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DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50–80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.
Background: After focal neuronal injury the endocannabinioid system becomes activated and protects or harms neurons depending on cannabinoid derivates and receptor subtypes. Endocannabinoids (eCBs) play a central role in controlling local responses and influencing neural plasticity and survival. However, little is known about the functional relevance of eCBs in long-range projection damage as observed in stroke or spinal cord injury (SCI).
Methods: In rat organotypic entorhino-hippocampal slice cultures (OHSC) as a relevant and suitable model for investigating projection fibers in the CNS we performed perforant pathway transection (PPT) and subsequently analyzed the spatial and temporal dynamics of eCB levels. This approach allows proper distinction of responses in originating neurons (entorhinal cortex), areas of deafferentiation/anterograde axonal degeneration (dentate gyrus) and putative changes in more distant but synaptically connected subfields (cornu ammonis (CA) 1 region).
Results: Using LC-MS/MS, we measured a strong increase in arachidonoylethanolamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) levels in the denervation zone (dentate gyrus) 24 hours post lesion (hpl), whereas entorhinal cortex and CA1 region exhibited little if any changes. NAPE-PLD, responsible for biosynthesis of eCBs, was increased early, whereas FAAH, a catabolizing enzyme, was up-regulated 48hpl.
Conclusion: Neuronal damage as assessed by transection of long-range projections apparently provides a strong time-dependent and area-confined signal for de novo synthesis of eCB, presumably to restrict neuronal damage. The present data underlines the importance of activation of the eCB system in CNS pathologies and identifies a novel site-specific intrinsic regulation of eCBs after long-range projection damage.
Accumulating evidence indicates that increased generation of reactive oxygen species (ROS) contributes to the development of exaggerated pain hypersensitivity during persistent pain. In the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin C and vitamin E in mouse models of inflammatory and neuropathic pain. We show that systemic administration of a combination of vitamins C and E inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by Complete Freund's Adjuvant. In contrast, vitamin C or vitamin E given alone failed to affect the nociceptive behavior in all tested models. The attenuated neuropathic pain behavior induced by the vitamin C and E combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. Moreover, the vitamin C and E combination ameliorated the allodynia induced by an intrathecally delivered ROS donor. Our results suggest that administration of vitamins C and E in combination may exert synergistic antinociceptive effects, and further indicate that ROS essentially contribute to nociceptive processing in special pain states.
BACKGROUND: The endocannabinoid 2-arachidonoyl glycerol (2-AG) acts as a retrograde messenger and modulates synaptic signaling e. g. in the hippocampus. 2-AG also exerts neuroprotective effects under pathological situations. To better understand the mechanism beyond physiological signaling we used Organotypic Entorhino-Hippocampal Slice Cultures (OHSC) and investigated the temporal regulation of 2-AG in different cell subsets during excitotoxic lesion and dendritic lesion of long range projections in the enthorhinal cortex (EC), dentate gyrus (DG) and the cornu ammonis region 1 (CA1).
RESULTS: 2-AG levels were elevated 24 h after excitotoxic lesion in CA1 and DG (but not EC) and 24 h after perforant pathway transection (PPT) in the DG only. After PPT diacylglycerol lipase alpha (DAGL) protein, the synthesizing enzyme of 2-AG was decreased when Dagl mRNA expression and 2-AG levels were enhanced. In contrast to DAGL, the 2-AG hydrolyzing enzyme monoacylglycerol lipase (MAGL) showed no alterations in total protein and mRNA expression after PPT in OHSC. MAGL immunoreaction underwent a redistribution after PPT and excitotoxic lesion since MAGL IR disappeared in astrocytes of lesioned OHSC. DAGL and MAGL immunoreactions were not detectable in microglia at all investigated time points. Thus, induction of the neuroprotective endocannabinoid 2-AG might be generally accomplished by down-regulation of MAGL in astrocytes after neuronal lesions.
CONCLUSION: Increase in 2-AG levels during secondary neuronal damage reflects a general neuroprotective mechanism since it occurred independently in both different lesion models. This intrinsic up-regulation of 2-AG is synergistically controlled by DAGL and MAGL in neurons and astrocytes and thus represents a protective system for neurons that is involved in dendritic reorganisation.
Ultraviolet-B (UVB)-induced inflammation produces a dose-dependent mechanical and thermal hyperalgesia in both humans and rats, most likely via inflammatory mediators acting at the site of injury. Previous work has shown that the gene expression of cytokines and chemokines is positively correlated between species and that these factors can contribute to UVB-induced pain. In order to investigate other potential pain mediators in this model we used RNA-seq to perform genome-wide transcriptional profiling in both human and rat skin at the peak of hyperalgesia. In addition we have also measured transcriptional changes in the L4 and L5 DRG of the rat model. Our data show that UVB irradiation produces a large number of transcriptional changes in the skin: 2186 and 3888 genes are significantly dysregulated in human and rat skin, respectively. The most highly up-regulated genes in human skin feature those encoding cytokines (IL6 and IL24), chemokines (CCL3, CCL20, CXCL1, CXCL2, CXCL3 and CXCL5), the prostanoid synthesising enzyme COX-2 and members of the keratin gene family. Overall there was a strong positive and significant correlation in gene expression between the human and rat (R = 0.8022). In contrast to the skin, only 39 genes were significantly dysregulated in the rat L4 and L5 DRGs, the majority of which had small fold change values. Amongst the most up-regulated genes in DRG were REG3B, CCL2 and VGF. Overall, our data shows that numerous genes were up-regulated in UVB irradiated skin at the peak of hyperalgesia in both human and rats. Many of the top up-regulated genes were cytokines and chemokines, highlighting again their potential as pain mediators. However many other genes were also up-regulated and might play a role in UVB-induced hyperalgesia. In addition, the strong gene expression correlation between species re-emphasises the value of the UVB model as translational tool to study inflammatory pain.
Oxaliplatin is a third-generation platinum-based anticancer drug that is widely used as first-line treatment for colorectal carcinoma. Patients treated with oxaliplatin develop an acute peripheral pain several hours after treatment, mostly characterized by cold allodynia as well as a long-term chronic neuropathy. These two phenomena seem to be causally connected. However, the underlying mechanisms that trigger the acute peripheral pain are still poorly understood. Here we show that the activity of the transient receptor potential melastatin 8 (TRPM8) channel but not the activity of any other member of the TRP channel family is transiently increased 1 h after oxaliplatin treatment and decreased 24 h after oxaliplatin treatment. Mechanistically, this is connected with activation of the phospholipase C (PLC) pathway and depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) after oxaliplatin treatment. Inhibition of the PLC pathway can reverse the decreased TRPM8 activity as well as the decreased PIP2-concentrations after oxaliplatin treatment. In summary, these results point out transient changes in TRPM8 activity early after oxaliplatin treatment and a later occurring TRPM8 channel desensitization in primary sensory neurons. These mechanisms may explain the transient cold allodynia after oxaliplatin treatment and highlight an important role of TRPM8 in oxaliplatin-induced acute and neuropathic pain.
Background: The quantification of global DNA methylation has been established in epigenetic screening. As more practicable alternatives to the HPLC-based gold standard, the methylation analysis of CpG islands in repeatable elements (LINE-1) and the luminometric methylation assay (LUMA) of overall 5-methylcytosine content in “CCGG” recognition sites are most widely used. Both methods are applied as virtually equivalent, despite the hints that their results only partly agree. This triggered the present agreement assessments.
Results: Three different human cell types (cultured MCF7 and SHSY5Y cell lines treated with different chemical modulators of DNA methylation and whole blood drawn from pain patients and healthy volunteers) were submitted to the global DNA methylation assays employing LINE-1 or LUMA-based pyrosequencing measurements. The agreement between the two bioassays was assessed using generally accepted approaches to the statistics for laboratory method comparison studies. Although global DNA methylation levels measured by the two methods correlated, five different lines of statistical evidence consistently rejected the assumption of complete agreement. Specifically, a bias was observed between the two methods. In addition, both the magnitude and direction of bias were tissue-dependent. Interassay differences could be grouped based on Bayesian statistics, and these groups allowed in turn to re-identify the originating tissue.
Conclusions: Although providing partly correlated measurements of DNA methylation, interchangeability of the quantitative results obtained with LINE-1 and LUMA was jeopardized by a consistent bias between the results. Moreover, the present analyses strongly indicate a tissue specificity of the differences between the two methods.
Background: R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. Methodology/Principal Findings: We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPAR gamma and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. Conclusion: Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.
Monoclonal antibodies (mAb) are promising therapeutics in multiple sclerosis and multiple new candidates have been developed, hence increasing the need for some agreement for preclinical mAb studies. We systematically analyzed publications of experimental autoimmune encephalomyelitis (EAE) studies showing effects of monoclonal antibodies. A PubMed search retrieved 570 records, out of which 122 studies with 253 experiments were eligible based on experimental design, number of animals and presentation of time courses of EAE scores. Analysis of EAE models, treatment schedules, single and total doses, routes of administration, and onset of treatment from pre-immunization up to 35 days after immunization revealed high heterogeneity. Total doses ranged from 0.1 to 360 mg/kg for observation times of up to 35 days after immunization. About half of experiments (142/253) used total doses of 10–70 mg/kg. Employing this range, we tested anti-Itga4 as a reference mAb at varying schedules and got no, mild or substantial EAE-score reductions, depending on the mouse strain and onset of the treatment. The result agrees with the range of outcomes achieved in 10 reported anti-Itga4 experiments. Studies comparing low and high doses of various mAbs or early vs. late onset of treatment did not reveal dose-effect or timing-effect associations, with a tendency towards better outcomes with preventive treatments starting within the first week after immunization. The systematic comparison allows for extraction of some “common” design characteristics, which may be helpful to further assess the efficacy of mAbs and role of specific targets in preclinical models of multiple sclerosis.
Based on accumulating evidence of a role of lipid signaling in many physiological and pathophysiological processes including psychiatric diseases, the present data driven analysis was designed to gather information needed to develop a prospective biomarker, using a targeted lipidomics approach covering different lipid mediators. Using unsupervised methods of data structure detection, implemented as hierarchal clustering, emergent self-organizing maps of neuronal networks, and principal component analysis, a cluster structure was found in the input data space comprising plasma concentrations of d = 35 different lipid-markers of various classes acquired in n = 94 subjects with the clinical diagnoses depression, bipolar disorder, ADHD, dementia, or in healthy controls. The structure separated patients with dementia from the other clinical groups, indicating that dementia is associated with a distinct lipid mediator plasma concentrations pattern possibly providing a basis for a future biomarker. This hypothesis was subsequently assessed using supervised machine-learning methods, implemented as random forests or principal component analysis followed by computed ABC analysis used for feature selection, and as random forests, k-nearest neighbors, support vector machines, multilayer perceptron, and naïve Bayesian classifiers to estimate whether the selected lipid mediators provide sufficient information that the diagnosis of dementia can be established at a higher accuracy than by guessing. This succeeded using a set of d = 7 markers comprising GluCerC16:0, Cer24:0, Cer20:0, Cer16:0, Cer24:1, C16 sphinganine, and LacCerC16:0, at an accuracy of 77%. By contrast, using random lipid markers reduced the diagnostic accuracy to values of 65% or less, whereas training the algorithms with randomly permuted data was followed by complete failure to diagnose dementia, emphasizing that the selected lipid mediators were display a particular pattern in this disease possibly qualifying as biomarkers.
Doping ist ein Thema, das den modernen Leistungssport – und nicht nur diesen – seit jeher begleitet. Immer wieder werden Sportler – oft noch nach Jahren – überführt, illegale Substanzen zur Leistungssteigerung eingenommen zu haben. Der Landessportbund Hessen e. V. hat im Sommer 2013 Professor Dr. Dr. Gerd Geißlinger zum Anti-Doping-Beauftragten berufen. Für den UniReport hat Dr. Beate Meichsner mit dem Direktor des Instituts für Klinische Pharmakologie am Klinikum der Goethe-Universität über die damit verbundenen Aufgaben, Ziele und
Möglichkeiten gesprochen.
Patient therapy is based mainly on a combination of diagnosis, suitable monitoring or support devices and drug treatment and is usually employed for a pre-existing disease condition. Therapy remains predominantly symptom-based, although it is increasingly clear that individual treatment is possible and beneficial. However, reasonable precision medicine can only be realized with the coordinated use of diagnostics, devices and drugs in combination with extensive databases (4Ds), an approach that has not yet found sufficient implementation. The practical combination of 4Ds in health care is progressing, but several obstacles still hamper their extended use in precision medicine.
Around 20% of the American population have chronic pain and estimates in other Western countries report similar numbers. This represents a major challenge for global health care systems. Additional problems for the treatment of chronic and persistent pain are the comparably low efficacy of existing therapies, the failure to translate effects observed in preclinical pain models to human patients and related setbacks in clinical trials from previous attempts to develop novel analgesics. Drug repurposing offers an alternative approach to identify novel analgesics as it can bypass various steps of classical drug development. In recent years, several approved drugs were attributed analgesic properties. Here, we review available data and discuss recent findings suggesting that the approved drugs minocycline, fingolimod, pioglitazone, nilotinib, telmisartan, and others, which were originally developed for the treatment of different pathologies, can have analgesic, antihyperalgesic, or neuroprotective effects in preclinical and clinical models of inflammatory or neuropathic pain. For our analysis, we subdivide the drugs into substances that can target neuroinflammation or substances that can act on peripheral sensory neurons, and highlight the proposed mechanisms. Finally, we discuss the merits and challenges of drug repurposing for the development of novel analgesics.
Class I and II histone deacetylases (HDAC) are considered important regulators of immunity and inflammation. Modulation of HDAC expression and activity is associated with altered inflammatory responses but reports are controversial and the specific impact of single HDACs is not clear. We examined class I and II HDACs in TLR-4 signaling pathways in murine macrophages with a focus on IκB kinase epsilon (IKKε) which has not been investigated in this context before. Therefore, we applied the pan-HDAC inhibitors (HDACi) trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) as well as HDAC-specific siRNA. Administration of HDACi reduced HDAC activity and decreased expression of IKKε although its acetylation was increased. Other pro-inflammatory genes (IL-1β, iNOS, TNFα) also decreased while COX-2 expression increased. HDAC 2, 3 and 4, respectively, might be involved in IKKε and iNOS downregulation with potential participation of NF-κB transcription factor inhibition. Suppression of HDAC 1–3, activation of NF-κB and RNA stabilization mechanisms might contribute to increased COX-2 expression. In conclusion, our results indicate that TSA and SAHA exert a number of histone- and HDAC-independent functions. Furthermore, the data show that different HDAC enzymes fulfill different functions in macrophages and might lead to both pro- and anti-inflammatory effects which have to be considered in therapeutic approaches.
Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.
Lysophosphatidic acid (LPA) is a synaptic phospholipid, which regulates cortical excitation/inhibition (E/I) balance and controls sensory information processing in mice and man. Altered synaptic LPA signaling was shown to be associated with psychiatric disorders. Here, we show that the LPA-synthesizing enzyme autotaxin (ATX) is expressed in the astrocytic compartment of excitatory synapses and modulates glutamatergic transmission. In astrocytes, ATX is sorted toward fine astrocytic processes and transported to excitatory but not inhibitory synapses. This ATX sorting, as well as the enzymatic activity of astrocyte-derived ATX are dynamically regulated by neuronal activity via astrocytic glutamate receptors. Pharmacological and genetic ATX inhibition both rescued schizophrenia-related hyperexcitability syndromes caused by altered bioactive lipid signaling in two genetic mouse models for psychiatric disorders. Interestingly, ATX inhibition did not affect naive animals. However, as our data suggested that pharmacological ATX inhibition is a general method to reverse cortical excitability, we applied ATX inhibition in a ketamine model of schizophrenia and rescued thereby the electrophysiological and behavioral schizophrenia-like phenotype. Our data show that astrocytic ATX is a novel modulator of glutamatergic transmission and that targeting ATX might be a versatile strategy for a novel drug therapy to treat cortical hyperexcitability in psychiatric disorders.
Pathophysiological role of prostanoids in coagulation of the portal venous system in liver cirrhosis
(2019)
Background: Prostanoids are important regulators of platelet aggregation and thrombotic arterial diseases. Their involvement in the development of portal vein thrombosis, frequent in decompensated liver cirrhosis, is still not investigated.
Methods: Therefore, we used pro-thrombotic venous milieu generation by bare metal stent transjugular intrahepatic portosystemic shunt insertion, to study the role of prostanoids in decompensated liver cirrhosis. Here, 89 patients receiving transjugular intrahepatic portosystemic shunt insertion were included in the study, and baseline levels of thromboxane B2, prostaglandin D2 and prostaglandin E2 were measured in the portal and the hepatic vein.
Results: While the hepatic vein contained higher levels of thromboxane B2 than the portal vein, levels of prostaglandin E2 and D2 were higher in the portal vein (all P<0.0001). Baseline concentrations of thromboxane B2 in the portal vein were independently associated with an increase of portal hepatic venous pressure gradient during short term follow-up, as an indirect sign of thrombogenic potential (multivariable P = 0.004). Moreover, severity of liver disease was inversely correlated with portal as well as hepatic vein levels of prostaglandin D2 and E2 (all P<0.0001).
Conclusions: Elevated portal venous thromboxane B2 concentrations are possibly associated with the extent of thrombogenic potential in patients with decompensated liver cirrhosis.
Trial registration: ClinicalTrials.gov identifier: NCT03584204.
The SARS-CoV-2 pandemic has challenged researchers at a global scale. The scientific community’s massive response has resulted in a flood of experiments, analyses, hypotheses, and publications, especially in the field of drug repurposing. However, many of the proposed therapeutic compounds obtained from SARS-CoV-2 specific assays are not in agreement and thus demonstrate the need for a singular source of COVID-19 related information from which a rational selection of drug repurposing candidates can be made. In this paper, we present the COVID-19 PHARMACOME, a comprehensive drug-target-mechanism graph generated from a compilation of 10 separate disease maps and sources of experimental data focused on SARS-CoV-2 / COVID-19 pathophysiology. By applying our systematic approach, we were able to predict the synergistic effect of specific drug pairs, such as Remdesivir and Thioguanosine or Nelfinavir and Raloxifene, on SARS-CoV-2 infection. Experimental validation of our results demonstrate that our graph can be used to not only explore the involved mechanistic pathways, but also to identify novel combinations of drug repurposing candidates.
Tissue injury and inflammation may result in chronic pain, a severe debilitating disease that is associated with great impairment of quality of life. An increasing body of evidence indicates that members of the Rab family of small GTPases contribute to pain processing; however, their specific functions remain poorly understood. Here, we found using immunofluorescence staining and in situ hybridization that the small GTPase Rab27a is highly expressed in sensory neurons and in the superficial dorsal horn of the spinal cord of mice. Rab27a mutant mice, which carry a single-nucleotide missense mutation of Rab27a leading to the expression of a nonfunctional protein, show reduced mechanical hyperalgesia and spontaneous pain behavior in inflammatory pain models, while their responses to acute noxious mechanical and thermal stimuli is not affected. Our study uncovers a previously unrecognized function of Rab27a in the processing of persistent inflammatory pain in mice.