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Biomedical subjects

J Michl

Publications and source records attributed to J Michl.

At least 19 recordsLinked to original sources
PubMed1991

The distribution of perforin in normal tissues.

We describe the production of monoclonal antibodies to murine and human forms of the lymphocyte pore-forming protein (perforin, PFP, or cytolysin), a major granule-localized cytolytic mediator of CTL and NK cells. Antibodies were raised against both murine perforin purified from a CTL line, and human perforin expressed in bacteria as a fusion protein with the Escherichia coli TrpE protein. Antibodies raised against either immunogen inhibited the hemolytic activity of murine perforin, and thus may enable us to identify the pore-forming or self-associative domain of perforin. One mAb, MP1, was used to study the distribution of perforin in murine tissues under physiological conditions. We found that perforin was expressed in the granular metrial gland (GMG) cells of the pregnant murine uterus, but not in other tissues examined. These results further support the view that perforin is induced only in activated cytolytic lymphocytes, and raise the question whether perforin-containing GMG cells represent an effector of a maternal immune response to the fetus.

Animals↗
PubMed1991

Homing receptors and addressins.

Molecular cloning of several homing receptors and their placement within unique families of adhesion receptors over the past 2 years will now permit detailed analyses of structure, function and regulation. Novel tools have significantly contributed to the characterization of carbohydrates as essential parts of the recognition site in addressins whose molecular structures remain to be elucidated.

Animals↗
PubMed1990

Induction of anti-idiotypic antibodies to a myeloma protein linked covalently to muramyl dipeptide.

BALB/c mice immunized with the MOPC-315 myeloma protein linked covalently to the adjuvant muramyl dipeptide developed antibodies against the MOPC protein. The anti-idiotypic nature of the antibodies was demonstrated by the ability of N epsilon-2,4-dinitrophenyl-l-lysine, a known MOPC-315 ligand, to block antibody binding. Immunized mice developed protection against in vivo challenge with MOPC-315 tumor cells. No anti-tumor cell-mediated cytotoxicity could be demonstrated in immunized and challenged mice.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1990

Fc and C3bi receptors and the differentiation antigen BH2-Ag are randomly distributed in the plasma membrane of locomoting neutrophils.

Reports from several laboratories suggest that neutrophils arrested during locomotion preferentially bind immune complexes at the front of the cell. Such asymmetry of binding has been interpreted as indicating an active modulation of phagocytic receptors to the anterior of the cell. To investigate this further, we have used digital analysis of fluorescence images to determine the binding patterns of mAbs directed against the Fc receptors, the receptors for the C3bi fragment of C3, and a neutrophil-specific antigen. We found that all three proteins are distributed nearly identically along the length of migrating neutrophils, and their distribution very closely parallels the anterior to posterior distribution of the plasma membrane. The use of mAbs offered an important advantage in that the binding of antireceptor antibodies, unlike the binding of ligands, should be independent of potential changes in the affinity of the receptors. We conclude that the anterior distribution of the phagocytic receptors in the plasma membrane of locomoting neutrophils parallels the overall increase in membrane area at the front of a migrating cell and that specific translocation of phagocytic receptors does not occur.

Antigens, CD↗
PubMed1989

5,5'-Dimethyloxazolidine-2,4-dione is a strong inducer of differentiation of human promyelocytic leukemia (HL-60) cells.

5,5'-Dimethyloxazolidine-2,4-dione (DMO), a weak non-metabolizable acid, is commonly utilized for determining intracellular pH. In these studies, DMO was tested as an inducer of differentiation on the basis that its uptake and subsequent dissociation might transiently raise intracellular pH and activate ion-fluxes critical for triggering maturation. After 5 days of exposure to 40 mM DMO, greater than 60% of HL-60 cells displayed phenotypic and functional changes characteristic of mature granulocytes. As with other inducers of HL-60 cell differentiation, commitment to differentiation required culture in the presence of DMO for more than 24 h, indicating that if transient effects on pH or ion-fluxes occurred, they were not sufficient to trigger this process. DMO was either weak or inactive as an inducer of murine erythroleukemia cell (FLC) differentiation. Although other weak acids and bases triggered differentiation of both HL-60 cells and FLC, the spectrum of response differed markedly between the two lines. These results suggest that: (1) a number of common buffering agents have the potential to alter cell phenotype, and (2) their effects must be evaluated for each individual cell type.

Animals↗
PubMed1988

A monoclonal antibody to a human neutrophil-specific plasma membrane antigen. Effect of the antibody on the C3bi-mediated adherence by neutrophils and expression of the antigen during myelopoiesis.

We have used mice selectively tolerized to antigens of human lymphocytes by treatment with cyclophosphamide to raise an mAb, BH2-C6, that reacts with a plasma membrane antigen specific for human neutrophils. This specificity is demonstrated by indirect immunofluorescence microscopy, cytochemical analysis of fluorescence-positive and -negative cell populations separated by flow cytometry, and by the selective, complement-mediated killing of mAb BH2-C6-treated neutrophils. Additional evidence for the neutrophil specificity of mAb BH2-C6 is shown by immunoelectron microscopy, which demonstrates a lack of reactivity with human eosinophils. Immunoblotting of SDS-PAGE-separated proteins of polymorphonuclear leukocytes with 125I-labeled BH2-C6 identifies protein with an average molecular mass of 157 kD. Binding studies show that, at saturation, neutrophils bind 214,000 molecules of 125I-BH2-C6 per cell. Addition of mAb BH2-C6 to neutrophils significantly reduces the number of C3bi-opsonized sheep erythrocytes (EIgMC3bi) bound by these cells. This reduction is partly reversed by the presence of soybean trypsin inhibitor (SBTI), indicating that at least one part of this inhibition is due to BH2-C6-stimulated secretion of a serine protease that may affect ligand binding. Cytochemical analysis of normal human bone marrow cells sorted by cytofluorimetry identifies the promyelocyte as the precursor cell that first expresses BH2-Ag on the plasma membrane. Using the leukemic cell line HL-60, we demonstrate that only inducers of granulocytic differentiation, cis-retinoic acid, and dimethyloxazolidine stimulate the expression of BH2-Ag. These results show that the expression of BH2-Ag during myelomonocytic differentiation is a property uniquely possessed by cells committed to the neutrophilic lineage.

Animals↗
PubMed1988

Treatment of experimental myasthenia with autologous idiotypes linked to muramyl dipeptide.

In order to develop a new treatment of experimental autoimmune myasthenia gravis (EAMG), rabbits were injected with purified acetylcholine receptor (AChR) from Torpedo californica. Polyclonal affinity-purified anti-AChR antibodies (idiotypes, Ids) were coupled covalently to muramyl dipeptide and injected back into the same (i.e. autologous) rabbits from which the Ids were obtained. Treated animals developed anti-Ids that bound to the F(ab')2 fragments of the Ids as demonstrated by ELISA and that also blocked binding of Ids to AChR in a radioimmunoassay. Treated animals showed a protective effect compared to control animals when challenged with a second injection of AChR. Anti-AChR titres in surviving animals achieved a steady-state equilibrium. No apparent toxicity from the treatment was noted.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1986

Evidence that excitatory amino acids not only activate the receptor channel complex but also lead to use-dependent block.

The effects of fast application of excitatory amino acids N-methyl-D-aspartate (NMDA), L-aspartate (ASP), L-glutamate (GLU), quisqualate (QU) and kainate (KAIN) were studied in neurons from the embryonic spinal cord of the chick in monolayer cultures by employing the 'patch clamp' technique in the 'whole cell' mode. It was found that NMDA, ASP, GLU and QU, but not KAIN, induced responses that exhibited several components. The early component decayed with a time constant of 2 s to a lower level of membrane current and discontinuation of the application was followed by an after-current which returned to the base-line with a time constant of about 7 s. It is suggested that NMDA, ASP, GLU and QU, but not KAIN, not only activate the receptor channel complex but also induce use-dependent block.

Amino Acids↗
PubMed1985

Cobalt ions block L-glutamate and L-aspartate-induced currents in cultured neurons from embryonic chick spinal cord.

The effects of Co2+ on L-glutamate and L-aspartate responses wee studied in cultured spinal cord neurons of the embryonic spinal cord of the chick by employing the patch-clamp technique in whole cell mode [9]. It was found that Co2+ blocks the responses at negative membrane potentials for both amino acids, while only partial inhibition was observed at positive membrane potentials. Co2+ alone decreases the resting membrane current which exhibits reversal close to zero. It is suggested that the effects of Co2+ are produced by non-specific interaction with negative charges on the outer side of the membrane.

Animals↗
PubMed1985

Ionic currents in neuroblastoma clone E-7 cells.

Ionic currents were studied in exponentially growing neuroblastoma cells (clone E-7) derived from mouse neuroblastoma C-1300 with the patch-clamp technique in the whole cell mode (Pflügers Arch., 391 (1981) 85-100). In differentiated cells, with one or several processes, an early inactivating inward current approximately equal to 50 microA/cm2 was observed in response to depolarizing steps from the holding potential -60 to -70 mV, which was insensitive to 2 microM tetrodotoxin but readily blocked by Co2+ (6 mM). This inward current was followed by a delayed outward current which was eliminated by 12 mM tetraethylammonium. In the undifferentiated cells, only delayed outward current was observed. It is suggested that, in differentiated cells both Ca2+ and delayed rectifier K+ channels exist, while only the latter are present in undifferentiated cells.

Animals↗
PubMed1985

Single non-inactivating K+ channels in the myotubes of the chick embryo in tissue culture.

Single-channel K+ currents were studied in the myotubes from the chick embryo grown in tissue culture for 4--9 days by employing the patch clamp technique. The "cell attached" configuration was used and the pipette was filled with a solution containing 3 mmol.l-1 K+. The channels exhibited a high conductance of approximately 90 pS and the probability of finding them open increased by an e-fold factor for 13 mV depolarization for low levels of activity. The channels did not inactivate during long-lasting depolarization. These channels have been suggested to contribute to delayed rectification.

Animals↗
PubMed1979

Effects of immobilized immune complexes on Fc- and complement-receptor function in resident and thioglycollate-elicited mouse peritoneal macrophages.

We have examined the Fc- and complement-receptor function of resident and thioglycollate-elicited mouse peritoneal macrophages plated on surfaces coated with rabbit antibody-antigen complexes and with complement. We derive four major conclusions from these studies. (a) The trypsin-resistant Fc receptors of resident and thioglycollate-elicited macrophages are completely modulated when these cells are plated on rabbit antibody-antigen complexes. Residual Fc receptor activity is a result of the incomplete modulation of trypsin-sensitive IgG2a receptors. (b) The complement receptors of thioglycollate-elicited macrophages, but not of resident macrophages, are modulated when these cells are plated on complement-coated surfaces. The capacity of the two cell types to modulate their complement receptors is correlated with their ability to ingest complement-coated erythrocytes. (c) The complement and Fc receptors of both types of macrophages move independently of one another. (d) Complement masks the Fc segments of IgG in immune complexes thereby rendering them ineffective as ligands for macrophage Fc receptors.

Animals↗
PubMed1978

The effect of growth-promoting alpha-globulin (GPAG) on the incorporation of exogenous DNA into L-cells. I. Incorporation of isologous and heterologous 3H-DNA.

Isologous and heterologous 3H-DNA (optimum concentration 25 microgram/ml of medium) are incorporated into L-cells from the medium during short-term incubation (up to 60 min). The incorporation of DNA is stimulated by a protein complex from calf serum--the growth-promoting alpha-globulin (GPAG) in the concentration 0.8 mg/ml of medium), which is rapidly taken into cells by pinocytosis. GPAG increases the frequency of incorporation of exogenous 3H-DNA into L-cells and the quantity of DNA incorporated. The quantity of 3H-DNA incorporated is further increased on the incubation of L-cells in a medium containing the complex 3H-DNA + GPAG, which is formed by the joint preincubation of the two components at 37 degrees C (20 hr); under these conditions the quantity of DNA incorporated is 150% greater than when 3H-DNA is used alone. GPAG acts as an activator of exogenous DNA transfer, stimulates its pinocytosis by the cells, and at the same time accelerates its intracellular transfer to the cell nuclei.

Alpha-Globulins↗
PubMed1978

Effect of growth-promoting alpha-globulin on the incorporation of exogenous DNA into L cells. II. Fate of exogenous 3H-DNA after incorporation into recipient cells.

GPAG stimulates the uptake of exogenous 3H-DNA by L cells and facilitates its retention in host cell nuclei during 48-h postcultivation; the nuclei contain 1.5 times more radioactivity than GPAG-free controls. Owing to the well-known properties of GPAG, the formation of the 3H-DNA-GPAG complex may affect the incorporation of exogenous 3-H-DNA and its intracellular transport and retention in the host cell nuclei.

Alpha-Globulins↗
PubMed1977

Proliferative capacity constant of metazoan cells in culture.

Proliferative capacity of metazoan cells in culture may be defined quantitatively by using GPAG-supplemented medium. For this purpose an expression called proliferative capacity constant (KPC) was introduced. KPC represents the logarithm of GPAG concentration over which the mitotic activity of cells is induced.

Alpha-Globulins↗
PubMed1976

2-Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages. I. Description of the inhibitory effect.

Incubation of normal or thioglycollate-elicited mouse peritoneal macrophages with 2-deoxy-D-glucose (2-dG) inhibits the capacity of these macrophages to phagocytize IgG- or complement-coated particles via their Fc and C3 receptors. 2-dG has no inhibitory effect on the capacity of these macrophages to phagocytize latex or zymosan particles, which are ingested in the absence of specific opsonins, and it does not inhibit binding of IgG- or C3-coated particles to their respective receptors on the macrophage's plasma membrane. 2-dG exerts its inhibitory effect on the macrophage and not on the opsonized particle. The inhibition is independent of particle size, occurs within 15-30 min of addition of this glucose analogue to the medium at 37 degrees C, cannot be overcome by supra-agglutinating amounts of opsonizing antibody, and is completely reversible by substitution of 5.5 mM glucose for 50 mM 2-dG in the medium. Addition of equimolar amounts of glucose or mannose, but not of fructose, galactose, fucose, or glucosamine, to medium containing 50 mM 2-dG results in substantial reversal of the inhibitory effect of 2-dG on Fc and C3 receptor mediated phagocytosis.

Animals↗