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Biomedical subjects

A Mackiewicz

Publications and source records attributed to A Mackiewicz.

At least 73 records · Page 4Linked to original sources
PubMed1992

Soluble human interleukin-6-receptor modulates interleukin-6-dependent N-glycosylation of alpha 1-protease inhibitor secreted by HepG2 cells.

Interleukin-6 (IL-6) induces changes in gene expression and the N-glycosylation pattern of acute-phase proteins in hepatocytes. IL-6 exerts its action via a cell surface receptor complex consisting of an 80 kDa IL-6 binding protein (gp80) and a 130 kDa glycoprotein (gp130) involved in signal transduction. A genetically engineered gp80-derived soluble human IL-6-receptor (shIL-6-R) significantly enhanced the IL-6 effect on N-glycosylation changes (revealed by reactivity with the lectin-concanavalin A) of a1-protease inhibitor (PI) secreted by human hepatoma cells (HepG2). Stable transfection of IL-6-cDNA into HepG2 cells (HepG2-IL-6) resulting in constitutive secretion of 2 micrograms of IL-6 per 10(6) cells in 24 h led to a down-regulation of surface-bound gp80 and subsequent homologous desensitization of HepG2-IL-6 cells towards IL-6. Soluble human IL-6-R functionally substituted membrane-bound gp80 resulting in a reconstitution of responsiveness of HepG2-IL-6 cells.

3T3 Cells↗
PubMed1992

Different capabilities of monocytes from patients with systemic lupus erythematosus and rheumatoid arthritis to induce glycosylation alterations of acute phase proteins in vitro.

The effect of conditioned medium on the biosynthesis and glycosylation profile of acute phase proteins secreted by the human hepatoma cell line Hep G2 was studied. Conditioned medium was prepared from nonactivated [CM-LPS(-)] and ex vivo lipopolysaccharide activated [CM-LPS(+)] monocytes from eight patients with active rheumatoid arthritis (RA), five patients with active systemic lupus erythematosus (SLE), and seven healthy subjects. The biosynthesis of albumin, alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor and the profile of glycosylation of proteinase inhibitor were analysed. CM-LPS(-) from patients with SLE had a similar effect to CM-LPS(-) from healthy subjects. In contrast, CM-LPS(-) from patients with RA had the same effect as CM-LPS(+) from healthy donors. A similar effect to that of CM-LPS(+) of healthy subjects was seen with CM-LPS(+) from patients with SLE and with CM-LPS(+) from patients with RA. The treatment of CM-LPS(+) with antibodies against interleukin 6 neutralised most of its ability to induce changes in the biosynthesis and glycosylation of acute phase proteins. Antibodies to interleukin 1 and tumour necrosis factor alpha had only a limited effect on the ability of CM-LPS(+) to induce changes of albumin and alpha 1-antichymotrypsin syntheses, whereas they had no effect on the biosynthesis and glycosylation of proteinase inhibitor. These results indicate that: (a) monocytes isolated from patients with active SLE and active RA have different capabilities of inducing alterations of acute phase proteins in vitro; (b) ex vivo activation of monocytes from patients with SLE leads to the full induction of its capabilities to change acute phase proteins, whereas the activation of monocytes from patients with RA has no additive effects; and (c) interleukin 6 seems to be a major cytokine involved in the regulation of the glycosylation pattern of acute phase proteins.

Acute-Phase Proteins↗
PubMed1991

Effects of cytokine combinations on acute phase protein production in two human hepatoma cell lines.

We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL-6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression.

Acute-Phase Proteins↗
PubMed1991

C-reactive protein and alpha 1-acid glycoprotein in monitoring of patients with acute arterial occlusion.

C-reactive protein (CRP) and alpha 1-acid glycoprotein (AGP) levels were studied in sera of 75 patients with acute arterial occlusion. Depending on the degree of ischaemia, patients were divided into two groups: grade I--26 patients--and grade II--49 patients. All patients were treated surgically; 42 embolectomies, 17 endarterectomies and 16 bypasses were performed. After surgery in 19 patients various complications were observed. The concentration of both proteins at the time of admission was higher in the serum of patients with grade II than with grade I ischaemia. Similarly the concentration of both proteins was significantly higher in the sera of the patients admitted after 8 h than in patients admitted within 8 h of the onset of ischaemia. CRP and AGP levels were significantly higher in the serum of patients with ischaemia of the lower limb than in those with ischaemia of the upper limb. In all patients 2-3 days after surgery a significant increase in serum CRP and AGP was observed. In uncomplicated cases on days 7-10 the values of both proteins decreased below the level observed at the time of admission. However, in patients who experienced postoperative complications high levels of both serum proteins (especially CRP) were found on days 7-10. Complications were detected with a sensitivity and specificity of 84 and 95%, respectively, using a CRP level of 49 mg l-1 as the cut-off point.

Acute Disease↗
PubMed1991

Acute phase mediated change in glycosylation of rat alpha 1-acid glycoprotein in transgenic mice.

Transgenic mouse lines carrying the gene for rat alpha 1-acid glycoprotein (AGP) express the protein in the plasma at concentrations equal to or exceeding that of acute phase rats. Owing to the high basal level, these transgenic mice represent a unique experimental system for defining the largely unknown function of AGP. Since the carbohydrate moiety of AGP has been found to be changed during acute phase and the oligosaccharide structure to be important for immunomodulating activity of the protein, the rat AGP in transgenic mice was characterized by lectin-affinity immuno-electrophoresis. Unlike in the rat, the AGP in the transgenic mouse plasma consisted primarily of strongly concanavalin A-reactive forms. Acute phase mediated a several-fold increase in the total plasma concentration of AGP concomitant with a shift toward moderately concanavalin A-reactive forms. A similar change in concanavalin A-reactive forms was observed for the endogenous acute phase plasma protein haptoglobin. To define the role of inflammatory factors in AGP production, primary cultures of hepatocytes were prepared. In contrast to in vivo, the AGP recovered from tissue culture medium represented primarily the concanavalin A-non-reactive form. Treatment of the cells with recombinant human interleukin-1, interleukin-6 and dexamethasone stimulated the production of concanavalin A-reactive AGP forms. The data indicate that the glycosylation pattern of plasma-resident AGP is modulated by acute phase, but that the profile of AGP forms does not coincide with that secreted by hepatocytes in tissue culture. This finding demands an assessment of which of the possible glycosylated forms of AGP is functionally significant in vivo.

Animals↗
PubMed1990

Induction of C-reactive protein by cytokines in human hepatoma cell lines is potentiated by caffeine.

Induction of C-reactive protein (CRP) by conditioned medium from lipopolysaccharide-stimulated human monocytes in two human hepatoma-cell lines, Hep 3B and NPLC/PRF/5, was potentiated 3-6-fold by the methylxanthine caffeine. The induction observed in the presence of conditioned medium plus caffeine was as much as 180-fold, comparable with that seen after many stimuli in vivo. This potentiation was accompanied by an increase in the levels of CRP mRNA. By contrast, no potentiating effect on CRP induction by conditioned medium was found when we tested theophylline, forskolin, 8-bromo cyclic AMP or two Ca2+ ionophores, namely ionomycin and A23187. None of the above compounds, including caffeine, when tested alone, had any detectable effect on the synthesis and secretion of CRP. Our previous study [Ganapathi, May, Schultz, Brabenec, Weinstein, Sehgal & Kushner (1988) Biochem. Biophys. Res. Commun. 157, 271-277], employing defined cytokines, had shown that induction of CRP in Hep 3B cells requires IL(interleukin)-6 plus IL-1, whereas, in the NPLC/PRF/5 cell line, IL-6 alone is effective. Caffeine similarly potentiated induction of CRP by these defined cytokine signals in these two cell lines. Changes in synthesis of other acute-phase proteins, including serum amyloid A (SAA), alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin and albumin, induced by conditioned medium or, in some cases, by IL-6 and/or IL-1 alpha, were only minimally affected by caffeine. Thus these results indicate that the mechanism by which caffeine potentiates CRP induction by cytokines appears to be independent of increases in intracellular concentrations of the two second messengers, cyclic AMP and Ca2+; the precise nature of this mechanism is unclear at the present time. Our results also indicate that the intracellular mechanisms by which cytokines regulate synthesis of CRP may differ from those regulating synthesis of some other acute-phase proteins. The differential response of CRP and SAA to caffeine is of particular interest, since induction of both of these two major acute-phase proteins can be accomplished by identical extracellular signals.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Transforming growth factor beta 1 influences glycosylation of alpha 1-protease inhibitor in human hepatoma cell lines.

We have previously shown that changes in acute-phase protein glycosylation result from alterations occurring within hepatocytes as a result of regulation by cytokines, that the glycosylation patterns of proteins secreted by Hep 3B and Hep G2 cells respond differently to the crude mixtures of cytokines found in conditioned medium from LPS-stimulated monocytes, and that interleukin-6 (IL-6) causes increased concanavalin A (Con A) binding of alpha 1 protease inhibitor in Hep 3B cells and decreased Con A binding of this protein in Hep G2 cells. In the present study we found that transforming growth factor beta 1 (TGF-beta), like IL-6, led to secretion of forms of alpha 1-protease inhibitor with increased Con A binding in Hep 3B cells, and that IL-6 and TGF-beta in combination were additive. In contrast, in Hep G2 cells, TGF-beta had an effect opposite to that produced by IL-6, leading to secretion of forms of alpha 1-protease inhibitor with increased Con A binding. When employed in combination with IL-6. TGF-beta abolished the effect of that cytokine. These studies indicate that TGF-beta influences glycosylation of alpha 1-protease inhibitor in two human hepatoma cell lines in a manner that can be differentiated from that of IL-6. The identification of TGF-beta as a second defined cytokine capable of influencing glycoprotein glycosylation and the demonstration that the effect of one cytokine can be modulated by another cytokine support the view that changes in glycosylation of plasma proteins are mediated by combinations of cytokines.

Carcinoma, Hepatocellular↗
PubMed1990

Transforming growth factor beta 1 regulates production of acute-phase proteins.

We explored the possible role of transforming growth factor beta 1 (TGF-beta), a cytokine that appears to be an important modulator of inflammation and tissue repair, in regulation of human plasma protein synthesis during the acute-phase response. In Hep 3B cells, TGF-beta led to increased secretion of the positive acute-phase proteins alpha 1-protease inhibitor and alpha 1-antichymotrypsin and decreased secretion of the negative acute-phase protein albumin. In Hep G2 cells, after incubation with TGF-beta, the same changes in secretion of alpha 1-protease inhibitor, alpha 1-antichymotrypsin, and albumin were observed, as well as decreased secretion of both the negative acute-phase protein alpha-fetoprotein and the positive acute-phase protein fibrinogen. In addition, TGF-beta modulated the effects of interleukin 6; these cytokines, in combination, were additive in inducing synthesis and secretion of alpha 1-protease inhibitor and alpha 1-antichymotrypsin and in decreasing secretion of albumin and alpha-fetoprotein. TGF-beta inhibited the induction of fibrinogen caused by interleukin 6. The effects on alpha 1-protease inhibitor were confirmed by metabolic labeling in Hep 3B cells and by demonstrating increased accumulation of specific mRNA in Hep G2 cells, and the effects on fibrinogen were confirmed in Hep 3B cells by studies of mRNA for the alpha chain of fibrinogen. TGF-beta had no effect on haptoglobin or alpha 1-acid glycoprotein secretion, either directly or in the presence of interleukin 6, which is capable of inducing these proteins. These studies demonstrate that TGF-beta can affect hepatic synthesis and secretion of a subset of acute-phase proteins, both directly and by modulating the effect of interleukin 6. The affected group of plasma proteins is distinct from those affected by other recognized acute-phase protein-inducing cytokines. These findings support the view that combinations of cytokines mediate the response of the hepatocyte to inflammatory stimuli.

Acute-Phase Proteins↗
PubMed1990

Direct quantitation of skin elasticity in systemic sclerosis.

A simple instrument, the "skin elastometer," was used to evaluate the elastic and plastic properties of volar forearm skin in 24 patients with systemic sclerosis and 24 healthy individuals matched for age, race and sex. Skin elastance in 17 patients with diffuse scleroderma was found to be significantly different from matched controls (p less than 0.001), and was associated with clinical skin scores independently determined by examination (r = 0.89, p less than 0.001). Seven patients with limited scleroderma (the CREST variant) had values for skin elastance which were intermediate between those of the patients with diffuse scleroderma and healthy persons. Plastic deformation of the stretched skin was similar in patients and controls. Quantitative measurement of skin elastance is a simple technique which may prove to be of value in the assessment of patients with systemic sclerosis.

Adult↗
PubMed1989

Microheterogeneity of alpha 1-acid glycoprotein in the detection of intercurrent infection in patients with rheumatoid arthritis.

We evaluated the clinical usefulness of determinations of alpha 1-acid glycoprotein (AGP) microheterogeneity in distinguishing patients who have active rheumatoid arthritis (RA) from those who have RA and an intercurrent infection. AGP microheterogeneity was studied by affinity electrophoresis with concanavalin A as the ligand, and the results were expressed as reactivity coefficients (RC). Significant differences were found between the mean RC (+/- SD) in healthy individuals (1.27 +/- 0.16) and the mean RC in RA patients with intercurrent infection (1.74 +/- 0.90), as well as with the mean RC in RA patients with grades III and IV disease activity (0.92 +/- 0.18 and 0.81 +/- 0.25, respectively). Moreover, an additional microheterogeneous form of AGP, similar to that observed in non-RA patients with infections, was noted in RA patients with infections (sensitivity 100%, specificity 100%). The results show that an increase in AGP reactivity with concanavalin A is a sensitive indicator of intercurrent infection in patients with RA.

Arthritis, Rheumatoid↗
PubMed1989

Affinity electrophoresis for studies of mechanisms regulating glycosylation of plasma proteins.

A model system for studies of mechanisms governing the alterations of glycosylation of plasma glycoproteins was developed. The system employs two human hepatoma cell lines, Hep 3B and Hep G2, as target cells and agarose affinity electrophoresis with lectins for studies of microheterogeneity of alpha 1-protease inhibitor (PI), a model glycoprotein synthesized by hepatocytes. As an example for the application of the system, the effect of cytokines on major microheterogeneity of plasma proteins is demonstrated. The results indicate that interleukin 6, transforming growth factor beta 1 and, to some extent, tumor necrosis factor alpha are directly involved in regulating the pattern of glycosylation of plasma proteins in vitro, but the major effect is obtained by using combinations of interleukin 6, transforming growth factor beta 1, tumor necrosis factor alpha and interleukin 1. In addition, the results underline the dissociation between alteration of gene expression and the changes in the pattern of plasma protein glycosylation.

Blood Proteins↗
PubMed1989

Interferon beta 2/B-cell stimulating factor 2/interleukin 6 affects glycosylation of acute phase proteins in human hepatoma cell lines.

Undefined monocyte-derived cytokines have previously been shown to affect glycan processing in glycoproteins secreted by human hepatoma cell lines. Hep 3B cells, when incubated with the cytokine interferon beta 2/B-cell stimulating factor 2/interleukin 6, secreted forms of alpha 1-protease inhibitor, ceruloplasmin, and alpha-fetoprotein with increased reactivity with concanavalin A (Con A) while incubation of Hep G2 cells with this cytokine led to secretion of forms of these proteins with decreased reactivity with Con A, reflecting changes in their oligosaccharide chains. The difference in response of these two transformed cell lines to this cytokine undoubtedly reflects differences in their intracellular glycan processing mechanisms. Changes in glycosylation patterns were dissociated from changes in rate of synthesis: this cytokine caused increased synthesis of alpha 1-protease inhibitor and ceruloplasmin, and decreased synthesis of alpha-fetoprotein in both cell lines.

Acute-Phase Proteins↗
PubMed1989

Serum IgA, acute phase proteins, and glycosylation of alpha 1-acid glycoprotein in ankylosing spondylitis.

Several investigators have suggested that gastrointestinal inflammation has a role in the pathogenesis of ankylosing spondylitis. To test this hypothesis markers of gastrointestinal immunostimulation, as manifested by serum IgA concentrations, were compared with serum markers of inflammation, as manifested by acute phase proteins. Serum samples from 45 unrelated Caucasian patients with ankylosing spondylitis (AS) were tested for correlation of serum IgA and six acute phase proteins: C reactive protein (CRP), alpha 1-antitrypsin, alpha 1-antichymotrypsin, caeruloplasmin, alpha 1-acid glycoprotein (AGP), and haptoglobin. Serum IgA was shown to be significantly positively correlated with four of these six acute phase proteins: CRP (r = 0.58, p less than 0.001), alpha 1-antitrypsin (r = 0.29, p less than 0.05), AGP (r = 0.61, p less than 0.01), and haptoglobin (r = 0.58, p less than 0.001), suggesting that gastrointestinal immunostimulation does have a role in the pathogenesis of inflammation in AS. In addition, the microheterogeneity of the pattern of glycosylation of AGP, expressed as reactivity coefficients, was examined. The AGP reactivity coefficient has been shown to increase in infection, remain the same in systemic lupus erythematosus, and decrease in rheumatoid arthritis. It was found that the AGP reactivity coefficient was significantly decreased in patients with AS as compared with healthy controls (p less than 0.006). As recent studies have indicated that patterns of glycosylation reflect intrahepatocellular biosynthetic processes induced by cytokines our data suggest that cytokine-hepatocellular mechanisms in AS may be similar to those occurring in rheumatoid arthritis, but different from those in systemic lupus erythematosus or infection.

Acute-Phase Proteins↗
PubMed1989

Effect of cytokines on glycosylation of acute phase proteins in human hepatoma cell lines.

The effects of various cytokines on synthesis and microheterogeneity of carbohydrate structure of alpha 1-proteinase inhibitor (PI) and alpha-fetoprotein (AFP) in the human hepatoma cell lines Hep 3B and Hep G2 were studied. In both lines, crude cytokine preparations from LPS-activated human monocytes (CM) and several cell lines led to increased PI and decreased AFP synthesis, while recombinant interleukin 1 (IL-1), recombinant tumor necrosis factor (TNF) and hepatocyte stimulating factor preparations (HSF) affected AFP but not PI production. Several of the crude cytokine preparations, but not IL-1, TNF, or HSF, caused Hep 3B cells to secrete forms of PI and AFP showing increased reactivity with Con A upon testing by affinity electrophoresis, while decreased reactivity with Con A was seen in these proteins secreted by Hep G2 cells. Determination of molecular size of PI inducing activity in CM showed a sharp peak at about 17 kD while AFP inhibiting activity was present in a very broad range of molecular size fractions maximal at 17-30 kD. Changes in patterns of glycosylation of these proteins were attributable to cytokines of about 30 kD in Hep 3B and 44 kD in Hep G2 cells. These findings demonstrate the existence of a family of glycosylation regulating cytokines, and suggest that distinct mechanisms within hepatocytes, responsive to different cytokines, may lead to increased or decreased Con A binding of glycoproteins and to altered gene expression.

Acute-Phase Proteins↗