Educating staff about pain management.
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Humans↗
Biomedical subjects
M Huston
Publications and source records attributed to M Huston.
TNF-alpha and IL-1beta cross-desensitization of astrocytes and astrocytoma cell lines.
Exposure of human astrocytes and astrocytoma cell lines to TNF-alpha, IL-1beta and gammaIFN induce expression of a specific member of the intercrine/chemokine family of cytokines, RANTES. Pre-incubation with non-stimulatory concentrations of TNF-alpha inhibit IL-1beta-stimulated RANTES expression and similarly, non-stimulatory concentrations of IL-1beta inhibits TNF-alpha induced RANTES expression. The lowered responsiveness of these cells is stably maintained for at least 24 h. The inhibitory effect of TNF-alpha on IL-1beta-induced responses was mediated by TNF receptor-1 since low concentrations of a specific anti-TNF receptor-1 antiserum mimicked the inhibitory effect. These results indicate that TNF and IL-1 receptors mediate pro- and antiinflammatory responses in a concentration dependent manner, suggesting that at low receptor occupancy, TNF and IL-1 receptors may share a common signaling pathway and behave as endogenous antiinflammatory cytokines.
Astrocytes↗
Induction of RANTES expression by astrocytes and astrocytoma cell lines.
The cellular infiltrate found during the acute phase of multiple sclerosis (MS) consists of monocytes and activated T cells, suggesting the presence of cell-specific chemotactic signals during the inflammatory response. We examined the ability of human astrocytoma cell lines, as well as primary human and rat astrocytes, to generate a specific member of the intercrine/chemokine family of cytokines, RANTES, when exposed to TNF-alpha, IL-1 beta and IFN-gamma. Astrocytoma cells as well as primary astrocytes produced RANTES upon incubation with TNF-alpha or IL-1 beta. IFN-gamma alone did not induce RANTES production by astrocytes, but it potentiated the effects of either TNF-alpha or IL-1 beta. Induction of RANTES by TNF-alpha was mediated by the p55 receptor since a specific anti-p55 antiserum mimicked the effect of TNF-alpha. These results indicate that human astrocytes are capable of generating a cell-specific chemokine that can account for the inflammatory cellular infiltrate observed during the acute phase of MS, in a process that is regulated by cytokines.
Animals↗
Interferon (IFN) beta acts downstream of IFN-gamma-induced class II transactivator messenger RNA accumulation to block major histocompatibility complex class II gene expression and requires the 48-kD DNA-binding protein, ISGF3-gamma.
Interferon (IFN) gamma, a cardinal proinflammatory cytokine, induces expression of the gene products of the class II locus of the major histocompatibility complex (MHC), whereas IFN-alpha or -beta suppresses MHC class II expression. The mechanism of IFN-beta-mediated MHC class II inhibition has been unclear. Recently, a novel factor termed class II transactivator (CIITA) has been identified as essential for IFN-gamma-induced MHC class II transcription. We studied the status of IFN-gamma-induced CIITA messenger RNA (mRNA) accumulation and CIITA-driven transactivation in IFN-beta-treated cells and used cell lines that had defined defects in the type I IFN response pathway to address the roles of IFN signaling components in the inhibition of MHC class II induction. IFN-beta treatment did not suppress IFN-gamma-induced accumulation of CIITA mRNA. After cells were stably transfected with CIITA, endogenous MHC class II genes were constitutively expressed, and MHC class II promoters, delivered by transfection, were actively transcribed in CIITA-expressing cells. Expression of these promoters was significantly impaired by pretreatment with IFN-beta. These results suggest that IFN-beta acts downstream of CIITA mRNA accumulation, and acts in part by reducing the functional competence of CIITA for transactivating MHC class II promoters. IFN stimulated gene factor 3 (ISGF3) gamma was essential for IFN-beta to mediate inhibition of MHC class II induction, regardless of whether MHC class II transcription was stimulated by IFN-gamma or directly by CIITA expression. Results of these experiments suggest that inhibition of MHC class II in IFN-beta-treated cells requires expression of gene(s) directed by the ISGF3-IFN-stimulated response element pathway, and that these gene product(s) may act by blocking CIITA-driven transcription of MHC class II promoters.
DNA-Binding Proteins↗
Role of protein kinase C and tyrosine kinase activity in IFN-gamma-induced expression of the class II MHC gene.
Astrocytes are induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. Our previous studies demonstrated that IFN-gamma-initiated signaling events important for class II expression include activation of protein kinase C (PKC) and the Na+/H+ antiporter. We have extended these studies and found that protein tyrosine kinase (PTK) activity is also required for class II expression. Treatment of astrocytes with inhibitors specific for PKC and PTK blocked INF-gamma-induced class II gene transcription, mRNA expression, and protein expression. Immunoblotting and immunoprecipitation experiments demonstrated that IFN-gamma induced tyrosine phosphorylation of the p91 component of ISGF3, which is blocked by preincubation of cells with PTK inhibitors. Treatment of astrocytes with IFN-gamma and either PKC and PTK inhibitors changed the mobility and intensity of a nuclear factor, IFN-gamma-enhanced factor X, which binds to the X box of the class II MHC promoter. Taken together, these data provide evidence that activation of both PTK and PKC is required for IFN-gamma-induced expression of the class II gene.
Animals↗
Implications of endotoxin contamination in the evaluation of antibodies to lipopolysaccharides in a murine model of gram-negative sepsis.
The pharmacological factors involved in lipopolysaccharide (LPS)-induced host resistance against infection were investigated in relation to the problem of endotoxin contamination of preparations of monoclonal antibody to the common structure of endotoxin. When administered prophylactically, purified LPS (as low as 4 ng/kg of mouse body weight) or antibody preparations contaminated with endotoxin (assayed by Limulus amoebocyte lysate test) were protective against lethal challenge with a clinical isolate of Escherichia coli (P less than .01). Antibodies that were nonreactive to LPS were similarly protective (P less than .001) when spiked with low doses of LPS (100 ng/mg of protein) but, as with LPS, were without effect when administered after infection. Endotoxin contamination of core-reactive antibody to LPS is mostly a problem associated with the large-scale production, purification, and concentration of the monoclonal antibody. The efficacy (as reported in studies in animal models of gram-negative bacterial sepsis) of antibody to LPS core is controversial. We suggest that endotoxin contamination is a likely factor in this controversy.
Animals↗
Serological characterization and gene localization of an Escherichia coli-expressed 37-kilodalton Treponema pallidum antigen.
A recombinant plasmid containing a 5.6-kilobase-pair DNA fragment of the Treponema pallidum genome was characterized by endonuclease mapping, and the encoded proteins were expressed in Escherichia coli and analyzed by use of in vitro transcription and translation. One of the proteins, identified as having a molecular weight of 37,000 (37K protein), was selected for further study. Initially, the seroreactivity of the partially purified 37K antigen was demonstrated by immunoblotting. After its purification to near homogeneity, the cloned T. pallidum protein was assessed for diagnostic significance by radioimmunoassay. Although first identified as seroreactive by screening with secondary syphilitic sera (T. E. Fehniger, A. M. Walfield, T. M. Cunningham, J. D. Radolf, J. N. Miller, and M. A. Lovett, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, B156, p. 44), the antigen was shown to be serologically reactive with antibodies in serum from all stages of syphilis but was not recognized by serum from controls by both immunoblotting and radioimmune assay. Further, a monospecific polyclonal rabbit antiserum generated to the 37K antigen recognized a polypeptide of the same molecular weight from T. pallidum but did not efficiently recognize proteins from five nonpathogenic treponemes tested. Therefore, because of reactivity with and specificity for T. pallidum antibodies, the 37K antigen may be of serodiagnostic value in the detection of syphilis.
Animals↗
Virosome-mediated implantation of red cell band 3 into the plasma membrane of cultured hepatoma cells.
A method for implanting exogenous membrane proteins into recipient hepatoma cells is described. Red cell band 3 and Sendai virus envelope proteins HN and F were extracted from their respective sources and purified by centrifugation to equilibrium through sucrose step gradients in the presence of octyl-beta-D-glucopyranoside. 0.05-0.15 micron vesicles were formed by adding lipid to combined detergent solubilized, isolated membrane proteins and removing detergent by dialysis. The vesicles were hybrid band 3-Sendai envelope vesicles and not a mixture of two distinct vesicle types as judged by (1) the ability of Sendai specific antibody to immunoprecipitate greater than 99% of band 3 from vesicle suspensions and (2) comigration of band 3 and Sendai envelope proteins on isopyknic sucrose density gradients. The hybrid vesicles (virosomes) were not fusogenic but did bind to cultured hepatoma cells in the cold. Subsequent treatment of virosomes absorbed onto cultured cells with polyethylene glycol resulted in a stable association of 2-10% of added band 3 and Sendai envelope proteins with the cells. Efficient transfer of virosome-associated band 3 to the cells was dependent on both lipid and Sendai envelope proteins. Fluid phase marker transfer, immunofluorescence, and protease digestion experiments demonstrate that the majority of the virosomes were implanted into recipient hepatoma membranes and not simply adsorbed onto their surface or immediately endocytosed. The hybrid membrane protein-viral envelope vesicles thus offer an efficient means for insertion of foreign proteins into the membranes of recipient cultured cells.
Animals↗
Degradation of exogenous membrane proteins implanted into the plasma membrane of cultured hepatoma cells.
The degradation of radiolabeled red cell band 3 and Sendai envelope proteins was studied after band 3 virosomes were fused with hepatoma cells as previously described (Hare, J E & Huston, M, Exp cell res 161 (1986) 317) [26]. 125I-band 3 (T1/2 = 13-14 h), Sendai HN (T1/2 = 37-40 h), and F (T1/2 = 21-23 h) envelope proteins were degraded by an apparent first-order process that was greater than 90% sensitive to 20 mM NH4Cl. 125I-Sendai envelope proteins were degraded at approximately similar rates when hepatoma cells were fused with intact virus, isolated viral membrane, or band 3 virosomes. There thus appears to be distinct heterogeneity among the degradation rates of implanted polypeptides dependent on structural aspects of each. To identify the subcellular site of membrane protein degradation, band 3 was labeled with membrane impermeant [14C]sucrose and implanted into hepatoma plasma membranes. After replating, trichloroacetic acid (TCA)-soluble label was found to accumulate in the lysosomal compartment of fractionated cells. The results identify the lysosome as the ultimate site of plasma membrane protein degradation, but suggest that plasma membrane proteins are selectively rather than non-selectively delivered to this compartment.
Animals↗
Degradation of surface-labeled hepatoma membrane polypeptides: effect of inhibitors.
When their membrane proteins were labeled with 125I by lactoperoxidase, dividing hepatoma cells lost radioactivity to the medium in a biphasic manner (T1/2 = 16-26 h, greater than 40 h). Lysosomotropic weak bases, chloroquine, and NH4Cl inhibited the rapid phase by 59%. More than 50% of the radioactivity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60-71% inhibited by lysosomotropic agents chloroquine and NH4Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH4Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-L-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by 21-24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 degrees C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lysosomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to agents which disrupt the cytoskeleton.
Ammonium Chloride↗
Comparative study of progress notes using problem-oriented and traditional methods of charting.
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Analysis of Variance↗