds, etc., do not affect chemiluminescence.

Arthritis, Rheumatoid↗

PubMedDate not supplied

[Initial results of the clinical use of the immunostimulant bestatin (3-amino-2-hydroxy-4-phenylbutyryl leucine) in patients with chronic polyarthritis].

We tested the efficacy of the immunomodulator Bestatin, which has been successfully applied in Japan, in a preliminary open study in ten patients with classical or definite rheumatoid arthritis. Patients whose arthritis was characterized by a low humoral activity showed a partial remission of the disease after six months' application of increasing doses of 10-60 mg in 48 hours. We found a significant increase in superoxide release from peripheral monocytes. Humoral parameters of inflammation such as erythrocyte sedimentation rate, C3a complement fraction, prostaglandin E2, and thromboxane B2 decreased. Concerning the subpopulations of lymphocytes we found a slight increase of activated T-lymphocytes and a decrease of O-cells and B-cells. The T-helper/T-suppressor ratio remained unchanged. The rheumatoid factor and the antinuclear antibodies showed no alteration under the treatment. Two weeks after withdrawal of Bestatin the improvement of the clinical and humoral parameter of the inflammation in rheumatoid arthritis vanished. No serious side effects were seen. Our results contribute to a long-term controlled study of the immunomodulator Bestatin as basic drug in the treatment of rheumatoid arthritis.

Adjuvants, Immunologic↗