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The antipsoriatic dimethylfumarate (DMF) has been anecdotically reported to reduce asthma symptoms and to improve quality of life of asthma patients. DMF decreases the expression of proinflammatory mediators by inhibiting the transcription factor NF-KB and might therefore be of interest for the therapy of inflammatory lung diseases. In this study, we determined the effect of DMF on platelet-derived growth factor (PDGF)-BB- and TNF[alpha]-induced asthma-relevant cytokines and NF-KB activation by primary human asthmatic and nonasthmatic airway smooth muscle ceils (ASMC). Confluent nonasthmatic and asthmatic ASMC were incubated with DMF (0.1-100 [micro]M) and/or dexamethasone (0.0001-0.1 [micro]M), NF-KB p65 siRNA (100 nM), the NF-[kappa]B inhibitor helenalin (1 [micro]M) before stimulation with PDGF-BB or TNF[alpha] (10 ng/ml). Cytokine release was measured by ELISA. NF-[kappa]B, mitogen and stress-activated kinase (MSK-1 ), and CREB activation was determined by immunoblotting and EMSA. TNF[alpha]-induced eotaxin, RANTES, and IL-6 as well as PDGF-BB-induced IL-6 expression was inhibited by DMF and by dexamethasone from asthmatic and nonasthmatic ASMC, but the combination of both drugs showed no glucocorticoid sparing effect in either of the two groups. NF-[kappa]B p65 siRNA and/or the NF-KB inhibitor helenalin reduced PDGF-BB- and TNF[alpha]-induced cytokine expression, suggesting the involvement of NF-[kappa]B signaling. DMF inhibited TNF[alpha]-induced NF-[kappa]B p65 phosphorylation, NF-[kappa]B nuclear entry, and NF-[kappa]B-DNA complex formation, whereas PDGF-BB appeared not to activate NF-[kappa]B within 60 min. Both stimuli induced the phosphorylation of MSK-1, NF-[kappa]B p65 at Ser276, and CREB, and all were inhibited by DMF. These data suggest that DMF downregulates cytokine secretion not only by inhibiting NF-[kappa]B but a wider range of NF-[kappa]B-linked signaling proteins, which may explain its potential beneficial effect in asthma. mitogen and stress-activated kinase; CREB; DMF anti-inflammatory action; chronic lung inflammation; DMF steroid sparing effect

Seidel P, Merfort I, Hughes JM, Oliver BG, Tamm M, Roth M. Dimethylfumarate inhibits NF-κB function at multiple levels to limit airway smooth muscle cell cytokine secretion. Am J Physiol Lung Cell Mo! Physiol 297: L326-L339, 2009. First published May 22, 2009; doi:10.1152/ajplung.90624.2008.-The antipsoriatic dimethylfumarate (DMF) has been anecdotically reported to reduce asthma symptoms and to improve quality of life of asthma patients. DMF decreases the expression of proinflammatory mediators by inhibiting the transcription factor NF-κB and might therefore be of interest for the therapy of inflammatory lung diseases. In this study, we determined the effect of DMF on platelet-derived growth factor (PDGF)-BBand TNFα-induced asthma-relevant cytokines and NF-κB activation by primary human asthmatic and nonasthmatic airway smooth muscle cells (ASMC). Confluent nonasthmatic and asthmatic ASMC were incubated with DMF (0.1-100 μM) and/or dexamethasone (0.00010.1 μM), NF-κB p65 siRNA (100 nM), the NF-κB inhibitor helenalin (1 μM) before stimulation with PDGF-BB or TNFa (10 ng/ml). Cytokine release was measured by ELISA. NF-κB, mitogen and stress-activated kinase (MSK-1), and CREB activation was determined by immunoblotting and EMSA. TNFα-induced eotaxin, RANTES, and IL-6 as well as PDGF-BB-induced IL-6 expression was inhibited by DMF and by dexamethasone from asthmatic and nonasthmatic ASMC, but the combination of both drugs showed no glucocorticoid sparing effect in either of the two groups. NF-κB p65 siRNA and/or the NF-κB inhibitor helenalin reduced PDGF-BBand TNFα-induced cytokine expression, suggesting the involvement of NF-κB signaling. DMF inhibited TNFα-induced NF-κB p65 phosphorylation, NF-κB nuclear entry, and NF-κB-DNA complex formation, whereas PDGF-BB appeared not to activate NF-κB within 60 mm. Both stimuli induced the phosphorylation of MSKI, NF-κB p65 at Ser276, and CREB, and all were inhibited by DMF. These data suggest that DMF downregulates cytokine Secretion not only by inhibiting NF-κB but a wider range of NF-κB-linked signaling proteins, which may explain its potential beneficial effect in asthma. [ABSTRACT FROM AUTHOR]

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-κB and Wnt/β-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFβ was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent β-catenin was monitored in response to the inhibition of GSK3β. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFβ-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach. [ABSTRACT FROM AUTHOR]

Abstract Background Cytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver. In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization. Results The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately up-regulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1beta proposed an "IL-1beta-like effect" of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase activity of the up-regulated Zc3h12a have to be considered. Conclusions Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signaling pathway. Gene expression profile suggests IL-17 alone and in concert with TNF-alpha a role in sustaining liver inflammatory processes. IL-17 might exceed this function by RNA stabilization.

Uncaria tomentosa Willd DC or Uña de gato (UG) is a Peruvian plant with antiinflammatory properties. We use a hidroalcoholic extract of UG enriched to 5.61 % in their content of total alkaloids, quantified by HPLC. We evaluated the potential acute toxicity and the anti-inflammatory activity in vivo using mice BALB/c and measured the inhibition of inflammation for the model of the oedema of the paw induced by carragenine (MPIC) and the production of TNF-alpha in the model of air pouch (MAP). In addition we used the electrophoretic mobility shift assay (EMSA) to quantify the inhibition of the transcription factor NF-kB and the inhibition capacity over cycleoxygenase (COX) 1 and 2 was evaluated by ELISA. The hidroalcoholic extract of UG showed a very low toxicity with LD50 located in the practically non-toxic level. A dose-dependent anti-inflammatory activity was demonstrated by the MPIC, which was higher than indomethacin at highest doses. In MAP the inhibition of TNF-alpha was 54.21% using a doses of 250 mg/kg of UG. In the inflammatory exudate the leukocytes diminished significantly to the third part of the group that did not receive treatment (UG 250 mg/Kg, 1.875 +/- 0.239 x 10 [6] cell vs control, 6.167 +/- 0.629 x 10 [6] cell). A moderate inhibitory activity of the activation of NF-kB was evidenced, as well as a discreet inhibition of the enzymes COX, predominantly over COX-2. This study demonstrates the activity anti-inflammatory of the hydro-alcoholic extract of UG and also outlines its mechanism of action through complementary molecular actions producing finally an anti-inflammatory activity. [ABSTRACT FROM AUTHOR]