Immunoadsorption (IA) has proven to be clinically effective in the treatment of steroid-refractory multiple sclerosis (MS) relapses, but its mechanism of action remains unclear. We used miniaturized ...adsorber devices with a tryptophan-immobilized polyvinyl alcohol (PVA) gel sorbent to mimic the IA treatment of patients with MS in vitro. The plasma was screened before and after adsorption with regard to disease-specific mediators, and the effect of the IA treatment on the migration of neutrophils and the integrity of the endothelial cell barrier was tested in cell-based models. The in vitro IA treatment with miniaturized adsorbers resulted in reduced plasma levels of cytokines and chemokines. We also found a reduced migration of neutrophils towards patient plasma treated with the adsorbers. Furthermore, the IA-treated plasma had a positive effect on the endothelial cell barrier's integrity in the cell culture model. Our findings suggest that IA results in a reduced infiltration of cells into the central nervous system by reducing leukocyte transmigration and preventing blood-brain barrier breakdown. This novel approach of performing in vitro blood purification therapies on actual patient samples with miniaturized adsorbers and testing their effects in cell-based assays that investigate specific hypotheses of the pathophysiology provides a promising platform for elucidating the mechanisms of action of those therapies in various diseases.
Staphylococcus aureus is a widespread opportunistic pathogen that can cause a wide variety of life-threatening diseases. Especially for the colonization of human tissues and the development of ...invasiveness, surface-exposed proteins are of major importance. In the present studies, we optimized a proteolytic shaving approach to identify those surface-exposed protein domains - the surfacome - of S. aureus that are accessible to extracellular bio-macromolecules, for example in the host milieu. Subsequently, this approach was applied to define the surfacomes of four strains with different genetic backgrounds. This resulted in the identification of 96 different proteins. Surprisingly, the overlap between the surfacomes of the four different strains was below 10% and each strain displayed its own characteristic set of surface-exposed proteins. The data were also evaluated at the peptide level and here we observed a similar phenomenon. From 190 unique peptides only five were commonly found in the four strains. Besides well known cell wall proteins, we also identified some essential proteins, several yet uncharacterized exported proteins and predicted intracellular proteins. These results show for the first time that the cell surface of different S. aureus strains is not only highly variable, but also that the displayed proteins are very heterogeneous.
Despite remarkable advances in the therapy of multiple sclerosis (MS), patients with MS may still experience relapses. High-dose short-term methylprednisolone (MP) remains the standard treatment in ...the acute management of MS relapses due to its potent anti-inflammatory and immunosuppressive properties. However, there is a lack of studies on the cell type-specific transcriptome changes that are induced by this synthetic glucocorticoid (GC). Moreover, it is not well understood why some patients do not benefit adequately from MP therapy.
We collected peripheral blood from MS patients in relapse immediately before and after ∼3–5 days of therapy with MP at 4 study centers. CD19+ B cells and CD4+ T cells were then isolated for profiling the transcriptome with high-density arrays. The patients' improvement of neurological symptoms was evaluated after ∼2 weeks by the treating physicians. We finally analyzed the data to identify genes that were differentially expressed in response to the therapy and whose expression differed between clinical responders and non-responders.
After MP treatment, a total of 33 genes in B cells and 55 genes in T helper cells were significantly up- or downregulated. The gene lists overlap in 10 genes and contain genes that have already been described as GC-responsive genes in the literature on other cell types and diseases. Their differential expression points to a rapid and coordinated modulation of multiple signaling pathways that influence transcription. Genes that were previously suggested as potential prognostic biomarkers of the clinical response to MP therapy could not be confirmed in our data. However, a greater increase in the expression of genes encoding proteins with antimicrobial activity was detected in CD4+ T cells from non-responders compared to responders.
Our study delved into the cell type-specific effects of MP at the transcriptional level. The data suggest a therapy-induced ectopic expression of some genes (e.g., AZU1, ELANE and MPO), especially in non-responders. The biological consequences of this remain to be explored in greater depth. A better understanding of the molecular mechanisms underlying clinical recovery from relapses in patients with MS will help to optimize future treatment decisions.
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•We explored gene expression changes during relapse therapy in multiple sclerosis.•Blood was collected before and after treatment with high-dose methylprednisolone (MP).•Ten genes were differentially expressed in both CD19+ B cells and CD4+ T helper cells.•In CD4+ T cells, some genes were particularly upregulated in clinical non-responders.•Our study provides novel insights into cell type-specific therapeutic effects of MP.
Reversible protein phosphorylation is an important and ubiquitous protein modification in all living cells. Here we report that protein phosphorylation on arginine residues plays a physiologically ...significant role. We detected 121 arginine phosphorylation sites in 87 proteins in the Gram-positive model organism Bacillus subtilis in vivo. Moreover, we provide evidence that protein arginine phosphorylation has a functional role and is involved in the regulation of many critical cellular processes, such as protein degradation, motility, competence, and stringent and stress responses. Our results suggest that in B. subtilis the combined activity of a protein arginine kinase and phosphatase allows a rapid and reversible regulation of protein activity and that protein arginine phosphorylation can play a physiologically important and regulatory role in bacteria.
Genome-wide association studies have identified more than 200 genetic variants to be associated with an increased risk of developing multiple sclerosis (MS). Still, little is known about the causal ...molecular mechanisms that underlie the genetic contribution to disease susceptibility. In this study, we investigated the role of the single-nucleotide polymorphism (SNP) rs1414273, which is located within the microRNA-548ac stem-loop sequence in the first intron of the CD58 gene. We conducted an expression quantitative trait locus (eQTL) analysis based on public RNA-sequencing and microarray data of blood-derived cells of more than 1000 subjects. Additionally, CD58 transcripts and mature hsa-miR-548ac molecules were measured using real-time PCR in peripheral blood samples of 32 MS patients. Cell culture experiments were performed to evaluate the efficiency of Drosha-mediated stem-loop processing dependent on genotype and to determine the target genes of this underexplored microRNA. Across different global populations and data sets, carriers of the MS risk allele showed reduced CD58 mRNA levels but increased hsa-miR-548ac levels. We provide evidence that the SNP rs1414273 might alter Drosha cleavage activity, thereby provoking partial uncoupling of CD58 gene expression and microRNA-548ac production from the shared primary transcript in immune cells. Moreover, the microRNA was found to regulate genes, which participate in inflammatory processes and in controlling the balance of protein folding and degradation. We thus uncovered new regulatory implications of the MS-associated haplotype of the CD58 gene locus, and we remind that paradoxical findings can be encountered in the analysis of eQTLs upon data aggregation. Our study illustrates that a better understanding of RNA processing events might help to establish the functional nature of genetic variants, which predispose to inflammatory and neurological diseases.
Multiple sclerosis is a demyelinating disease affecting the central nervous system. T cells are known to contribute to this immune-mediated condition. Fingolimod modulates sphingosine-1-phosphate ...receptors, thereby preventing the egress of lymphocytes, especially CCR7-expressing CD8+ and CD4+ T cells, from lymphoid tissues. Using Affymetrix Human Transcriptome Arrays (HTA 2.0), we performed a transcriptome profiling analysis of CD4+ cells obtained from the peripheral blood of patients with highly active relapsing-remitting multiple sclerosis. The samples were drawn before the first administration of fingolimod as well as 24 hours and 3 months after the start of therapy. Three months after treatment initiation, 890 genes were found to be differentially expressed with fold-change >2.0 and t-test p-value < 0.001, among them several microRNA precursors. A subset of 272 genes were expressed at lower levels, including CCR7 as expected, while 618 genes showed an increase in expression, e.g., CCR2, CX3CR1, CD39, CD58 as well as LYN, PAK1 and TLR2. To conclude, we studied the gene expression of CD4+ cells to evaluate the effects of fingolimod treatment, and we identified 890 genes to be altered in expression after continuous drug administration. T helper cells circulating in the blood during fingolimod therapy present a distinct gene expression signature.
The Gram-positive soil bacterium Bacillus subtilis responds to oxidative stress by the activation of different cellular defence mechanisms. These are composed of scavenging enzymes as well as ...protection and repair systems organized in highly sophisticated networks. In this study, the peroxide and the superoxide stress stimulons of B. subtilis were characterized by means of transcriptomics and proteomics. The results demonstrate that oxidative-stress-responsive genes can be classified into two groups. One group encompasses genes which show similar expression patterns in the presence of both reactive oxygen species. Examples are members of the PerR and the Fur regulon which were induced by peroxide and superoxide stress. Similarly, both kinds of stress stimulated the activation of the stringent response. The second group is composed of genes primarily responding to one stimulus, like the members of the SOS regulon which were particularly upregulated in the presence of peroxide, and many genes involved in sulfate assimilation and methionine biosynthesis which were only induced by superoxide. Several genes encoding proteins of unknown function could be assigned to one of these groups.
possesses two glyceraldehyde-3-phosphate dehydrogenases with opposite roles, the glycolytic NAD-dependent GapA and the NADP-dependent GapB enzyme, which is exclusively required during gluconeogenesis ...but not active under conditions promoting glycolysis. We propose that proteins that are no longer needed will be recognized and proteolyzed by Clp proteases and thereby recycled. To test this postulation, we analyzed the stability of the glycolytic enzyme GapA and the gluconeogenetic enzyme GapB in the presence and absence of glucose. It turned out that GapA remained rather stable under both glycolytic and gluconeogenetic conditions. In contrast, the gluconeogenetic enzyme GapB was degraded after a shift from malate to glucose (i.e., from gluconeogenesis to glycolysis), displaying an estimated half-life of approximately 3 h. Comparative
pulse-chase labeling and immunoprecipitation experiments of the wild-type strain and isogenic mutants identified the ATP-dependent ClpCP protease as the enzyme responsible for the degradation of GapB. However, arginine protein phosphorylation, which was recently described as a general tagging mechanism for protein degradation, did not seem to play a role in GapB proteolysis, because GapB was also degraded in a
mutant, lacking arginine kinase, in the same manner as in the wild type.
GapB, the NADP-dependent glyceraldehyde-3-phosphosphate dehydrogenase, is essential for
under gluconeogenetic conditions. However, after a shift to glycolytic conditions, GapB loses its physiological function within the cell and becomes susceptible to degradation, in contrast to GapA, the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, which remains stable under glycolytic and gluconeogenetic conditions. Subsequently, GapB is proteolyzed in a ClpCP-dependent manner. According to our data, the arginine kinase McsB is not involved as adaptor protein in this process. ClpCP appears to be in charge in the removal of inoperable enzymes in
, which is a strictly regulated process in which the precise recognition mechanism(s) remains to be identified.
Background:
Patients with multiple sclerosis (MS) often undergo complex treatment regimens, resulting in an increased risk of polypharmacy and potential drug-drug interactions (pDDIs). Drug ...interaction databases are useful for identifying pDDIs to support safer medication use.
Objective:
To compare three different screening tools regarding the detection and classification of pDDIs in a cohort of MS patients. Furthermore, we aimed at ascertaining sociodemographic and clinical factors that are associated with the occurrence of severe pDDIs.
Methods:
The databases Stockley’s, Drugs.com and MediQ were used to identify pDDIs by screening the medication schedules of 627 patients. We determined the overlap of the identified pDDIs and the level of agreement in pDDI severity ratings between the three databases. Logistic regression analyses were conducted to determine patient risk factors of having a severe pDDI.
Results:
The most different pDDIs were identified using MediQ (
n
= 1,161), followed by Drugs.com (
n
= 923) and Stockley’s (
n
= 706). The proportion of pDDIs classified as severe was much higher for Stockley’s (37.4%) than for Drugs.com (14.4%) and MediQ (0.9%). Overall, 1,684 different pDDIs were identified by at least one database, of which 318 pDDIs (18.9%) were detected with all three databases. Only 55 pDDIs (3.3%) have been reported with the same severity level across all databases. A total of 336 pDDIs were classified as severe (271 pDDIs by one database, 59 by two databases and 6 by three databases). Stockley’s and Drugs.com revealed 47 and 23 severe pDDIs, respectively, that were not included in the other databases. At least one severe pDDI was found for 35.2% of the patients. The most common severe pDDI was the combination of acetylsalicylic acid with enoxaparin, and citalopram was the drug most frequently involved in different severe pDDIs. The strongest predictors of having a severe pDDI were a greater number of drugs taken, an older age, living alone, a higher number of comorbidities and a lower educational level.
Conclusions:
The information on pDDIs are heterogeneous between the databases examined. More than one resource should be used in clinical practice to evaluate pDDIs. Regular medication reviews and exchange of information between treating physicians can help avoid severe pDDIs.
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