Gut microbiota composition during a 12-week intervention with delayed-release dimethyl fumarate in multiple sclerosis - a pilot trial.

INTRODUCTION: Patients with multiple sclerosis may have a distinct gut microbiota profile. Delayed-release dimethyl fumarate is an orally administered drug for relapsing-remitting multiple sclerosis, which has been associated with gastrointestinal side-effects in some patients. OBJECTIVES: The purpose of this study was to determine if dimethyl fumarate alters the abundance and diversity of commensal gut bacteria, and if these changes are associated with gastrointestinal side-effects. METHODS: Thirty-six patients with relapsing-remitting multiple sclerosis received either dimethyl fumarate (n = 27) or an injectable multiple sclerosis disease-modifying therapy (glatiramer acetate or interferons, n = 9) for 12 weeks. Stool samples were collected at baseline, two and 12 weeks. We included 165 healthy individuals as controls. RESULTS: At baseline, 16 microbial genera were altered in multiple sclerosis patients compared with healthy controls. In the dimethyl fumarate-treated patients (n = 21) we observed a trend of reduced Actinobacteria (p = 0.03, QFDR = 0.24) at two weeks, mainly driven by Bifidobacterium (p = 0.06, QFDR = 0.69). At 12 weeks, we observed an increased abundance of Firmicutes (p = 0.02, QFDR = 0.09), mostly driven by Faecalibacterium (p = 0.01, QFDR = 0.48). CONCLUSIONS: This pilot study did not detect a major effect of dimethyl fumarate on the gut microbiota composition, but we observed a trend towards normalization of the low abundance of butyrate-producing Faecalibacterium after 12 weeks treatment. The study was underpowered to link microbiota to gastrointestinal symptoms.

Differential transcriptional profiling of damaged and intact adjacent dorsal root ganglia neurons in neuropathic pain.

Neuropathic pain, caused by a lesion in the somatosensory system, is a severely impairing mostly chronic disease. While its underlying molecular mechanisms are not thoroughly understood, neuroimmune interactions as well as changes in the pain pathway such as sensitization of nociceptors have been implicated. It has been shown that not only are different cell types involved in generation and maintenance of neuropathic pain, like neurons, immune and glial cells, but, also, intact adjacent neurons are relevant to the process. Here, we describe an experimental approach to discriminate damaged from intact adjacent neurons in the same dorsal root ganglion (DRG) using differential fluorescent neuronal labelling and fluorescence-activated cell sorting (FACS). Two fluorescent tracers, Fluoroemerald (FE) and 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI), were used, whose properties allow us to distinguish between damaged and intact neurons. Subsequent sorting permitted transcriptional analysis of both groups. Results and qPCR validation show a strong regulation in damaged neurons versus contralateral controls as well as a moderate regulation in adjacent neurons. Data for damaged neurons reveal an mRNA expression pattern consistent with established upregulated genes like galanin, which supports our approach. Moreover, novel genes were found strongly regulated such as corticotropin-releasing hormone (CRH), providing novel targets for further research. Differential fluorescent neuronal labelling and sorting allows for a clear distinction between primarily damaged neuropathic neurons and "bystanders," thereby facilitating a more detailed understanding of their respective roles in neuropathic processes in the DRG.

A label-free, impedance-based real time assay to identify drug-induced toxicities and differentiate cytostatic from cytotoxic effects.

Cell-based assays are key tools in drug safety assessment. However, they usually provide only limited information about time-kinetics of a toxic effect and implementing multiple measurements is often complex. To overcome these issues we established an impedance-based approach which is able to differentiate cytostatic from cytotoxic drugs by recording time-kinetics of compound-effects on cells. NIH 3T3 fibroblasts were seeded on xCELLigence® E-plates and impedance was continuously measured over 5 days. The obtained results reflected cytotoxicity and cell proliferation, as confirmed by neutral red uptake in vitro. Based on known toxicants, we established an algorithm able to discriminate cytostatic, cytotoxic and non-toxic compounds based on the shape of the impedance curves. Analyzing impedance curve patterns of additional 37 compounds allowed the identification and differentiation of these distinct effects as results correlated well with previous in vivo findings. We show that impedance-based real-time cell analysis is a convenient tool to characterize and discriminate effects of compounds on cells in a time-dependent and label-free manner. The presented impedance assay could be used to further characterize toxicities observed in vivo or in vitro. Due to the ease of performance it may also be a suitable screening tool.

Serum cardiac troponin I concentrations transiently increase in rats given rosiglitazone.

Rosiglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist of the thiazolidinedione class, is a major insulin-sensitizing drug widely used to treat type-2 diabetes. Rosiglitazone causes myocardial hypertrophy in rodents and increases the risk of cardiac events in man. To better characterize its cardiac effects, male Wistar rats were orally administered 0, 10 or 80 mg/kg/day rosiglitazone. Myocardial gene expression profiling, hematology, histopathology and clinical chemistry, including measurement of serum cardiac troponin (cTn) I concentration with the ultrasensitive assay, were evaluated after 6 and 24h and 7 and 14 days of dosing. Heart weight was increased 10% after 7 days and 16% after 14 days of dosing at 80 mg/kg/day in the absence of microscopic changes. At the transcriptomic level, the number of differentially expressed probes was small: it was most at 24h in rats given 80 mg/kg rosiglitazone with 356 differentially regulated probes (fold change >1.3 fold, p<0.05). Also, gene categories typically associated with myocardial damage were not over-represented. Most importantly, serum cTnI concentrations in 5/9 rats after 7 days of dosing at 80 mg/kg/day were above the upper limit of serum cTnI concentration. cTnI concentrations after 14 days of dosing were similar between rats given the vehicle and rosiglitazone at 80 mg/kg. This is the first study to detect increases of serum cTnI concentrations in rats administered rosiglitazone. In light of reported cardiac events in patients chronically dosed with PPARγ agonists, our results support serum cTnI concentrations as an early biomarker of cardiac liability.

Discrimination of direct and indirect interactions in a network of regulatory effects.

The matter of concern are algorithms for the discrimination of direct from indirect regulatory effects from an interaction graph built up by error-prone measurements. Many of these algorithms can be cast as a rule for the removal of a single edge of the graph, such that the remaining graph is still consistent with the data. A set of mild conditions is given under which iterated application of such a rule leads to a unique minimal consistent graph. We show that three of the common methods for direct interactions search fulfill these conditions, thus providing a justification of their use. The main issues a reconstruction algorithm has to deal with, are the noise in the data, the presence of regulatory cycles, and the direction of the regulatory effects. We introduce a novel rule that, in contrast to the previously mentioned methods, simultaneously takes into account all these aspects. An efficient algorithm for the computation of the minimal graph is given, whose time complexity is cubic in the number of vertices of the graph. Finally, we demonstrate the utility of our method in a simulation study.