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Biomedical subjects

J Michl

Publications and source records attributed to J Michl.

At least 37 records · Page 2Linked to original sources
PubMed1999

Glucocorticoid receptors are down-regulated in inflamed colonic mucosa but not in peripheral blood mononuclear cells from patients with inflammatory bowel disease.

BACKGROUND: Growing evidence indicates that the immune system and the hypothalamic-pituitary-adrenal system are linked by several mechanisms, for example intracellular glucocorticoid receptors (hGR). Glucocorticoids are the standard treatment of acute attacks of inflammatory bowel disease (IBD). Binding of glucocorticoids to hGR down-regulates the transcription of inflammatory genes that can propagate IBD. PATIENTS AND METHODS: IBD patients were either treated with 5-60 mg of prednisolone for more than 1 week or were without glucocorticoid treatment for more than 4 weeks. hGR levels were determined from isolated cytosol of peripheral blood mononuclear cells (PBMCs) or mucosal biopsies using a radioassay with [3H]-dexamethasone. Interleukin (IL) 6 levels were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The systemic (PBMC) hGR levels of corticosteroid-treated IBD patients were significantly lower than those of control subjects (59.6 +/- 57.1 dpm mg-1 cytosol protein vs. 227.0 +/- 90.8 dpm mg-1 cytosol protein, P = 0.007) and IBD patients not receiving glucocorticoid treatment (179.7 +/- 171.3 dpm mg-1 cytosol protein, P = 0.002). Systemic hGR levels in untreated IBD patients did not differ significantly from those in control subjects. In patients with connective tissue diseases, systemic hGR levels were also found to be decreased in the absence of glucocorticoid treatment. Systemic hGR levels in patients with Crohn's disease (CD) treated with steroids (66.6 +/- 61.0 dpm mg-1 cytosol protein) were not different from those in patients with ulcerative colitis (UC) (56.1 +/- 51.6 dpm mg-1 cytosol protein). In contrast to these findings, mucosal hGR levels were significantly decreased in both steroid-treated (18.0 +/- 15.5) and not steroid-treated (37.8 +/- 30.5) patients compared with control subjects (125.6 +/- 97.1; P = 0.00009 and P = 0.0008 respectively). IL-6 levels in all IBD groups with and without steroids were significantly different from those in control subjects. CONCLUSION: In IBD there is no difference in systemic hGR levels between not steroid-treated patients and control subjects, in spite of inflammatory activity (IL-6). Mucosal hGR levels were decreased independently of treatment, probably leading to a decreased protection against NF-kappaB action in the intestinal mucosa.

Binding, Competitive↗
PubMed1998

Protein kinase C isoform expression and function in transformed and non-transformed pancreatic acinar cell lines.

Members of the protein kinase C (PKC) family of multifunctional serine/threonine phosphorylating enzymes are believed to play a role in regulating cellular differentiation and proliferation in many cell types. In the present study, we examined the expression of PKC isoforms in non-transformed (BMRPA.430) and transformed (TUC3) rat pancreatic acinar cell lines and compared this to PKC expression in freshly dispersed acini from rat pancreas. BMRPA.430 cells maintain characteristics of normal acini and are not tumorigenic, whereas TUC3 cells do not express tight junctions or polygonal morphology and are tumorigenic. As reported previously, PKC alpha, delta, epsilon, and zeta are expressed in freshly prepared acini. Likewise, these isoforms were detected in both the BMRPA.430 and TUC3 cell lines. In addition, PKC theta, a novel isoform, was detected in all three cell types at low levels. We used two PKC inhibitors to examine the role of PKC in acinar cell proliferation. CGP 41 251, a selective PKC inhibitor, and Go 6976, an agent which specifically inhibits calcium-dependent PKC isoforms, inhibited cell proliferation of both cell lines. Translocation of PKC alpha to the membrane was not observed in either cell line. Hence, our data indicate that ras-induced transformation does not alter PKC isoform expression in pancreatic acinar cells and that activation of PKC alpha is involved with acinar cell growth.

Animals↗
PubMed1998

Antioxidant therapy of cobalt and vitamin E in hemosiderosis.

The protective effects of cobalt and vitamin E in iron overloaded rats were investigated. Rats were divided into four groups: group 1 as control, group 2 received only iron; group 3 iron and cobalt, group 4 iron and vitamin E. All injections were given 3 times per week for 3 weeks. Biochemical and histopathologic studies were done on samples of blood and liver, spleen, and intestine. The results showed that the administration of iron with cobalt or vitamin E decreased lipid peroxidation and the levels of hypoxanthine in all tissues (P < .001). Tissue associated myeloperoxidase (MPO) activity was increased in all iron-overloaded animals. However, vitamin E and cobalt decreased MPO activity (P < .001) in all tissues with the exception of the intestines, where cobalt was ineffective. Cobalt therapy increased hemoglobin, hematocrit, and MCV (P < .05). In contrast to SGPT activity, SGOT activity was significantly increased in all groups but more so in group 3 animals. The increased activity of serum SGOT levels might be related to the mechanical injury by cardiac puncture. The most striking histopathologic finding was the presence of granulomas in the livers of 71% of the animals of group 2 and in 66.6% of group 3. Interestingly, granulomas developed in only 33.3% of group 4 animals, whereas no granulomas were found in the livers of control animals (group 1). In this article we report that cobalt is as effective as vitamin E in significantly reducing iron-induced biochemical changes in an iron-overload in vivo model. We further describe for the first time the presence of extensive granuloma formation in iron-overloaded liver tissue and the greater efficiency of vitamin E over cobalt in protecting against granuloma formation in iron overload.

Alanine Transaminase↗
PubMed1993

Neutrophils do not bind to or phagocytize human immune complexes formed with influenza virus.

Neutrophils appear to form the first line of defense against influenza virus, yet it is unclear how these leukocytes recognize influenza-infected cells. While demonstrating that neutrophils adhere specifically to the sialic acid-binding site on the hemagglutinin molecule (HA) on the surface of influenza-infected (WSN[H1N1]) epithelial cells and not to other viral or epithelial cell antigens, it was observed that human neutrophils do not recognize immune complexes formed with influenza virus. Intact antibodies (mouse monoclonal antibodies [MoAbs] IgG1 and IgG2b, human immune heat-inactivated serum [predominantly IgG1], and IgG purified from human immune serum) that block the sialic acid-binding site on HA significantly reduced (> 80%) neutrophil adherence to influenza-infected epithelial cells. Binding and phagocytosis of free influenza virions and neutrophil agglutination by influenza virus were completely prevented by these antibodies. Intact and F(ab')2 fragments of mouse MoAbs to other viral epitopes caused increased neutrophil adherence to infected cells. This binding was eliminated by F(ab'2) fragments of MoAbs against the sialic acid-binding site on HA, but not by saturating amounts of MoAbs, which block the neutrophil Fc receptors. Thus, it appears that human neutrophils show little ability to bind via their Fc receptors to the immune complexes formed with antibody and either influenza-infected epithelial cells or the free virion. These findings are in contrast to the general dogma, and are the first example of antibody opsonization reducing, rather than enhancing, neutrophil binding and phagocytosis of a pathogen.

Antibodies, Monoclonal↗
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↗
PubMed1984

Interferon suppresses pinocytosis but stimulates phagocytosis in mouse peritoneal macrophages: related changes in cytoskeletal organization.

Treatment of thioglycolate-elicited macrophages with mouse beta-interferon markedly reduces pinocytosis of horseradish peroxidase and fluorescein isothiocyanate (FITC)-dextran but stimulates phagocytosis of IgG-coated sheep erythrocytes. Experiments with FITC-dextran have revealed that the overall decrease in pinocytosis is due to a nearly complete inhibition of pinocytosis in a large fraction of interferon-treated macrophages. In the remaining cells pinocytosis continues at a rate similar to that in untreated control cells. A considerable reduction in the number of cells pinocytosing FITC-dextran was observed within 12 h from the beginning of interferon treatment. Measurement of the overall level of pinocytic activity with horseradish peroxidase showed a progressive decline through 72 h of treatment. In the interferon-sensitive subpopulation, there were marked changes in cytoskeletal organization. Microtubules and 10-nm filaments were aggregated in the perinuclear region while most of the peripheral cytoplasm became devoid of these cytoskeletal structures as observed by fluorescence and electron microscopy. In addition, interferon treatment of macrophages appeared to disrupt the close topological association between bundles of 10-nm filaments and organelles such as mitochondria, lysosomes, and elements of the Golgi apparatus and endoplasmic reticulum. Such alterations in the distribution of microtubules and 10-nm filaments were not seen in the interferon-insensitive subpopulation. We have investigated the mechanism of the interferon-induced enhancement of phagocytic activity by binding IgG-coated sheep erythrocytes to mouse peritoneal macrophages at 4 degrees C and then initiating a synchronous round of ingestion by warming the cells to 37 degrees C. Thioglycolate-elicited macrophages that had been treated with mouse beta-interferon ingested IgG-coated erythrocytes faster and to a higher level than control cells in a single round of phagocytosis. In interferon-treated cultures, phagocytic cups became evident within 30 s of the shift of cultures from 4 degrees to 37 degrees C, whereas in control cultures, they appeared in 2 min. Cytochalasin D, an inhibitor of actin assembly and polymerization, abolished phagocytic activity in both control and beta-interferon-treated macrophages. However, to inhibit phagocytosis completely in thioglycolate-elicited interferon-treated macrophages, twice as much cytochalasin D was required in the treated as in control cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗
PubMed1984

Establishment of mouse neuroblastoma clone E 7 in serum-free medium.

Ninety-six clonal populations were derived from a wild mouse neuroblastoma cell population C 1300 in a serum-free medium containing commercially available serum growth-promoting proteins (GPP). From among these 96 lines the clonal population E 7 was chosen for further work because it displayed maximum spontaneous morphological differentiation. The neuroblastoma clonal population differs morphologically from the original population; it was defined both cytogenetically and by means of growth parameters. The cells of the neuroblastoma clone E 7 are hypertetraploid with two chromosome number modals - 88 and approximately 180-200. The majority of telocentric chromosomes in metaphases with a modal number of 88 chromosomes are identical with the chromosomes of mouse diploid cells. The cell generation time is 22 hours. The cells of the clonal population E 7 are highly sensitive to the action of ethanolamine, which induces morphological differentiation, so that the processes of 30% of the cells in the population are over 40 micron long. Electrophysiological studies showed that the cells of the neuroblastoma clonal population E 7 retain the character of excitable cells and they are thus suitable for studying some of the properties of nervous tissue cells.

Animals↗