De-escalation and Discontinuation of Disease-Modifying Therapies in Multiple Sclerosis.

PURPOSE OF REVIEW: Long-term use of multiple sclerosis (MS) disease-modifying therapies (DMTs) is standard practice to prevent accumulation of disability. Immunosenescence and other age-related changes lead to an altered risk-benefit ratio for older patients on DMTs. This article reviews recent research on the topic of de-escalation and discontinuation of MS DMTs. RECENT FINDINGS: Observational and interventional studies have shed light on what happens to patients who de-escalate or discontinue DMTs and the factors, such as age, treatment type, and presence of recent disease activity, that influence outcomes. Though many questions remain, recent findings have been valuable for the development of an evidence-based approach to making de-escalation and discontinuation decisions in MS.

Processing speed and memory test performance are associated with different brain region volumes in Veterans and others with progressive multiple sclerosis.

Background: Cognitive dysfunction and brain atrophy are both common in progressive multiple sclerosis (MS) but are seldom examined comprehensively in clinical trials. Antioxidant treatment may affect the neurodegeneration characteristic of progressive MS and slow its symptomatic and radiographic correlates. Objectives: This study aims to evaluate cross-sectional associations between cognitive battery components of the Brief International Cognitive Assessment for Multiple Sclerosis with whole and segmented brain volumes and to determine if associations differ between secondary progressive (SPMS) and primary progressive (PPMS) MS subtypes. Design: The study was based on a baseline analysis from a multi-site randomized controlled trial of the antioxidant lipoic acid in veterans and other people with progressive MS (NCT03161028). Methods: Cognitive batteries were conducted by trained research personnel. MRIs were processed at a central processing site for maximum harmonization. Semi-partial Pearson's adjustments evaluated associations between cognitive tests and MRI volumes. Regression analyses evaluated differences in association patterns between SPMS and PPMS cohorts. Results: Of the 114 participants, 70% had SPMS. Veterans with MS made up 26% (n = 30) of the total sample and 73% had SPMS. Participants had a mean age of 59.2 and sd 8.5 years, and 54% of them were women, had a disease duration of 22.4 (sd 11.3) years, and had a median Expanded Disability Status Scale of 6.0 (with an interquartile range of 4.0-6.0, moderate disability). The Symbol Digit Modalities Test (processing speed) correlated with whole brain volume (R = 0.29, p = 0.01) and total white matter volume (R = 0.33, p < 0.01). Both the California Verbal Learning Test (verbal memory) and Brief Visuospatial Memory Test-Revised (visual memory) correlated with mean cortical thickness (R = 0.27, p = 0.02 and R = 0.35, p < 0.01, respectively). Correlation patterns were similar in subgroup analyses. Conclusion: Brain volumes showed differing patterns of correlation across cognitive tasks in progressive MS. Similar results between SPMS and PPMS cohorts suggest combining progressive MS subtypes in studies involving cognition and brain atrophy in these populations. Longitudinal assessment will determine the therapeutic effects of lipoic acid on cognitive tasks, brain atrophy, and their associations.

Monitoring, Switching, and Stopping Multiple Sclerosis Disease-Modifying Therapies.

PURPOSE OF REVIEW: This article reviews appropriate monitoring of the various multiple sclerosis (MS) disease-modifying therapies, summarizes the reasons patients switch or stop treatment, and provides a framework for making these management decisions. RECENT FINDINGS: With the increasing number of highly effective immunotherapies available for MS, the possibility of better control of the disease has increased, but with it, the potential for side effects has rendered treatment decisions more complicated. Starting treatment early with more effective and better-tolerated disease-modifying therapies reduces the likelihood of switching because of breakthrough disease or lack of compliance. Clinical and radiographic surveillance, and often blood and other paraclinical tests, should be performed periodically, depending on the disease-modifying therapy. Helping patients navigate the uncertainty around switching or stopping treatment, either temporarily or permanently, is one of the most important things we do as providers of MS care. SUMMARY: Ongoing monitoring of drug therapy is a crucial component of long-term MS care. Switching treatments may be necessary for a variety of reasons. Permanent discontinuation of treatment may be appropriate for some patients with MS, although more study is needed in this area.

Alemtuzumab in multiple sclerosis: an update.

Since the introduction of IFN-ß, disease-modifying treatments, acting through various immune mechanisms, have been shown to reduce disease activity and severity in relapsing multiple sclerosis. Nevertheless, there remain patients for whom these treatments are incompletely effective, poorly tolerated or contraindicated. Alemtuzumab is a humanized monoclonal antibody that works by selectively depleting circulating lymphocytes. It is given as an intravenous infusion of 12 mg daily for 5 days, then a year later for 3 days. Effectiveness in patients with active relapsing-remitting multiple sclerosis has been demonstrated in two Phase III clinical trials, where it outperformed IFN-ß-1a 44 mcg on clinical and radiographic efficacy measures. Its side effect profile, including infusion-associated reactions, infections and secondary autoimmunity, coupled with its long-lasting biological effect, requires patients to commit to close monitoring while on the drug and for 4 years after the final infusion. For select patients with active disease, alemtuzumab offers a powerful therapeutic option.

Natural selection canalizes expression variation of environmentally induced plasticity-enabling genes.

Many organisms survive fluctuating and extreme environmental conditions by manifesting multiple distinct phenotypes during adulthood by means of developmental processes that enable phenotypic plasticity. We report on the discovery of putative plasticity-enabling genes that are involved in transforming the gill of the euryhaline teleost fish, Fundulus heteroclitus, from its freshwater to its seawater gill-type, a process that alters both morphology and function. Gene expression that normally enables osmotic plasticity is inhibited by arsenic. Gene sets defined by antagonistic interactions between arsenic and salinity show reduced transcriptional variation among individual fish, suggesting unusually accurate and precise regulatory control of these genes, consistent with the hypothesis that they participate in a canalized developmental response. We observe that natural selection acts to preserve canalized gene expression in populations of killifish that are most tolerant to abrupt salinity change and that these populations show the least variability in their transcription of genes enabling plasticity of the gill. We found that genes participating in this highly canalized and conserved plasticity-enabling response had significantly fewer and less complex associations with transcriptional regulators than genes that respond only to arsenic or salinity. Collectively these findings, which are drawn from the relationships between environmental challenge, plasticity, and canalization among populations, suggest that the selective processes that facilitate phenotypic plasticity do so by targeting the regulatory networks that gives rise to the response. These findings also provide a generalized, conceptual framework of how genes might interact with the environment and evolve toward the development of plastic traits.

Computational discovery of transcriptional regulatory modules in fungal ribosome biogenesis genes reveals novel sequence and function patterns.

Genes involved in ribosome biogenesis and assembly (RBA) are responsible for ribosome formation. In Saccharomyces cerevisiae, their transcription is regulated by two dissimilar DNA motifs. We were interested in analyzing conservation and divergence of RBA transcription regulation machinery throughout fungal evolution. We have identified orthologs of S. cerevisiae RBA genes in 39 species across fungal phylogeny and searched upstream regions of these gene sets for DNA sequences significantly similar to S. cerevisiae RBA regulatory motifs. In addition to confirming known motif arrangements comprising two different motifs in a set of S. cerevisiae close relatives or two instances of the same motif (that we refer to as modules), we have also discovered novel modules in a group of fungi closely related to Neurospora crassa. Despite a single nucleotide difference between consensus sequences of RBA motifs, modules associated with S, cerevisiae group and N. crassa group displayed consistently different characteristics with respect to preferred module organization and several other module properties. For a given species, we have found a correlation between the configuration of the RBA module and significant enrichment in a set of specific Gene Ontology biological processes. We have identified several likely new candidates for a role in ribosome biogenesis in S. cerevisiae based on the combined evidence of RBA module presence in the upstream regions, functional annotation information and microarray expression profiles. We believe that this approach will be useful in terms of generating hypotheses about functional roles of genes for which only fragmentary data from a single source are available.

Dis3- and exosome subunit-responsive 3' mRNA instability elements.

Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3'-5' exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3' untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of mRNA turnover that involves direct Dis3 and other exosome subunit recruitment to and/or regulation on mRNA substrates.

Using SCOPE to identify potential regulatory motifs in coregulated genes.

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data. In this article, we utilize a web version of SCOPE to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs and has been used in other studies. The three algorithms that comprise SCOPE are BEAM, which finds non-degenerate motifs (ACCGGT), PRISM, which finds degenerate motifs (ASCGWT), and SPACER, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well. Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor. Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail.

Identifying functional relationships within sets of co-expressed genes by combining upstream regulatory motif analysis and gene expression information.

BACKGROUND: Existing clustering approaches for microarray data do not adequately differentiate between subsets of co-expressed genes. We devised a novel approach that integrates expression and sequence data in order to generate functionally coherent and biologically meaningful subclusters of genes. Specifically, the approach clusters co-expressed genes on the basis of similar content and distributions of predicted statistically significant sequence motifs in their upstream regions. RESULTS: We applied our method to several sets of co-expressed genes and were able to define subsets with enrichment in particular biological processes and specific upstream regulatory motifs. CONCLUSIONS: These results show the potential of our technique for functional prediction and regulatory motif identification from microarray data.

Genome-wide analysis of light-inducible responses reveals hierarchical light signalling in Neurospora.

White collar-1 (WC-1) and white collar-2 (WC-2) are essential for light-mediated responses in Neurospora crassa, but the molecular mechanisms underlying gene induction and the roles of other real and putative photoreceptors remain poorly characterized. Unsupervised hierarchical clustering of genome-wide microarrays reveals 5.6% of detectable transcripts, including several novel mediators, that are either early or late light responsive. Evidence is shown for photoreception in the absence of the dominant, and here confirmed, white collar complex (WCC) that regulates both types of light responses. VVD primarily modulates late responses, whereas light-responsive submerged protoperithecia-1 (SUB-1), a GATA family transcription factor, is essential for most late light gene expression. After a 15-min light stimulus, the WCC directly binds the sub-1 promoter. Bioinformatics analysis detects many early light response elements (ELREs), as well as identifying a late light response element (LLRE) required for wild-type activity of late light response promoters. The data provide a global picture of transcriptional response to light, as well as illuminating the cis- and trans-acting elements comprising the regulatory signalling cascade that governs the photobiological response.

Identification of TTP mRNA targets in human dendritic cells reveals TTP as a critical regulator of dendritic cell maturation.

Dendritic cells provide a critical link between innate and adaptive immunity and are essential to prime a naive T-cell response. The transition from immature dendritic cells to mature dendritic cells involves numerous changes in gene expression; however, the role of post-transcriptional changes in this process has been largely ignored. Tristetraprolin is an AU-rich element mRNA-binding protein that has been shown to regulate the stability of a number of cytokines and chemokines of mRNAs. Using TTP immunoprecipitations and Affymetrix GeneChips, we identified 393 messages as putative TTP mRNA targets in human dendritic cells. Gene ontology analysis revealed that approximately 25% of the identified mRNAs are associated with protein synthesis. We also identified six MHC Class I alleles, five MHC Class II alleles, seven chemokine and chemokine receptor genes, indoleamine 2,3 dioxygenase, and CD86 as putative TTP ligands. Real-time PCR was used to validate the GeneChip data for 15 putative target genes and functional studies performed for six target genes. These data establish that TTP regulates the expression of DUSP1, IDO, SOD2, CD86, and MHC Class I-B and F via the 3'-untranslated region of each gene. A novel finding is the demonstration that TTP can interact with and regulate the expression of non-AU-rich element-containing messages. The data implicate TTP as having a broader role in regulating and limiting the immune response than previously suspected.

A novel ensemble learning method for de novo computational identification of DNA binding sites.

BACKGROUND: Despite the diversity of motif representations and search algorithms, the de novo computational identification of transcription factor binding sites remains constrained by the limited accuracy of existing algorithms and the need for user-specified input parameters that describe the motif being sought. RESULTS: We present a novel ensemble learning method, SCOPE, that is based on the assumption that transcription factor binding sites belong to one of three broad classes of motifs: non-degenerate, degenerate and gapped motifs. SCOPE employs a unified scoring metric to combine the results from three motif finding algorithms each aimed at the discovery of one of these classes of motifs. We found that SCOPE's performance on 78 experimentally characterized regulons from four species was a substantial and statistically significant improvement over that of its component algorithms. SCOPE outperformed a broad range of existing motif discovery algorithms on the same dataset by a statistically significant margin. CONCLUSION: SCOPE demonstrates that combining multiple, focused motif discovery algorithms can provide a significant gain in performance. By building on components that efficiently search for motifs without user-defined parameters, SCOPE requires as input only a set of upstream sequences and a species designation, making it a practical choice for non-expert users. A user-friendly web interface, Java source code and executables are available at http://genie.dartmouth.edu/scope.

SCOPE: a web server for practical de novo motif discovery.

SCOPE is a novel parameter-free method for the de novo identification of potential regulatory motifs in sets of coordinately regulated genes. The SCOPE algorithm combines the output of three component algorithms, each designed to identify a particular class of motifs. Using an ensemble learning approach, SCOPE identifies the best candidate motifs from its component algorithms. In tests on experimentally determined datasets, SCOPE identified motifs with a significantly higher level of accuracy than a number of other web-based motif finders run with their default parameters. Because SCOPE has no adjustable parameters, the web server has an intuitive interface, requiring only a set of gene names or FASTA sequences and a choice of species. The most significant motifs found by SCOPE are displayed graphically on the main results page with a table containing summary statistics for each motif. Detailed motif information, including the sequence logo, PWM, consensus sequence and specific matching sites can be viewed through a single click on a motif. SCOPE's efficient, parameter-free search strategy has enabled the development of a web server that is readily accessible to the practising biologist while providing results that compare favorably with those of other motif finders. The SCOPE web server is at .

SPACER: identification of cis-regulatory elements with non-contiguous critical residues.

MOTIVATION: Many transcription factors bind to sites that are long and loosely related to each other. De novo identification of such motifs is computationally challenging. In this article, we propose a novel semi-greedy algorithm over the space of all IUPAC degenerate strings to identify the most over-represented highly degenerate motifs. RESULTS: We present an implementation of this algorithm, named SPACER (Separated Pattern-based Algorithm for cis-Element Recognition) and demonstrate its effectiveness in identifying 'gapped' and highly degenerate motifs. We compare SPACER's performance against ten motif finders on 42 experimentally defined regulons from Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae. These motif finders cover a wide range of both enumerative and statistical approaches, including programs specifically designed for prokaryotic and 'gapped' motifs. AVAILABILITY: A Java 1.4 implementation is freely available on the Web at http://genie.Dartmouth.edu/SPACER/

Phosphate-dependent modulation of c-di-GMP levels regulates Pseudomonas fluorescens Pf0-1 biofilm formation by controlling secretion of the adhesin LapA.

Biofilm formation is commonly described as a developmental process regulated by environmental cues. In the current study we present a mechanistic model to explain regulation of Pseudomonas fluorescens biofilm formation by the environmentally relevant signal inorganic phosphate (P(i)). We show that activation of the Pho regulon, the major pathway for adaptation to phosphate limitation, results in conditional expression of a c-di-GMP phosphodiesterase referred to as RapA. Genetic analysis indicated that RapA is an inhibitor of biofilm formation and required for loss of biofilm formation in response to limiting P(i). Our results suggest that RapA lowers the level of c-di-GMP, which in turn inhibits the secretion of LapA, a large adhesion required for biofilm formation by P. fluorescens. The ability of c-di-GMP to modulate protein secretion is a novel finding and further extends the biological influence of c-di-GMP beyond that of regulating exopolysaccharide synthesis and motility. Interestingly, Pho regulon expression does not impinge on the rate of attachment to a surface but rather inhibits the transition of cells to a more stable interaction with the surface. We hypothesize that Pho regulon expression confers a surface-sensing mode on P. fluorescens and suggest this strategy may be broadly applicable to other bacteria.

Bounded search for de novo identification of degenerate cis-regulatory elements.

BACKGROUND: The identification of statistically overrepresented sequences in the upstream regions of coregulated genes should theoretically permit the identification of potential cis-regulatory elements. However, in practice many cis-regulatory elements are highly degenerate, precluding the use of an exhaustive word-counting strategy for their identification. While numerous methods exist for inferring base distributions using a position weight matrix, recent studies suggest that the independence assumptions inherent in the model, as well as the inability to reach a global optimum, limit this approach. RESULTS: In this paper, we report PRISM, a degenerate motif finder that leverages the relationship between the statistical significance of a set of binding sites and that of the individual binding sites. PRISM first identifies overrepresented, non-degenerate consensus motifs, then iteratively relaxes each one into a high-scoring degenerate motif. This approach requires no tunable parameters, thereby lending itself to unbiased performance comparisons. We therefore compare PRISM's performance against nine popular motif finders on 28 well-characterized S. cerevisiae regulons. PRISM consistently outperforms all other programs. Finally, we use PRISM to predict the binding sites of uncharacterized regulons. Our results support a proposed mechanism of action for the yeast cell-cycle transcription factor Stb1, whose binding site has not been determined experimentally. CONCLUSION: The relationship between statistical measures of the binding sites and the set as a whole leads to a simple means of identifying the diverse range of cis-regulatory elements to which a protein binds. This approach leverages the advantages of word-counting, in that position dependencies are implicitly accounted for and local optima are more easily avoided. While we sacrifice guaranteed optimality to prevent the exponential blowup of exhaustive search, we prove that the error is bounded and experimentally show that the performance is superior to other methods. A Java implementation of this algorithm can be downloaded from our web server at http://genie.dartmouth.edu/prism.

BEAM: a beam search algorithm for the identification of cis-regulatory elements in groups of genes.

The identification of potential protein binding sites (cis-regulatory elements) in the upstream regions of genes is key to understanding the mechanisms that regulate gene expression. To this end, we present a simple, efficient algorithm, BEAM (beam-search enumerative algorithm for motif finding), aimed at the discovery of cis-regulatory elements in the DNA sequences upstream of a related group of genes. This algorithm dramatically limits the search space of expanded sequences, converting the problem from one that is exponential in the length of motifs sought to one that is linear. Unlike sampling algorithms, our algorithm converges and is capable of finding statistically overrepresented motifs with a low failure rate. Further, our algorithm is not dependent on the objective function or the organism used. Limiting the space of candidate motifs enables the algorithm to focus only on those motifs that are most likely to be biologically relevant and enables the algorithm to use direct evaluations of background frequencies instead of resorting to probabilistic estimates. In addition, limiting the space of candidate motifs makes it possible to use computationally expensive objective functions that are able to correctly identify biologically relevant motifs.

The impact of topical cyclosporine A emulsion 0.05% on the outcomes of patients with keratoconjunctivitis sicca.

OBJECTIVE: To assess patients' experiences with topical cyclosporine A (tCSA) 0.05% ophthalmic emulsion (Restasis) to treat keratoconjunctivitis sicca (KCS) in a real-world setting. METHODS: A total of 4504 ophthalmologists, optometrists and primary care physicians from throughout the United States participated in the study. Individual physicians identified patients from their practice who were appropriate candidates for treatment with tCSA and provided them with free sample medication and study materials. Patients voluntarily enrolled in the program by following the instructions provided in the study materials. Data for this study were obtained from patient surveys that included questions related to patients' experiences using topical cyclosporine A 0.05% ophthalmic emulsion (tCSA). Using automated surveys at baseline, 30-days and 60-days post-medication initiation, patients rated symptom severity, symptom impact on daily activities, and use of artificial tears pre- and post-treatment with study medication. Participants also reported on the rapidity of symptom relief and satisfaction with tCSA. RESULTS: A total of 5884 patients completed the study. The vast majority (84%) was female; average age was 63 years. Patients who completed the study (n = 5884) achieved significant reductions (p < 0.001) of 30% in symptom severity and 31%-36% in activity impairments relative to baseline. A positive association (p < 0.001) was observed between number of years with dry eye and the average ratings for symptom severity and impact on everyday activities. Onset of relief was noted within 1 week by 32% of patients and within 3 weeks by 73%. More than 60% reported decreased use of artificial tears at both 30 days and 60 days post-treatment initiation. CONCLUSIONS: Results suggest that in a real-world setting tCSA is an effective treatment for patients suffering from KCS. Onset of relief may be more rapid than in previously published reports.

Single-cell analysis reveals cell-specific patterns of expression of a family of putative voltage-gated sodium channel genes in the leech.

To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel alpha subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel alpha subunits, including the "S4 region" involved in activation, the "pore loops" responsible for ion selectivity, and the "inactivation loop" between the third and fourth domains, though the latter lacks the highly conserved "IFM" motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based "real time" RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system.