Crucial role of interferon consensus sequence binding protein, but neither of interferon regulatory factor 1 nor of nitric oxide synthesis for protection against murine listeriosis.

Listeria monocytogenes is widely used as a model to study immune responses against intracellular bacteria. It has been shown that neutrophils and macrophages play an important role to restrict bacterial replication in the early phase of primary infection in mice, and that the cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) are essential for protection. However, the involved signaling pathways and effector mechanisms are still poorly understood. This study investigated mouse strains deficient for the IFN-dependent transcription factors interferon consensus sequence binding protein (ICSBP), interferon regulatory factor (IRF) 1 or 2 for their capacity to eliminate Listeria in vivo and in vitro and for production of inducible reactive nitrogen intermediates (RNI) or reactive oxygen intermediates (ROI) in macrophages. ICSBP-/- and to a lesser degree also IRF2-/- mice were highly susceptible to Listeria infection. This correlated with impaired elimination of Listeria from infected peritoneal macrophage (PEM) cultures stimulated with IFN-gamma in vitro; in addition these cultures showed reduced and delayed oxidative burst upon IFN-gamma stimulation, whereas nitric oxide production was normal. In contrast, mice deficient for IRF1 were not able to produce nitric oxide, but they efficiently controlled Listeria in vivo and in vitro. These results indicate that (a) the ICSBP/IRF2 complex is essential for IFN-gamma-mediated protection against Listeria and that (b) ROI together with additional still unknown effector mechanisms may be responsible for the anti-Listeria activity of macrophages, whereas IRF1-induced RNI are not limiting.

Modulation of the antilisterial activity of human blood-derived macrophages by activating and deactivating cytokines.

A concept of macrophage deactivation by hormones and cytokines that opposes activation was recently proposed. Deactivation of the antilisterial activity of macrophages by IL-4, IL-10, and TGF-beta, as well as by dexamethasone, was studied here. IL-4, IL-10, and dexamethasone, but not TGF-beta, caused a complete loss of the competence of human blood-derived macrophages infected with Listeria monocytogenes to control or eliminate ingested bacteria. IL-10 and, to a lesser degree, dexamethasone lessened in parallel the capacity of macrophages to secrete H2O2. The antilisterial activity of cells simultaneously exposed to deactivating agents could be significantly augmented by IFN-gamma. Likewise, TNF-alpha and to a limited degree GM-CSF increased the antilisterial activity of cells treated with IL-10 and dexamethasone but not that of cells treated with IL-4. Suppression of TNF-alpha secretion in response to Listeria by TGF-beta, IL-10, dexamethasone, or pentoxifylline did not closely parallel antilisterial activity. Studies by transmission electron microscopy and actin staining suggested that deactivation by IL-10, IL-4, and dexamethasone of human blood-derived macrophages resulted in intraphagosomal multiplication of Listeria followed only then by an escape of bacteria into the cytoplasm. The antibacterial competence of human macrophages is lessened by IL-4 and IL-10 and augmented by IFN-gamma, TNF-alpha, and GM-CSF. The success of human macrophages in controlling intracellular pathogens appears to depend on the balance of activating and deactivating mediators modulating their activity.

In vitro immunization with antigen directly blotted from SDS-polyacrylamide gels to polyvinylidene difluoride membranes.

A new immunization method has been developed for the production of monoclonal antibodies. This technique uses small amounts of partially purified and weak immunogenic antigen, bound to membranes after blotting from SDS-PAGE. For this purpose two different membranes have been tested. Immobilon-P polyvinylidene difluoride (PVDF) membranes were less mitogenic than nitrocellulose membranes, and were therefore selected for the in vitro immunization using 6-pyruvoyl tetrahydropterin synthase as antigen. The in vitro immunization method was then used for the production of monoclonal antibodies against 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes on the biosynthetic pathway of tetrahydrobiopterin, the natural cofactor of the mammalian aromatic amino acid hydroxylases. The antibodies obtained were mainly of the IgM type.

Immunovascular communication: activation and deactivation of murine endothelial cell nitric oxide synthase by cytokines.

A murine endothelial cell line, send1, was found to produce substantial amounts of nitric oxide, particularly after activation with cytokines. The endothelial cell activation paralleled that of macrophages. Macrophage deactivation opposing activation has recently been brought into focus. We therefore studied the cytokine-mediated deactivation of endothelial cells in send1 and vascular strips. Our observations document that activation of nitric oxide synthase of endothelial cells can be counterbalanced by deactivating cytokines such as interleukin-4, interleukin-8, interleukin-10 and transforming growth factor-beta. Deactivation of nitric oxide synthase in endothelial cells might be an essential mechanism for the control of immune-mediated vasodilatation or septic shock and represents a novel mechanism of communication between the immune and the vascular systems.

Effects of activating and deactivating cytokines on the functionally linked tetrahydrobiopterin. No pathways in vascular smooth muscle cells.

The functional relationship of nitric oxide (NO) production and synthesis of tetrahydrobiopterin (BH4), the requisite cofactor for NO synthase, was investigated in rat aortic smooth muscles cells (SMC). Inflammatory cytokines induced BH4 and NO synthesis in different ratios, IL-1 beta induced mainly NO synthesis with concomitant but limiting amounts of BH4 for maximal NO production. TNF alpha did not induce NO synthesis but induced BH4 synthesis. IFN gamma was ineffective on both the induction of NO and BH4 synthesis. TGF beta downregulated NO production but did not affect BH4 biosynthesis. IL-4 and IL-10 had no effect on both BH4 and NO synthesis. Activating cytokines strongly synergized in induction of NO production, whereas endogenous BH4 production became insufficient for maximal NO synthesis. Exogenous cofactor in the form of sepiapterin or authentic BH4, but not the natural isomer 7-BH4, enhanced NO production twofold. Inhibition of BH4 synthesis with dicumarol abolished NO production that could be restored in the presence of BH4.

Induction of tetrahydrobiopterin synthesis in human umbilical vein smooth muscle cells by inflammatory stimuli.

Tetrahydrobiopterin (BH4) is an obligatory cofactor and regulator of nitric oxide synthases (NOS). We evaluated the biosynthesis of BH4 in human umbilical vein smooth muscle cells (HUVSMC). Trace amounts of BH4 were found intra- and extracellularly in untreated cells. When HUVSMC were activated by individual inflammatory stimuli (IL-1beta, TNFalpha, IFNgamma or LPS), both intra- and extracellular levels of BH4 increased significantly, with TNFalpha being the most potent single stimulus. Combined inflammatory cytokines synergized in the induction of an up to 600-fold increase of BH4 synthesis. Addition of LPS to the cytokine mixture led to a further increase of BH4 synthesis. Neopterin, a product of the first intermediate in BH4 biosynthesis, was also raised, but to a much lesser extent. The increase of BH4 synthesis was paralleled by an enhanced expression of isoform-1 (the only isoform coding for the active enzyme) of GTP cyclohydrolase I in cytokine treated cells. Our results show for the first time that BH4 biosynthesis is strongly induced by combinations of inflammatory stimuli in HUVSMC. The importance of BH4-dependent NO synthesis in HUVSMC needs, however, additional detailed studies.

The nitric oxide synthase cofactor tetrahydrobiopterin reduces allograft ischemia-reperfusion injury after lung transplantation.

OBJECTIVE: Exogenous nitric oxide reduces ischemia-reperfusion injury after solid organ transplantation. Tetrahydrobiopterin, an essential cofactor for nitric oxide synthases, may restore impaired endothelium-dependent nitric oxide synthesis. We evaluated whether tetrahydrobiopterin administration to the recipient attenuates lung reperfusion injury after transplantation in swine. METHODS: Unilateral left lung transplantation was performed in 15 weight-matched pigs (24-31 kg). Donor lungs were flushed with 1.5 L cold (1 degrees C) low-potassium-dextran solution and preserved for 20 hours. Group I animals served as controls. Group II and III animals were treated with a bolus of tetrahydrobiopterin (20 mg/kg). In addition, in group III a continuous infusion of tetrahydrobiopterin (10 mg/kg per hour over 5 hours) was given. One hour after reperfusion, the recipient right lung was occluded. Cyclic guanosine monophosphate levels were measured in the pulmonary venous and central venous blood. Extravascular lung water index, hemodynamic variables, lipid peroxidation, and neutrophil migration to the allograft were assessed. RESULTS: In group III a significant reduction of extravascular lung water was noted in comparison with the controls (P =.0047). Lipid peroxidation in lung allograft tissue was significantly reduced in group II (P =.0021) and group III ( P =. 0077) in comparison with group I. Pulmonary venous levels of cyclic guanosine monophosphate increased up to 23 +/- 1 pmol/mL at 5 hours in group II and up to 40 +/- 1 pmol/mL in group III (group I, 4.1 +/- 0.5 pmol/mL [I vs III]; P <.001), whereas central venous levels of cyclic guanosine monophosphate were unchanged in all groups. CONCLUSION: Tetrahydrobiopterin administration during lung allograft reperfusion may reduce posttransplantation lung edema and oxygen-derived free radical injury in the graft. This effect is mediated by local enhancement of the nitric oxide/cyclic guanosine monophosphate pathway.

8-Br-cyclic GMP given during reperfusion improves post-transplant lung edema and free radical injury.

UNLABELLED: Substitution of the NO-pathway reduces ischemia/reperfusion injury following lung transplantation. 8-Br-cGMP is a membrane permeable analogue of cGMP, the second messenger of NO. In this study the effect of continuous administration of 8-Br-cGMP on early graft function was evaluated. METHODS: Unilateral left lung transplantation was performed in 10 weight-matched pigs (23-30 kg). Donor lungs were flushed with 1.51 cold (1 degree C) LPD solution and preserved for 20 hours. In Group I (n = 5), 8-Br-cGMP (0.2 mg/kg/h) was given continuously over the entire observation time starting 15 min before reperfusion. Group II served as control, no 8-Br-cGMP was administered. In both groups, 250 microg PGE1 was injected into the pulmonary artery (PA) before flush. One hour after reperfusion the recipients contralateral right PA and bronchus were ligated to assess isolated graft function only. Extravascular lung water index (EVLWI), pulmonary vascular resistance, mean PA pressure, mean systemic arterial pressure and gas exchange were assessed during a 5-hour observation period. Lipid peroxidation as indicator for free radical mediated injury and neutrophil migration to the allograft were measured at the end of the assessment. RESULTS: EVLWI was significantly reduced in animals treated with 8-Br-cGMP (overall difference P = 0.024) with a peak 2 hours after reperfusion (Group I, 8.2+/-0.3 mg/ml vs Group II, 10.1+/-0.6 mg/ml; P = 0.039). Also in Group I the free radical mediated tissue injury was significantly lower when compared to Group II (Group I, 61.8+/-12.3 pmol/g vs Group II, 120.7+/-7.2 pmol/g; P = 0.006). A tendency towards a reduced neutrophil migration after 8-Br-cGMP infusion was shown; however, the changes in comparison to the control animals were not statistically significant (Group I, 1.0+/-0.2 deltaOD/mg/min vs Group II, 1.7+/-0.3 deltaOD/mg/min; P = 0.13). Pulmonary- and systemic hemodynamics, and allograft gas exchange did not differ between groups. CONCLUSIONS: The results indicate that substitution of the NO pathway by administration of the second messenger cGMP at the time of reperfusion improves post-transplant lung allograft function.

8-Br-cGMP is superior to prostaglandin E1 for lung preservation.

BACKGROUND: Substitution of the nitric oxide (NO) pathway reduces ischemia/reperfusion injury after lung transplantation. 8-Br-cGMP is a membrane-permeable analogue of cGMP, the second messenger of NO. In this study, we evaluated the effect of administration of 8-Br-cGMP in the flush solution on early graft function. METHODS: Unilateral left lung transplantation was performed in 10 weight-matched pairs of outbred pigs (24 to 31 kg). Donor lungs were flushed with 1.5 L cold (1 degree C) low potassium dextrane (LPD) solution and preserved for 20 hours. In group I (n = 5), 8-Br-cGMP (1 mg/kg) was added to the flush solution. In group II (n = 5), 8 microg/kg prostaglandin E1 (PGE1) was injected into the pulmonary artery (PA) before flush. One hour after reperfusion, the recipients' contralateral right PA and bronchus were ligated to assess graft function only. cGMP levels in the PA and pulmonary vein were measured. Extravascular lung water index (EVLWI), pulmonary vascular resistance, mean PA pressure, and gas exchange (PaO2) were assessed during a 5-hour observation period. Lipid peroxidation (thiobarbituric acid-reactive substance) and neutrophil migration to the allograft (myeloperoxidase activity) were measured at the end of the assessment. RESULTS: In group I, a significant reduction of EVLWI (group I, 6.7 +/- 1.0 mL/kg vs group II, 10.1 +/- 0.6 ml/kg after 2 hours of reperfusion; p = 0.022), TBARS (group I, 65.6 +/- 10.0 pmol/g vs group II, 120.8 +/- 7.2 pmol/g, p = 0.0039), and MPO activity (group I, 0.8 +/- 0.1 change in optical density, (deltaOD)/mg/min vs group II, 1.7 +/- 0.3 deltaOD/mg/min, p = 0.036) was noted in comparison with group II. PaO2 levels tended to be higher in cGMP-treated animals, but the changes were not significant. Hemodynamic parameters did not differ between groups. CONCLUSIONS: In this large animal model of lung allograft ischemia/reperfusion injury, 8-Br-cGMP as additive to the flush solution improves posttransplant lung edema, lipid peroxidation, and neutrophil migration to the allograft. This effect is not attributable to improved flush by vasodilation, as we compared 8-Br-cGMP with PGE1 given before flush in control animals.

Survival and graft function in a large animal lung transplant model after 30 h preservation and substitution of the nitric oxide pathway.

OBJECTIVE: Substitution of the nitric oxide- (NO-) pathway improves early graft function following lung transplantation. We previously demonstrated that 8-Br-cGMP (second messenger of NO) to the flush solution and tetrahydrobiopterin (BH4, coenzyme of NO synthase) given as additive during reperfusion improve post-transplant graft function. In the present study, the combined treatment with 8-Br-cGMP and BH4 was evaluated. METHODS: Unilateral left lung transplantation was performed in weight matched outbred pigs (24-31 kg). In group I, grafts were preserved for 30 h (n=5). 8-Br-cGMP (1mg/kg) was added to the flush solution (Perfadex, 1.5l, 1 degrees C) and BH4 (10mg/kg/h) was given to the recipient for 5h after reperfusion. In group II, lungs were transplanted after a preservation time of 30 h (n=3) and prostaglandin E(1) (250 g) was given into the pulmonary artery (PA) prior to flush. In all recipients 1h after reperfusion the contralateral right PA and bronchus were ligated to assess graft function only. Survival time after reperfusion, extravascular lung water index (EVLWI), hemodynamic variables, and gas exchange (PaO(2)) were assessed during a 12h observation period. RESULTS: All recipients in group I survived the 12h assessment, whereas none of the group II animals survived more than 4h after reperfusion with a rapid increase of EVLWI up to 24.8+/-6.7 ml/kg. In contrast, in group I EVLWI reached up to 8.9+/-1.5 ml/kg and returned to nearly normal levels at 12h (6.1+/-0.8 ml/kg). In two animals of group I the gas exchange deteriorated slightly. The other three animals showed normal arterial oxygenation over the entire observation time. CONCLUSION: Our data indicate that the combined substitution of the NO pathway during preservation and reperfusion reduces ischemia/reperfusion injury substantially and that this treatment even allows lung transplantation after 30 h preservation in this model.

Tetrahydrobiopterin increases myocardial blood flow in healthy volunteers: a double-blind, placebo-controlled study.

OBJECTIVES: Tetrahydrobiopterin (BH4) is a regulatory cofactor for the activity of nitric oxide synthases. Vasodilating properties of BH4 have been reported in vitro and in vivo. The influence of BH4 on myocardial blood flow (MBF), however, is largely unknown. We therefore performed a double-blind, placebo-controlled study to investigate the effect of intravenous BH4 on MBF in healthy volunteers. METHODS AND RESULTS: Resting MBF was assessed in 15 subjects receiving either intravenous BH4 (10 mg/kg) or placebo using positron emission tomography (PET) and [13N]ammonia. From a mean baseline MBF of 0.91 +/- 0.09 ml/min/g, MBF increased to 1.18 +/- 0.10 ml/min/g after BH4 (n = 10; p = 0.0042). In contrast, in the group receiving placebo mean MBF remained unchanged (non-significant decrease from 0.97 +/- 0.19 to 0.84 +/- 0.11 ml/min/g; n = 5; p = 0.36). Systemic haemodynamics and ECGs remained unaffected in both groups. BH4 was very well tolerated. CONCLUSION: Systemically administered BH4 is safe and effectively increases resting MBF in healthy volunteers.

Mechanisms of haptoglobin protection against hemoglobin peroxidation triggered endothelial damage.

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are found within the cardiovascular system and Hb-triggered oxidative toxicity toward the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study, we systematically investigated the structural, biochemical, and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb's oxidative reactions with lipoproteins, providing dramatic protection against both pathways of toxicity. By these mechanisms, Hp shifts Hb's destructive pseudo-peroxidative reaction to a potential anti-oxidative function during peroxidative stress.

Differential regulation of constitutive and inducible nitric oxide production by inflammatory stimuli in murine endothelial cells.

The murine vascular endothelial cell line send1 expresses both constitutive and inducible nitric oxide (NO) synthases. Interferon-gamma (IFN gamma) or endotoxin (LPS) alone inhibited constitutive NO production in a dose and time dependent manner. Addition of L-arginine had no influence on the decrease of NO production caused by IFN gamma or LPS. On the other hand, IFN gamma and LPS synergized in the induction of high output NO production. Successive incubations with IFN gamma and LPS in different sequences revealed IFN gamma as the time setting signal for the induction of NO synthesis. These results demonstrate that LPS and IFN gamma have a direct suppressive effect on constitutive NO synthase while at the same time they strongly activate inducible NO production. Thus inflammatory stimuli trigger murine vascular endothelial cells to switch from constitutive to inducible NO synthase activity.

Modulation of inducible nitric oxide synthase mRNA stability by tetrahydrobiopterin in vascular smooth muscle cells.

Tetrahydrobiopterin (BH4) regulates inducible nitric oxide synthase (iNOS) as cofactor and allosteric effector. The present paper describes a novel function of BH4 in vascular smooth muscle cells (SMC). By varying BH4 levels with dicumarol (an inhibitor of BH4 synthesis) and sepiapterin (an exogenous source of co-factor), we investigated iNOS expression in activated rat aortic SMC. In sepiapterin-supplemented cells, iNOS protein levels were increased while in dicumarol-treated cells, iNOS levels were diminished. Time-kinetic experiments revealed that inhibition or supplementation of BH4 synthesis had no effects on iNOS induction or transcription rate. However, iNOS mRNA was present over a prolonged time in sepiapterin-supplemented SMC. Analysis of iNOS mRNA levels showed stable iNOS mRNA in sepiapterin-treated cells 8 hours after transcription inhibition, while in dicumarol-treated cells iNOS mRNA disappeared. The decrease of iNOS mRNA by dicumarol was abolished by sepiapterin. These data indicate that BH4 post-transcriptionally stabilizes iNOS mRNA in SMC. By this way BH4 modulates iNOS expression in the vascular system.

Production of monoclonal antibodies against human 6-pyruvoyl tetrahydropterin synthase and immunocytochemical localization of the enzyme.

Monoclonal antibodies were produced against human pituitary gland 6-pyruvoyl tetrahydropterin synthase, one of the key enzymes in the biosynthesis of tetrahydrobiopterin, by in vitro immunization with the antigen directly blotted from SDS-PAGE to polyvinylidene difluoride membranes. The antibodies produced show crossreactivity in the enzyme linked immunosorbent assay, not only with the human 6-pyruvoyl tetrahydropterin synthase but some also with the same enzyme isolated from salmon liver. 6-Pyruvoyl tetrahydropterin synthase was localized immuno-enzymatically in peripheral blood smears and in skin fibroblasts by the use of these monoclonal antibodies and the alkaline phosphatase monoclonal anti-alkaline phosphatase labeling technique.

Purification of GTP cyclohydrolase I from human liver and production of specific monoclonal antibodies.

GTP cyclohydrolase I, the first enzyme in the de novo biosynthesis of tetrahydrobiopterin, was enriched more than 13,000-fold from human liver by preparative isoelectric focusing using Sephadex G-200 SF gels. The pI of the active enzyme was determined as 5.6 by analytical isoelectric focusing in the same matrix. The native enzyme has an apparent molecular mass of 440 kDa and appears to be composed of eight 50-kDa subunits as estimated from SDS/PAGE. The enriched enzyme preparation was used to produce specific monoclonal antibodies. From 11 monoclonal antibodies obtained, one was extensively characterized for further applications. This monoclonal antibody belongs to the IgM class and shows immunoreactivity with GTP cyclohydrolase I both from man and from Escherichia coli. It is capable of highly sensitive detection of GTP cyclohydrolase I by ELISA and by Western blot analysis. The monoclonal antibody was used for the immunoenzymatic localisation of GTP cyclohydrolase I in human peripheral blood mononuclear cells. Furthermore, it was possible to demonstrate the absence of immunoreactivity in cells with GTP cyclohydrolase I deficiency. The antibody's use as a tool either for differential diagnosis of atypical phenylketonuria due to GTP cyclohydrolase I deficiency or prenatal diagnosis of this severe inherited metabolic disease is now under investigation.

Regulation of 6-pyruvoyltetrahydropterin synthase activity and messenger RNA abundance in human vascular endothelial cells.

BACKGROUND: The nitric oxide synthase cofactor tetrahydrobiopterin (BH4) is involved in the regulation of endothelium-dependent vascular functions mediated by nitric oxide. Vascular endothelial cells synthesize and secrete large amounts of BH4 on cytokine activation. There is scant knowledge about molecular mechanisms of cytokine-triggered BH4 production in endothelial cells. METHODS AND RESULTS: Pteridine production, mRNA expression of GTP cyclohydrolase (GTPCH) and 6-pyruvoyltetrahydropterin synthase (PTPS) (both key enzymes of BH4 biosynthesis), and PTPS activity were studied in human umbilical vein endothelial cells (HUVECs) exposed to inflammatory cytokines. BH4 levels were

Tetrahydrobiopterin synthesis precedes nitric oxide-dependent inhibition of insulin secretion in INS-1 rat pancreatic beta-cells.

Interleukin 1 (IL-1) induces pancreatic beta-cell dysfunction mainly due to overproduction of nitric oxide (NO). Since tetrahydrobiopterin (BH4) is a obligatory cofactor of NO synthases, we examined the temporal relationship of BH4 synthesis, NO production and insulin secretion in a pancreatic beta-cell line (INS-1) which was exposed to IL-1. IL-1 affected BH4 synthesis in a time- and concentration-dependent manner. At a concentration of 10 ng/ml IL-1 caused an increase in intracellular BH4 with peak levels being observed at 6 hours followed by a steady decline in the cellular BH4 content. The increase in BH4 synthesis was followed by enhanced NO production and, consecutively, inhibition of insulin secretion. The concentration-dependent regulation of BH4 synthesis, NO production and suppression of insulin secretion indicate a functional link between these parameters in pancreatic beta-cells.

Two novel genes FIND and LIND differentially expressed in deactivated and Listeria-infected human macrophages.

Deactivation of macrophage functions plays an important role in human infectious and inflammatory diseases. In this study, differential-display RT-PCR was used to analyze the gene expression of human mononuclear phagocytes deactivated with interleukin (IL)-4, IL-10, and dexamethasone (DEX), in the absence and presence of infection with Listeria monocytogenes (Listeria). Two novel differentially expressed mRNA species were discovered: FIND (IL-Four INDuced) was upregulated with IL-4 but down-regulated with DEX, and is predicted to code for an M(r) 53,000 transmembrane protein. LIND (Listeria INDuced) was induced by Listeria infection, and is predicted to code for an M(r) 39,000 nuclear or cytoplasmic protein containing three coiled-coil domains. In addition, we report several novel effects of deactivators and infection on the expression of known genes: (1) IL-4 caused pronounced upregulation of ABCG2, coding for an ATP-binding cassette transporter highly expressed in the placenta, which mediates multidrug resistance of cancer cells, but is otherwise of unknown function; (2) both DEX and IL-4 downregulated osteopontin, an important factor of host resistance against intracellular infections; (3) inhibition of the CC-chemokine I-309 mRNA expression by all three deactivators in the presence of Listeria infection, and (4) upregulation by Listeria infection of the interferon-stimulated gene ISG20 of unknown function, whose product localizes with nuclear dots/PML bodies.