There has been a consistent gap in understanding how TNF-α neutralization affects the disease state of arthritis patients so rapidly, considering that joint inflammation in rheumatoid arthritis is a chronic condition with structural changes. We thus hypothesized that neutralization of TNF-α acts through the CNS before directly affecting joint inflammation. Through use of functional MRI (fMRI), we demonstrate that within 24 h after neutralization of TNF-α, nociceptive CNS activity in the thalamus and somatosensoric cortex, but also the activation of the limbic system, is blocked. Brain areas showing blood-oxygen level-dependent signals, a validated method to assess neuronal activity elicited by pain, were significantly reduced as early as 24 h after an infusion of a monoclonal antibody to TNF-α. In contrast, clinical and laboratory markers of inflammation, such as joint swelling and acute phase reactants, were not affected by anti-TNF-α at these early time points. Moreover, arthritic mice overexpressing human TNF-α showed an altered pain behavior and a more intensive, widespread, and prolonged brain activity upon nociceptive stimuli compared with wild-type mice. Similar to humans, these changes, as well as the rewiring of CNS activity resulting in tight clustering in the thalamus, were rapidly reversed after neutralization of TNF-α. These results suggest that neutralization of TNF-α affects nociceptive brain activity in the context of arthritis, long before it achieves antiinflammatory effects in the joints.cytokines | antiinflammatory therapy

1 Docosahexaenoic acid (DHA) and arachidonic acid (AA), polyunsaturated fatty acids (PUFAs), are important for central nervous system function during development and in various pathological states. Astrocytes are involved in the biosynthesis of PUFAs in neuronal tissue. Here, we investigated the mechanism of DHA and AA release in cultured rat brain astrocytes. 2 Primary astrocytes were cultured under standard conditions and prelabeled with [ 14 C]DHA or with [ 3 H]AA. Adenosine 5 0 -triphosphate (ATP) (20 mm applied for 15 min), the P2Y receptor agonist, stimulates release of both DHA (289% of control) and AA (266% of control) from astrocytes. DHA release stimulated by ATP is mediated by Ca 2 þ -independent phospholipase A 2 (iPLA 2 ), since it is blocked by the selective iPLA 2 inhibitor 4-bromoenol lactone (BEL, 5 mm) and is not affected either by removal of Ca 2 þ from extracellular medium or by suppression of intracellular Ca 2 þ release through PLC inhibitor (U73122, 5 mm). 3 AA release, on the other hand, which is stimulated by ATP, is attributed to Ca 2 þ -dependent cytosolic PLA 2 (cPLA 2 ). AA release is abolished by U73122 and, by removal of extracellular Ca 2 þ , is insensitive to BEL and can be selectively suppressed by methyl arachidonyl fluorophosphonate (3 mm), a general inhibitor of intracellular PLA 2 s. 4 Western blot analysis confirms the presence in rat brain astrocytes of 85 kDa cPLA 2 and 40 kDa protein reactive to iPLA 2 antibodies. 5 The influence of cAMP on regulation of PUFA release was investigated. Release of DHA is strongly amplified by the adenylyl cyclase activator forskolin (10 mm), and by the protein kinase A (PKA) activator dibutyryl-cAMP (1 mm). In contrast, release of AA is not affected by forskolin or dibutyryl-cAMP, but is almost completely blocked by 2,3-dideoxyadenosine (20 mm) and inhibited by 34% by H89 (10 mm), inhibitors of adenylyl cyclase and PKA, respectively. 6 Other neuromediators, such as bradykinin, glutamate and thrombin, stimulate release of DHA and AA, which is comparable to the release stimulated by ATP. 7 Different sensitivities of iPLA 2 and cPLA 2 to Ca 2 þ and cAMP reveal new pathways for the regulation of fatty acid release and reflect the significance of astrocytes in control of DHA and AA metabolism under normal and pathological conditions in brain.

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Cilia are cell organelles that play important roles in cell motility, sensory and developmental functions and are involved in a range of human diseases, known as ciliopathies. Here, we search for novel human genes related to cilia using a strategy that exploits the previously reported tendency of cell type-specific genes to be coexpressed in the transcriptome of complex tissues. Gene coexpression networks were constructed using the noise-resistant WGCNA algorithm in 12 publicly available microarray datasets from human tissues rich in motile cilia: airways, fallopian tubes and brain. A cilia-related coexpression module was detected in 10 out of the 12 datasets. A consensus analysis of this module's gene composition recapitulated 297 known and predicted 74 novel cilia-related genes. 82% of the novel candidates were supported by tissue-specificity expression data from GEO and/or proteomic data from the Human Protein Atlas. The novel findings included a set of genes (DCDC2, DYX1C1, KIAA0319) related to a neurological disease dyslexia suggesting their potential involvement in ciliary functions. Furthermore, we searched for differences in gene composition of the ciliary module between the tissues. A multidrug-and-toxin extrusion transporter MATE2 (SLC47A2) was found as a brain-specific central gene in the ciliary module. We confirm the localization of MATE2 in cilia by immunofluorescence staining using MDCK cells as a model. While MATE2 has previously gained attention as a pharmacologically relevant transporter, its potential relation to cilia is suggested for the first time. Taken together, our large-scale analysis of gene coexpression networks identifies novel genes related to human cell cilia.

Gliomas are primary brain tumors with high mortality and heterogeneous biology that is insufficiently understood. In this study, we performed a systematic analysis of the intrinsic organization of complex glioma transcriptome to gain deeper knowledge of the tumor biology. Gene coexpression relationships were explored in 790 glioma samples from 5 published patient cohorts treated at different institutions. We identified 20 coexpression modules that were common to all the data sets and associated with proliferation, angiogenesis, hypoxia, immune response, genomic alterations, cell differentiation phenotypes, and other features inherent to glial tumors. A collection of high-quality signatures for the respective processes was obtained using cross-data set summarization of the modules' gene composition. Individual modules were found to be organized into higher order coexpression groups, the two largest of them associated with glioblastoma and oligodendroglioma, respectively. We identified a novel prognostic gene expression signature (185 genes) linked to a proastrocytic pattern of tumor cell differentiation. This "proastrocytic" signature was associated with long survival and defined a subgroup of the previously established "proneural" class of gliomas. A strong negative correlation between proastrocytic and proneural markers across differentiated tumors underscored the distinction between these subtypes of glioma. Interestingly, one further novel signature in glioma was identified that was associated with EGFR (epidermal growth factor receptor) gene amplification and suggested that EGF signaling in glioma may be a subject to regulation by Sprouty family proteins. In summary, this integrated analysis of the glioma transcriptome provided several novel insights into molecular heterogeneity and pathogenesis of glial tumors.

Peroxisome proliferator-activated receptor (PPAR) transcription factors are pharmaceutical drug targets for treating diabetes, atherosclerosis, and inflammatory degenerative diseases. The possible mechanism of interaction between the three PPAR isotypes (␣, ␤/␦, and ␥) is not yet clear. However, this is important both for understanding transcription factor regulation and for the development of new drugs. The present study was designed to compare the effects of combinations of synthetic agonists of PPAR␣ [2-[4-[2-[4-cyclohexylbutyl (cyclohexylcarbamoyl), and PPAR␥ (rosiglitazone, ciglitazone) on inflammatory gene regulation in rat primary astrocytes. We measured cyclooxygenase-2 (COX-2) expression and prostaglandin E 2 synthesis in lipopolysaccharide (LPS)-stimulated cells. PPAR␣, PPAR␤/␦, and PPAR␥ knockdown models served to delineate the contribution of each PPAR isotype. Thiazolidinediones enhanced the LPSinduced COX-2 expression via PPAR␥-dependent pathway, whereas L-165041 and GW7647 had no influence. However, the addition of L-165041 potentiated the effect of PPAR␥ activation through PPAR␤/␦-dependent mechanism. On the contrary, PPAR␣ activation (GW7647) suppressed the effect of the combined L-165041/rosiglitazone application. The mechanism of the interplay arising from combined applications of PPAR agonists involves changes in PPAR expression levels. A PPAR␤/␦ overexpression model confirmed that PPAR␤/␦ expression level is the point at which PPAR␥ and PPAR␣ pathways converge in control of COX-2 gene expression. Thus, we discovered that in primary astrocytes, PPAR␥ has a positive influence and PPAR␣ has a negative influence on PPAR␤/␦ expression and activity. A positive/negative-feedback loop is formed by PPAR␤/␦-dependent increase in PPAR␣ expression level. These findings elucidate a novel principle of regulation in the signaling by synthetic PPAR agonists that involves modulating the interaction between PPAR␣, -␤/␦, and -␥ isoforms on the level of their expression.

Various diseases of the central nervous system are characterized by induction of inflammatory events, which involve formation of prostaglandins. Production of prostaglandins is regulated by activity of phospholipases A(2) and cyclooxygenases. These enzymes release the prostaglandin precursor, the n-6 polyunsaturated fatty acid, arachidonic acid and oxidize it into prostaglandin H(2). Docosahexaenoic acid, which belongs to the n-3 class of polyunsaturated fatty acids, was shown to reduce production of prostaglandins after in vivo and in vitro administration. Nevertheless, the fact that in brain tissue cellular phospholipids naturally have a uniquely high content of docosahexaenoic acid was ignored so far in studies of prostaglandin formation in brain tissue. We consider the following possibilities: docosahexaenoic acid might attenuate production of prostaglandins by direct inhibition of cyclooxygenases. Such inhibition was found with the isolated enzyme. Another possibility, which has been already shown is reduction of expression of inducible cyclooxygenase-2. Additionally, we propose that docosahexaenoic acid could influence intracellular Ca(2+) signaling, which results in changes of activity of Ca(2+)-dependent phospholipase A(2), hence reducing the amount of arachidonic acid available for prostaglandin production. Astrocytes, the main type of glial cells in the brain control the release of arachidonic acid, docosahexaenoic acid and the formation of prostaglandins. Our recently obtained data revealed that the release of arachidonic and docosahexaenoic acids in astrocytes is controlled by different isoforms of phospholipase A(2), i.e. Ca(2+)-dependent phospholipase A(2) and Ca(2+)-independent phospholipase A(2), respectively. Moreover, the release of arachidonic and docosahexaenoic acids is differently regulated through Ca(2+)- and cAMP-dependent signal transduction pathways. Based on analysis of the current literature and our own data we put forward the hypothesis that Ca(2+)-independent phospholipase A(2) and docosahexaenoic acid are promising targets for treatment of inflammatory related disorders in brain. We suggest that Ca(2+)-independent phospholipase A(2) and docosahexaenoic acid might be crucially involved in brain-specific regulation of prostaglandins.

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