Isorhamnetin inhibits Prevotella intermedia lipopolysaccharide-induced production of interleukin-6 in murine macrophages via anti-inflammatory heme oxygenase-1 induction and inhibition of nuclear factor-κB and signal transducer and activator of transcription 1 activation.

BACKGROUND AND OBJECTIVE: Interleukin-6 (IL-6) is a key proinflammatory cytokine that has been considered to be important in the pathogenesis of periodontal disease. Therefore, host-modulatory agents directed at inhibiting IL-6 appear to be beneficial in terms of attenuating periodontal disease progression and potentially improving disease susceptibility. In the current study, we investigated the effect of the flavonoid isorhamnetin on the production of IL-6 in murine macrophages stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in inflammatory periodontal disease, and its mechanisms of action. MATERIAL AND METHODS: Lipopolysaccharide from P. intermedia ATCC 25611 was isolated using the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time PCR to quantify IL-6 and heme oxygenase-1 (HO-1) mRNA expression. The expression of HO-1 protein and the levels of signaling proteins were monitored using immunoblot analyses. The DNA-binding activity of nuclear factor-κB (NF-κB) was analyzed using ELISA-based assay kits. RESULTS: Isorhamnetin significantly down-regulated P. intermedia LPS-induced production of IL-6 as well as its mRNA expression in RAW264.7 cells. Isorhamnetin up-regulated the expression of HO-1 at both gene transcription and translation levels in cells stimulated with P. intermedia LPS. In addition, inhibition of HO-1 activity by tin protoporphyrin IX blocked the inhibitory effect of isorhamnetin on IL-6 production. Isorhamnetin failed to prevent LPS from activating either c-Jun N-terminal kinase or p38 pathways. Isorhamnetin did not inhibit NF-κB transcriptional activity at the level of inhibitory κB-α degradation. Isorhamnetin suppressed NF-κB signaling through inhibition of nuclear translocation and DNA binding activity of NF-κB p50 subunit and attenuated signal transducer and activator of transcription 1 signaling. CONCLUSION: Although further research is required to clarify the detailed mechanism of action, we propose that isorhamnetin may contribute to blockade of the host-destructive processes mediated by IL-6 and could be a highly efficient modulator of the host response in the treatment of inflammatory periodontal disease. Further research in animal models of periodontitis is required to better evaluate, the potential of isorhamnetin as a novel agent for treating periodontal disease.

Dexamethasone enhances 1alpha,25-dihydroxyvitamin D3 effects by increasing vitamin D receptor transcription.

Calcitriol, the active form of vitamin D, in combination with the glucocorticoid dexamethasone (Dex) has been shown to increase the antitumor effects of calcitriol in squamous cell carcinoma. In this study we found that pretreatment with Dex potentiates calcitriol effects by inhibiting cell growth and increasing vitamin D receptor (VDR) and VDR-mediated transcription. Treatment with actinomycin D inhibits Vdr mRNA synthesis, indicating that Dex regulates VDR expression at transcriptional level. Real time PCR shows that treatment with Dex increases Vdr transcripts in a time- and a dose-dependent manner, indicating that Dex directly regulates expression of Vdr. RU486, an inhibitor of glucocorticoids, inhibits Dex-induced Vdr expression. In addition, the silencing of glucocorticoid receptor (GR) abolishes the induction of Vdr by Dex, indicating that Dex increases Vdr transcripts in a GR-dependent manner. A fragment located 5.2 kb upstream of Vdr transcription start site containing two putative glucocorticoid response elements (GREs) was evaluated using a luciferase-based reporter assay. Treatment with 100 nm Dex induces transcription of luciferase driven by the fragment. Deletion of the GRE distal to transcription start site was sufficient to abolish Dex induction of luciferase. Also, chromatin immunoprecipitation reveals recruitment of GR to distal GRE with Dex treatment. We conclude that Dex increases VDR and vitamin D effects by increasing Vdr de novo transcription in a GR-dependent manner.

Nonenzymatic glycation of high-density lipoprotein impairs its anti-inflammatory effects in innate immunity.

AIMS/HYPOTHESIS: In type 2 diabetes mellitus (T2DM), the abnormal protein and lipid composition of diabetic high-density lipoprotein (HDL) could impair its anti-inflammatory functions. Whether nonenzymatic glycation directly impaired the anti-inflammatory effects of HDL in innate immunity remained unclear. METHODS: Human acute monocytic leukemia cell line (THP-1) cells, mouse RAW 264.7 macrophages and primary human monocytes derived macrophages were pre-incubated with native HDL, diabetic HDL isolated from T2DM patients or HDL glycated with different doses of d-glucose in vitro and then challenged with lipopolysaccharide (LPS). The release of tumor necrosis factor (TNF)-α and IL-1ß was assayed by enzyme-linked immunosorbent assay (ELISA). Phosphorylation of Iκ-Bα in cytoplasm and nuclear translocation of NF-κB were detected by western blot. Glycation levels of native HDL, glycated HDL and diabetic HDL were determined using LC-MS/MS. RESULTS: The potency of diabetic HDL to inhibit the release of TNF-α (p < 0.05) and IL-1ß (p < 0.001) was dramatically attenuated compared with that of native HDL. Similarly, glycation of HDL in vitro impaired its ability to inhibit TNF-α and IL-1ß release in a glucose dose-dependent manner. Moreover, apoHDL still effectively inhibited the release of TNF-α and IL-1ß induced by LPS, but glycated apoHDL partly lost such abilities. Nonenzymatic glycation levels of glycated HDL and diabetic HDL increased 28 fold (p < 0.001) and 4 fold (p < 0.001), respectively compared with that of native HDL. CONCLUSIONS: In this study, we observed that diabetic HDL and HDL glycated in vitro both partly lose their protective effects to inhibit cytokines release induced by LPS in macrophages, and nonenzymatic glycation of the protein components of HDL plays key roles in these impairments.

In vivo GM-CSF prevents dexamethasone suppression of killing of Aspergillus fumigatus conidia by bronchoalveolar macrophages.

Dexamethasone (DEX) is a potent immunosuppressive agent used in the treatment of several disorders. However, despite its beneficial effects, DEX puts patients at risk for opportunistic infections, especially pulmonary aspergillosis. Previously we reported that in vitro granulocyte-macrophage colony-stimulating factor (GM-CSF) blocks the immunosuppressive action of DEX on bronchoalveolar macrophages (BAMs). Here we report that BAMs freshly isolated from mice treated intraperitoneally with DEX for 24 h had significantly (P<0.01) reduced killing of conidia, i.e., 15 +/- 5% conidia killed by BAMs from DEX-treated mice versus 35 +/- 3% by BAMs from mice given saline, 38 +/- 5% by BAMs from mice given GM-CSF, and 39 +/- 1% by BAMs from mice given both DEX and GM-CSF. On the other hand, in another compartment GM-CSF could not block the DEX reduction of spleen weight and spleen cellularity. Unlike GM-CSF, granulocyte colony-stimulating factor did not block DEX suppression of BAMs. GM-CSF given 24 h before DEX resulted in blocking of DEX suppression of BAM conidiacidal activity. However, when DEX was given 24 h before GM-CSF, DEX suppression of BAM was not reversed. These data show that GM-CSF in vivo blocks the in vivo immunosuppressive effects of DEX on BAM killing of conidia and suggest a potential use of GM-CSF in patients at risk for aspergillosis due to immunosuppressive DEX treatment.

Inhibition of glucocorticoid-mediated, caspase-independent dendritic cell death by CD40 activation.

Glucocorticoids (GC) are potent anti-inflammatory and immunosuppressive agents that act on a variety of immune cells, including T cells, monocytes/macrophages, osteoclasts, and dendritic cells (DC). However, the mechanism(s) by which GC exert anti-inflammatory effects is still largely unknown. It is already well known that GC treatment inhibits DC maturation and interleukin (IL)-12 production by DC. In this study, we investigated the apoptosis induction of DC by a synthetic GC, dexamethasone (Dex). The stimulation with Dex resulted in DC apoptosis in a dose- and time-dependent manner as it was measured by determining annexin V-positive cells and mitochondrial potential. In contrast, monocytes that are precursor cells of DC are resistant to Dex-mediated apoptosis. The Dex-induced apoptosis of DC was independent of caspase activation because it was not inhibited by the broad caspase inhibitor, Z-VAD-fmk. It is interesting that agonistic CD40 antibody completely inhibited Dex-induced cell death, whereas other inflammatory stimuli did not show the same effect, suggesting that CD40 signaling may selectively modulate GC-mediated DC apoptosis. Taken together, our findings revealed an important role of GC and CD40 signaling in the regulation of immune responses in which DC play a key role in the inflammatory process of various immunomediated diseases.

Anti-inflammatory drugs block cytokine mRNA accumulation in the skin and improve the clinical condition of reactional leprosy patients.

The aim of this study was to investigate in what ways in vivo anti-inflammatory treatment affects cytokine mRNA expression in situ in both erythema nodosum leprosum and reversal reaction patients. Serial biopsies were collected from the patients undergoing leprosy reactions before and during pentoxifylline (n = 7) or thalidomide (n = 3) treatment for erythema nodosum leprosum and prednisone (n = 3) for reversal reaction. Clinical evolution of the skin lesion was assessed during the study and semiquantitative reverse transcription-polymerase chain reaction was used to investigate cytokine mRNA expression at the lesion site. Results showed expression of interferon-gamma, interleukin-6, interleukin-10, interleukin-12 p40, and tumor necrosis factor-alpha in all patients tested at the onset of reactional episodes, but interleukin-4 mRNA was rarely detected in the lesions (n = 4). Follow-up analysis showed that, irrespective of the drugs used, tumor necrosis factor-alpha mRNA was diminished in 10 of the 13 patients tested. A concomitant decrease of mRNA accumulation was also observed for interferon-gamma (nine of 11 patients), interleukin-6 (nine of 11), and interleukin-12 p40 (six of eight). An inhibitory effect on interleukin-10 mRNA was likewise seen after thalidomide and pentoxifylline, but not subsequent to prednisone treatment. The data also demonstrated that cytokine mRNA inhibition correlates to the resolution of the inflammatory response in situ (n = 10), whereas the persistence/enhancement of cytokine message expression after treatment was associated with worsening of the skin condition, as seen in three erythema nodosum leprosum patients whose maintenance of local inflammation was accompanied by the appearance/persistence of interleukin-4 gene expression in situ subsequent to anti-inflammatory treatment. In summary, the participation of cytokines in leprosy inflammatory episodes seems to be directly associated with the patients' clinical evolution following therapy for reaction.

RU 486 inhibits peripheral effects of glucocorticoids in humans.

RU 486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl)17 alpha-(prop-1-ynyl)estra-4,9-dien-3-one] is a synthetic steroid receptor antagonist. To evaluate the peripheral antiglucocorticoid action of this compound, we investigated its ability to antagonize cutaneous steroid-induced vasoconstriction. This phenomenon, produced by three different topical steroids in six normal men, was consistently and significantly attenuated or abolished by oral administration of 6 mg/kg RU 486. This demonstration of a peripheral action of RU 486 is important in relation to the potential therapeutic use of this well tolerated drug in states of hypercortisolism. It also indicates that the cutaneous vasoconstrictor effects of topical steroids are mediated by occupancy of glucocorticoid receptors.

Raloxifene concurrently stimulates osteoprotegerin and inhibits interleukin-6 production by human trabecular osteoblasts.

Raloxifene reduces bone loss and prevents vertebral fractures in postmenopausal women. Its skeletal effects are mediated by estrogen receptors (ER) and their modulation of paracrine osteoblastic factors. Receptor activator of nuclear factor-kappa B ligand is essential for osteoclasts and enhances bone resorption, whereas osteoprotegerin (OPG) neutralizes receptor activator of nuclear factor-kappa B ligand. Here, we assessed the effects of raloxifene on OPG production in human osteoblasts (hOB). Raloxifene enhanced gene expression of ER-alpha and progesterone receptor. Moreover, raloxifene increased OPG mRNA levels and protein secretion by hOB in a dose- and time-dependent fashion by 2- to 4-fold with a maximum effect at 10(-7) M and after 72 h (P < 0.001). Treatment with the ER antagonist ICI 182,780 abrogated the effects of raloxifene on OPG production. Moreover, raloxifene enhanced osteoblastic differentiation markers, type 1 collagen secretion, and alkaline phosphatase activity by 3- and 2-fold, respectively (P < 0.001). In addition, raloxifene inhibited expression of the bone-resorbing cytokine IL-6 by 25-45% (P < 0.001). In conclusion, our data suggest that raloxifene stimulates OPG production and inhibits IL-6 production by hOB. Because OPG production increases with osteoblastic maturation, enhancement of OPG production by raloxifene could be related to its stimulatory effects on osteoblastic differentiation.

Reduction of the inhibitory effect of ketoconazole on budesonide pharmacokinetics by separation of their time of administration.

BACKGROUND: Budesonide is a glucocorticosteroid used in the treatment of, for example, inflammatory bowel diseases, with a recommended once-daily morning dosing regimen. Ketoconazole is a potent inhibitor of the cytochrome P450 3A (CYP3A) activities and known to inhibit the elimination of drugs metabolized by CYP3A, including budesonide. It is of therapeutic interest to know whether the influence of ketoconazole can be reduced by administration on an occasion different in time to CYP3A substrates. METHODS: Eight healthy men completed this randomized, open crossover study that comprised three different periods. In period 1, a single oral dose of 3 mg budesonide was given in the morning. In period 2, a 200-mg ketoconazole tablet was administered once daily in the morning on 4 consecutive days. On the fourth day, 3 mg budesonide was administered at the same time as the ketoconazole. In period 3, 200 mg ketoconazole was administered once daily in the evening on 4 consecutive days. On the fourth day, 3 mg budesonide was administered 12 hours before the ketoconazole. One-week washout periods separated the budesonide administrations. RESULTS: The mean area under the plasma drug concentration-time curve [AUC(0-24)] for budesonide was increased by 6.5 times when it was given simultaneously with ketoconazole. When the administrations of the two drugs were separated by 12 hours, the mean AUC(0-24) for budesonide was increased by only 3.8 times. CONCLUSION: This study shows that the capability of ketoconazole to inhibit the elimination of budesonide is significantly reduced (by 50%) by a 12-hour separation of the administration times.

Inhibition of dexamethasone-induced cytoskeletal changes in cultured human trabecular meshwork cells by tetrahydrocortisol.

PURPOSE: To determine the cellular mechanism of action of the intraocular pressure (IOP) lowering steroid tetrahydrocortisol (THF). METHODS: Tetrahydrocortisol was evaluated for glucocorticoid antagonist activity using in vitro and in vivo assays. Systemically administered THF was evaluated for its ability to inhibit dexamethasone-induced body weight loss and systemic hypertension in rats. In vitro receptor antagonism was tested using the supernatant fraction of IM9 cells as the source of soluble glucocorticoid receptor in 3H-dexamethasone displacement binding assays. In addition, six different primary human trabecular meshwork (TM) cell lines were cultured for 0 to 14 days in the absence or presence of dexamethasone (10(-7) M) and/or THF (10(-6) to 10(-8) M). The effects of these steroids on the TM cytoskeleton were determined by epifluorescent microscopy and by transmission electron microscopy. RESULTS: Tetrahydrocortisol was unable to inhibit the dexamethasone (DEX)-induced systemic hypertension and decrease in body mass in rats and was unable to displace 3H-DEX from the soluble human glucocorticoid receptor. However, THF inhibited the DEX-induced formation of cross-linked actin networks in cultured human TM cells in a progressive and dose-dependent manner (IC50 = 5.7 x 10(-7) M). Dexamethasone caused changes in the TM cell microtubules that were reversed partially by concomitant treatment with THF. Tetrahydrocortisol alone appeared to increase microfilament bundling in TM cells. CONCLUSIONS: Tetrahydrocortisol was not a glucocorticoid antagonist at the level of the classical glucocorticoid receptor and did not appear to antagonize systemically mediated glucocorticoid activity in the rat. Tetrahydrocortisol inhibited DEX-induced changes in the TM microfilaments and microtubules. These results may explain partially the IOP lowering activity of THF because glucocorticoid-mediated changes in the TM cytoskeleton have been proposed to be involved in the generation of ocular hypertension.

Regulation of histamine H1 receptor coupling by dexamethasone in human cultured airway smooth muscle.

1. The regulation of histamine-induced [3H]-inositol phosphate and intracellular calcium responses in human cultured airway smooth muscle cells was studied. 2. Histamine induced concentration-dependent [3H]-inositol phosphate formation (EC50 4 microM). This response was inhibited by a range of selective H1 receptor antagonists but not by the H2-selective antagonist, tiotidone or the H3 receptor-selective antagonist, thioperamide, indicating that an H1 receptor is involved in this response in human cultured airway smooth muscle cells. 3. Preincubation of human cultured airway smooth muscle cells with concentrations of dexamethasone > 10 nM for 22 h produced concentration-dependent inhibition of histamine-induced inositol phosphate formation. The maximum inhibition observed was 45% of the response in control cells. The inhibitory effect of dexamethasone was itself reversed by prior exposure to the glucocorticoid receptor antagonist, RU38486 (10 microM). Preincubation for 22 h with 1 microM dexamethasone produced inhibition of the inositol phosphate response to histamine to all concentrations of histamine inducing significant inositol phosphate formation in these cells. In contrast, the response to the G protein activator, NaF (0.1-20 mM) was unaltered by preincubation with dexamethasone. 4. Preincubation of human airway smooth muscle cells with 1 microM dexamethasone for time periods of < 6 h failed to inhibit histamine-induced inositol phosphate formation in human airway smooth muscle cells. 5. Histamine also induced concentration-dependent elevation of intracellular calcium levels in Fura 2-loaded human airway smooth muscle cells. This response was inhibited by preincubation with 1 microM dexamethasone. 6. We conclude that signal transduction through the H1 receptor in human airway smooth muscle is subject to regulation by dexamethasone and that this may in part account for the protective effect of dexamethasone against spasmogen-induced contractile responses in the airways.

The inhibition of histamine release from rat peritoneal mast cells by non-steroid anti-inflammatory drugs and its reversal by calcium.

1 The non-steroid anti-inflammatory drugs, indomethacin, flufenamate and meclofenamate, inhibited the release of histamine from rat peritoneal mast cells induced by pharmacological or immunological challenge in vitro. 2 Anti-inflammatory steroids had little effect on histamine release from the mast cells. 3 Th inhibition of histamine release by the aspirin-like drugs was not prevented by incubation with glucose, unlike the inhibition of 2,4,dinitrophenol or antimycin-A. This suggests that the non-steroid anti-inflammatory compounds do not act by preventing the energy production from oxidative metabolism, required for histamine release. 4 The inhibition of the calcium ionophore A23187-induced histamine release by the aspirin-like drugs was reversed by an increase in the calcium concentration of the incubation medium. 5 The results suggest that the non-steroid anti-inflammatory compounds inhibit histamine release by actions on calcium influx into the mast cell, or on calcium mobilization or utilization within the mast cell.

Anti-inflammatory activity of creatine supplementation in endothelial cells in vitro.

1 Creatine (CR) supplementation augments muscle strength in skeletal muscle cells by increasing intracellular energy pools. However, the effect of CR supplementation on endothelial cells remains to be clarified. 2 In this study, we investigated whether CR supplementation had any anti-inflammatory activity against human pulmonary endothelial cells in culture. 3 We confirmed that supplementation with 0.5 mM CR significantly increased both intracellular CR and phosphocreatine (PC) through a CR transporter while keeping intracellular ATP levels constant independent of CR supplementation and a CR transporter antagonist. 4 In the assay system of endothelial permeability, supplementation with 5 mM CR significantly suppressed the endothelial permeability induced by serotonin and H(2)O(2). 5 In cell adhesion experiments, supplementation with 5 mM CR significantly suppressed neutrophil adhesion to endothelial cells. 6 In the measurement of adhesion molecules, CR supplementation with more than 0.5 mM CR significantly inhibited the expressions of ICAM-1 and E-selectin on endothelial cells, and the inhibition was significantly suppressed by an adenosine A(2A) receptor antagonist. 7 The present study suggests that CR supplementation has anti-inflammatory activities against endothelial cells.

A new HMG-CoA reductase inhibitor, rosuvastatin, exerts anti-inflammatory effects on the microvascular endothelium: the role of mevalonic acid.

Recent studies have reported that hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have vasculoprotective effects independent of their lipid-lowering properties, including anti-inflammatory actions. We used intravital microscopy of the rat mesenteric microvasculature to examine the effects of rosuvastatin, a new HMG-CoA reductase inhibitor, on leukocyte-endothelium interactions induced by thrombin. Intraperitoneal administration of 0.5 and 1.25 mg kg(-1) rosuvastatin 18 h prior to the study, significantly and dose-dependently attenuated leukocyte rolling, adherence, and transmigration in the rat mesenteric microvasculature superfused with 0.5 u ml(-1) thrombin. This protective effect of rosuvastatin was reversed by intraperitoneal injection of 25 mg kg(-1) mevalonic acid 18 h before the study. Immunohistochemical detection of the endothelial cell adhesion molecule P-selectin showed a 70% decrease in endothelial cell surface expression of P-selectin in thrombin-stimulated rats given 1.25 mg kg(-1) rosuvastatin. In addition, rosuvastatin enhanced release of nitric oxide (NO) from the vascular endothelium as measured directly in rat aortic segments. Moreover, rosuvastatin failed to attenuate leukocyte-endothelium interactions in peri-intestinal venules of eNOS(-/-) mice. These data indicate that rosuvastatin exerts important anti-inflammatory effects via inhibition of endothelial cell adhesion molecule expression, and that this protective action of rosuvastatin requires release of nitric oxide by the vascular endothelium. These data also demonstrate that the mechanism of the non-lipid lowering actions of HMG-CoA reductase inhibitors in vivo may be due to reduced formation or availability of mevalonic acid within endothelial cells.

Superoxide is an antagonist of antiinflammatory drugs that inhibit hypochlorous acid production by myeloperoxidase.

Myeloperoxidase, the most abundant enzyme in neutrophils, catalyses the conversion of hydrogen peroxide and chloride to hypochlorous acid. This potent oxidant has the potential to cause considerable tissue damage in many inflammatory diseases. We have investigated the ability of dapsone, diclofenac, primaquine, sulfapyridine and benzocaine to inhibit hypochlorous acid production by stimulated human neutrophils. The drugs were also tested against purified myeloperoxidase using xanthine oxidase to generate hydrogen peroxide and superoxide. The inhibitory effects of the drugs on hypochlorous acid production, either by cells stimulated with phorbol myristate acetate or by myeloperoxidase and xanthine oxidase, were significantly less than those determined with myeloperoxidase and reagent hydrogen peroxide. Comparable potency was observed only when superoxide dismutase was present to remove superoxide. We also observed that with the xanthine oxidase system, inhibition of hypochlorous acid production by dapsone decreased markedly as the concentration of myeloperoxidase increased. Dapsone was a poor inhibitor of hypochlorous acid production by neutrophils stimulated with opsonized zymosan, regardless of the presence of superoxide dismutase. With this phagocytic stimulus, catalase inhibited hypochlorous acid formation by only 60%, which indicates that a substantial amount of the hypochlorous acid detected originated from within phagosomes. Thus, it is apparent that dapsone is unable to affect intraphagosomal conversion of hydrogen peroxide to hypochlorous acid. All the drugs inhibit myeloperoxidase reversibly by trapping it as its inactive redox intermediate, compound II. We propose that superoxide limits the potency of the drugs by reducing compound II back to the active enzyme. Furthermore, under conditions where the activity of myeloperoxidase exceeds that of the hydrogen peroxide-generating system, which is most likely to occur in phagosomes, partial inhibition of myeloperoxidase need not affect hypochlorous acid production. We conclude that drugs that inhibit myeloperoxidase by converting it to compound II are unlikely to be effective against hypochlorous acid-mediating tissue damage.

Tumor necrosis factor-alpha production in whole blood after cardiopulmonary bypass: downregulation caused by circulating cytokine-inhibitory activities.

OBJECTIVES: Cardiopulmonary bypass is associated with the release of proinflammatory cytokines (tumor necrosis factor alpha, interleukin 1beta, interleukin 6, and interleukin 8) and anti-inflammatory cytokines (interleukin 10 and transforming growth factor beta(1)). On the one hand this cytokine release is related to the postoperative systemic inflammatory response syndrome, and on the other hand it is related to deterioration of the immune system, for example in monocyte or polymorphonuclear neutrophil function, leading to an increased susceptibility to infections. To gain further insight into the alterations of immune cell reactivity and possible regulatory mechanisms, we studied lipopolysaccharide-induced tumor necrosis factor alpha synthesis in whole blood from cardiac surgical patients. METHODS: Fifteen patients undergoing elective heart surgery with cardiopulmonary bypass were included in the study. Ex vivo lipopolysaccharide-induced tumor necrosis factor alpha synthesis was measured in a whole blood assay before, during, and after bypass. Corresponding tumor necrosis factor alpha messenger RNA levels were determined by semiquantitative reverse transcriptase-polymerase chain reaction. In addition, the influence of patient serum on whole blood responsiveness and its relationship to anti-inflammatory cytokines were evaluated in vitro. RESULTS: Tumor necrosis factor alpha synthesis was significantly reduced after 30 minutes of cardiopulmonary bypass and showed the lowest values at the end of bypass (mean +/- SD 0.109 +/- 0.105 ng/10(6) white blood cells after 30 minutes of bypass and 0.050 +/- 0.065 ng/10(6) white blood cells at the end of bypass, vs 0.450 +/- 0.159 ng/10(6) white blood cells preoperatively, P <.001). As a further indication of reduced cytokine biosynthesis, diminished messenger RNA levels for tumor necrosis factor alpha were detected. Serum withdrawn from patients at the end of cardiopulmonary bypass reduced tumor necrosis factor alpha synthesis in heterologous blood from healthy volunteers highly significantly to 39.93% +/- 23.18% relative to control serum (P =.005) and preoperatively drawn serum (P =.024). This effect was dose dependent and was not specific for lipopolysaccharide-induced tumor necrosis factor alpha synthesis. Anesthesia and heparin administration did not influence tumor necrosis factor alpha production significantly. Ex vivo tumor necrosis factor alpha synthesis was negatively related to interleukin 10 serum levels, positively but weakly related to interleukin 4, and was not related to transforming growth factor beta(1) (Spearman correlation coefficients -0.565, P <.001, 0.362, P <.001, and -0.062, P =.460, respectively). However, interleukin 10 levels in patient serum after cardiopulmonary bypass were 300-fold below the quantities needed for half-maximal inhibition of tumor necrosis factor alpha synthesis in vitro. Moreover, the inhibitory activity could not be removed by immune absorption of interleukin 10. CONCLUSIONS: These results suggest that during cardiac operations cytokine-inhibitory serum activities are released or newly formed. These activities could not be explained by the actions of interleukins 4 and 10 or transforming growth factor beta(1). Although their exact nature remains undetermined, these substances may contribute to the diminished immune cell functions after cardiopulmonary bypass and thus need further characterization.

Immunological properties of dehydroepiandrosterone, its conjugates, and metabolites.

The immunological response to the administration of various C-19 steroids has been of increasing interest. Although the action of dehydroepiandrosterone has been studied, the responses to its metabolites have not been explored. In the present study the ability of dehydroepiandrosterone, its conjugates, and metabolites to oppose the anti-inflammatory action of glucocorticoids on the inflammatory response to 2,4-dinitrochlorobenzene in the sensitized mouse was examined. A clear difference was seen between the antiglucocorticoid activity of dehydroepiandrosterone, its conjugates, and its 5 beta-metabolites on the one hand and the planar 5 alpha- and delta 4-metabolites, which were devoid of antiglucocorticoid activity. The mechanism of this antiglucocorticoid activity remains to be established.

Clonidine-induced antinociception and locomotor hypoactivity are reduced by dexamethasone in mice.

The effects of dexamethasone pretreatment on clonidine-induced antinociception and locomotor hypoactivity were investigated in mice. In the hot-plate and the tail-flick tests, dexamethasone administered intraperitoneally at a dose of 1 mg kg(-1), 30 or 60 min before clonidine, reduced clonidine antinociception in both tests and reduced clonidine-induced locomotor hypoactivity in the activity cage. When administered 15 min before clonidine, dexamethasone had no effect on clonidine antinociception. A higher dexamethasone dose (10 mg kg(-1)) induced the same effects observed at a dose of 1 mg kg(-1) in the hot-plate and the tail-flick tests, but the former dose had a stronger effect on locomotor hypoactivity. Dexamethasone (10 ng/mouse) administered intracerebroventricularly 30 min before clonidine was also able to reduce both clonidine-induced antinociception and locomotor hypoactivity. The protein synthesis inhibitor, cycloheximide, administered intraperitoneally at the dose of 10 mg kg(-1), 2 h before clonidine, was able to prevent dexamethasone effects on clonidine-induced antinociception. The glucocorticoid receptor antagonist RU-38486, administered intracerebroventricularly at the dose of 1 ng/mouse, was also able to block dexamethasone effects on clonidine-induced antinociception and locomotor hypoactivity, whereas both cycloheximide and RU-38486 per se did not influence pain sensitivity or locomotor activity. These results suggest that the dexamethasone effects on clonidine-induced antinociception and locomotor hypoactivity depend on the stimulating effects that dexamethasone exert, on the protein synthesis via the glucocorticoid receptor in the brain.

[Effect of anti-inflammatory drugs, alone and combined with ofloxacin, on the respiratory burst of human polymorphonuclear leukocytes]. / Efecto de los antiinflamatorios, solos y asociados con ofloxacino, en la explosión respiratoria de los leucocitos polimorfonucleares humanos.

The antibacterial activity of polymorphonuclear leukocytes (PMNs) is based on the production of superoxide anion and H(2)O(2) in the respiratory burst and can be impaired in different ways. The combination of an antibacterial agent and an antiinflammatory drug is quite common in immunodepressed patients whose respiratory burst of PMN could be impaired. In this study we examine in vitro the effect of pretreating (35 degrees C for 30 min) PMNs with the antiinflammatory drugs dexamethasone (0.4, 4 and 40 microgram/ml), methylprednisolone (0.37, 3.7 and 37 microgram/ ml), hydrocortisone (0.048, 0.48 and 4.8 microgram/ml), betamethasone (0.1, 1, 5 and 10 mg/ml), phenylbutazone (1000 microgram/ml) and acetylsalicylic acid (25, 250, 2500 microgram/ml) alone, and combined with 10 mg/ml of ofloxacin on the respiratory burst. Superoxide anion was measured by the cytochrome c reduction microtechnique and H(2)O(2) by phenol red. The antiinflammatory drugs alone decreased the production of H(2)O(2) (except dexamethasone and methylprednisolone) and superoxide anion (except betamethasone) from 15-45%, depending on the antiinflammatory drug and concentration, while ofloxacin increased the production of superoxide anion (20.2 +/- 6.7%). The combination of antiinflammatory drugs with ofloxacin neutralizes the inhibitory effect of the former on the respiratory burst of PMNs. It is therefore important to know the effect of drugs on the respiratory burst in order to choose those that have the same therapeutic effect without interfering with PMN functions.

Epileptogenic activity induced by combined treatment with antiinflammatory drugs and enoxacin and its inhibition by a calcium antagonist, nicardipine.

Epileptogenic activity induced by combined treatment with antiinflammatory drugs and enoxacin was investigated in chronic electrode-implanted rats. Ferubinac ethyl and aspirin DL-lysine showed a spike and wave complex in EEG without showing remarkable behavioral changes when they were injected intraventricularly, although a relatively high dose was needed. Enoxacin, on the other hand, elicited potent epileptogenic activity characterized by uninterrupted high voltage spike and wave complex at doses of 50 and 100 micrograms. At the same time, rats showed hyperactivity, jumping and violent convulsion. Combined treatment with enoxacin (p.o.) and ferubinac ethyl (i.v.) caused potent epileptogenic activity characterized by uninterrupted burst of high voltage spike and wave complex. Behaviorally, animals showed forelimb clonus, head nodding and generalized convulsion. High voltage spike and wave complex was also observed after combined treatment with enoxacin (i. vent.) and ferubinac ethyl (i.v. or i. vent.) in association with hyperactivity and jumping and violent convulsion. Nicardipine remarkably inhibited epileptic seizures induced by combined treatment with enoxacin (p.o.) and ferubinac ethyl (i.v.). It is concluded that simultaneous treatment with enoxacin and ferubinac ethyl produced epileptogenic activity when injected intraventricularly, and nicardipine inhibited convulsions induced by combined use of enoxacin (p.o.) and ferubinac ethyl (i.v.).