Table of Contents

Table of contents for Volume 10, Issue 3 of the Linfield Magazin

The role of arginine 47 in the cyclization and coupling reactions of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 - Implications for product inhibition and product specificity

Cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) is used for the industrial production of cyclodextrins. Its application, however, is hampered by the limited cyclodextrin product specificity and the strong inhibitory effect of cyclodextrins on CGTase activity. Recent structural studies have identified Arg47 in the Bacillus circulans strain 251 CGTase as an active-site residue interacting with cyclodextrins, but not with linear oligosaccharides. Arg47 thus may specifically affect CGTase reactions with cyclic substrates or products. Here we show that mutations in Arg47 (to Leu or Gln) indeed have a negative effect on the cyclization and coupling activities; Arg47 specifically stabilizes the oligosaccharide chain in the transition state for these reactions. As a result, the mutant proteins display a shift in product specificity towards formation of larger cyclodextrins. As expected, both mutants also showed lower affinities for cyclodextrins in the coupling reaction, and a reduced competitive (product) inhibition of the disproportionation reaction by cyclodextrins. Both mutants also provide valuable information about the processes taking place during cyclodextrin production assays. Mutant Arg47-->Leu displayed an increased hydrolyzing activity, causing accumulation of increasing amounts of short oligosaccharides in the reaction mixture, which resulted in lower final amounts of cyclodextrins produced from starch. Interestingly, mutant Arg47-->Gln displayed an increased ratio of cyclization/coupling and a decreased hydrolyzing activity. Due to the decreased coupling activity, which especially affects the production of larger cyclodextrins, this CGTase variant produced the various cyclodextrins in a stable ratio in time. This feature is very promising for the industrial application of CGTase enzymes with improved product specificity

MEK and PI3K-AKT inhibitors synergistically block activated IL7 receptor signaling in T-cell acute lymphoblastic leukemia

We identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually exclusive, suggesting that they each cause the aberrant activation of a common downstream target. Expressing these mutant signaling molecules—but not their wild-type counterparts—rendered Ba/F3 cells independent of IL3 by activating the RAS-MEK-ERK and PI3K-AKT pathways. Interestingly, cells expressing either IL7Ra or JAK mutants are sensitive to JAK inhibitors, but respond less robustly to inhibitors of the downstream RAS-MEK-ERK and PI3K-AKT-mTOR pathways, indicating that inhibiting only one downstream pathway is not sufficient. Here, we show that inhibiting both the MEK and PI3K-AKT pathways synergistically prevents the proliferation of BaF3 cells expressing mutant IL7Ra, JAK and RAS. Furthermore, combined inhibition of MEK and PI3K/AKT was cytotoxic to samples obtained from 6 out of 11 primary T-ALL patients, including 1 patient who had no mutations in the IL7R signaling pathway. Taken together, these results suggest that the potent cytotoxic effects of inhibiting both MEK and PI3K/AKT should be investigated further as a therapeutic option using leukemia xenograft models.Leukemia advance online publication, 13 May 2016; doi:10.1038/leu.2016.83

Development and evaluation of a manual segmentation protocol for deep grey matter in multiple sclerosis: Towards accelerated semi-automated references

Background: Deep grey matter (dGM) structures, particularly the thalamus, are clinically relevant in multiple sclerosis (MS). However, segmentation of dGM in MS is challenging; labeled MS-specific reference sets are needed for objective evaluation and training of new methods. Objectives: This study aimed to (i) create a standardized protocol for manual delineations of dGM; (ii) evaluate the reliability of the protocol with multiple raters; and (iii) evaluate the accuracy of a fast-semi-automated segmentation approach (FASTSURF). Methods: A standardized manual segmentation protocol for caudate nucleus, putamen, and thalamus was created, and applied by three raters on multi-center 3D T1-weighted MRI scans of 23 MS patients and 12 controls. Intra- and inter-rater agreement was assessed through intra-class correlation coefficient (ICC); spatial overlap through Jaccard Index (JI) and generalized conformity index (CIgen). From sparse delineations, FASTSURF reconstructed full segmentations; accuracy was assessed both volumetrically and spatially. Results: All structures showed excellent agreement on expert manual outlines: intra-rater JI > 0.83; inter-rater ICC ≥ 0.76 and CIgen ≥ 0.74. FASTSURF reproduced manual references excellently, with ICC ≥ 0.97 and JI ≥ 0.92. Conclusions: The manual dGM segmentation protocol showed excellent reproducibility within and between raters. Moreover, combined with FASTSURF a reliable reference set of dGM segmentations can be produced with lower workload

Kappa free light chains is a valid tool in the diagnostics of MS: A large multicenter study

Objective: To validate kappa free light chain (KFLC) and lambda free light chain (LFLC) indices as a diagnostic biomarker in multiple sclerosis (MS). Methods: We performed a multicenter study including 745 patients from 18 centers (219 controls and 526 clinically isolated syndrome (CIS)/MS patients) with a known oligoclonal IgG band (OCB) status. KFLC and LFLC were measured in paired cerebrospinal fluid (CSF) and serum samples. Gaussian mix- ture modeling was used to define a cut-off for KFLC and LFLC indexes. Results: The cut-off for the KFLC index was 6.6 (95% confidence interval (CI) = 5.2-138.1). The cut-off for the LFLC index was 6.9 (95% CI=4.5-22.2). For CIS/MS patients, sensitivity of the KFLC index (0.88; 95% CI = 0.85-0.90) was higher than OCB (0.82; 95%CI = 0.79-0.85; p < 0.001), but specificity (0.83; 95% CI = 0.78-0.88) was lower (OCB = 0.92; 95% CI = 0.89-0.96; p < 0.001). Both sensitivity and specificity for the LFLC index were lower than OCB. Conclusion: Compared with OCB, the KFLC index is more sensitive but less specific for diagnosing CIS/MS. Lacking an elevated KFLC index is more powerful for excluding MS compared with OCB but the latter is more important for ruling in a diagnosis of CIS/MS

Efficacy and Safety of Alemtuzumab Through 9 Years of Follow-up in Patients with Highly Active Disease: Post Hoc Analysis of CARE-MS I and II Patients in the TOPAZ Extension Study

Background: Alemtuzumab efficacy versus subcutaneous interferon-β-1a (SC IFNB-1a) was demonstrated over 2 years in patients with relapsing-remitting multiple sclerosis, with continued efficacy over 7 additional years. Alemtuzumab is included as a recommended treatment for patients with highly active disease (HAD) by the American Academy of Neurology Practice Guidelines, and the label indication in Europe was recently restricted to the treatment of HAD patients. There is currently no consensus definition for HAD, and alemtuzumab efficacy across various HAD definitions has not been explored previously. Objectives: In this post hoc analysis, we assess the efficacy and safety of alemtuzumab in Comparison of Alemtuzumab and Rebif® Efficacy in Multiple Sclerosis (CARE-MS) trial patients who met criteria for at least one of four separate definitions of HAD (one primary and three alternatives). Over 2 years, alemtuzumab-treated HAD patients were compared with SC IFNB-1a-treated HAD patients, with additional 7-year follow-up in patients from the alemtuzumab arm. Methods: Patients in the CARE-MS studies received either alemtuzumab (baseline: 5&nbsp;days; 12&nbsp;months later: 3&nbsp;days) or SC IFNB-1a (3 times weekly). Alemtuzumab-treated patients who enrolled in the extensions could receive additional courses ≥ 12&nbsp;months apart. Four definitions of HAD were applied to assess alemtuzumab efficacy: the pre-specified primary definition (two or more relapses in the year prior to baseline and at least one gadolinium [Gd]-enhancing lesion at baseline) and three alternative definitions that focused on relapse, magnetic resonance imaging (MRI), or prior treatment response criteria. Efficacy outcomes were annualized relapse rate, change in Expanded Disability Status Scale score, 6-month confirmed disability worsening, 6-month confirmed disability improvement, MRI disease activity, and brain volume change. Adverse events were summarized for HAD patients meeting the primary definition. Results: In the pooled CARE-MS population, 208 alemtuzumab-treated patients met the primary HAD definition. Annualized relapse rate was 0.27 in years 0–2 and 0.16 in years 3–9. Over 9 years, 62% of patients were free of 6-month confirmed disability worsening, 50% had 6-month confirmed disability improvement, and median cumulative change in brain volume was − 2.15%. During year 9, 62% had no evidence of disease activity, and 69% were free of MRI disease activity. Similar efficacy outcomes were observed using an alternative relapse-driven HAD definition. For patients meeting alternative HAD definitions focused on either higher MRI lesion counts or disease activity while on prior therapy, reduced efficacy for some endpoints was seen. Safety was consistent with the overall CARE-MS population through year 9. Conclusions: Over 9&nbsp;years, alemtuzumab efficacy was maintained in CARE-MS HAD patients based on four HAD definitions. These results support intervention with alemtuzumab in patients with early indicators of HAD, including frequent relapse without high MRI activity. No safety signals were observed over 9&nbsp;years that were unique to the HAD populations. ClinicalTrials.gov Identifiers: NCT00530348; NCT00548405; NCT00930553; NCT02255656

Longitudinal assessment of multiple sclerosis with the brain-age paradigm

OBJECTIVE: During the natural course of MS, the brain is exposed to ageing as well as disease effects. Brain ageing can be modelled statistically; the so-called 'brain-age' paradigm. Here, we evaluated whether brain-predicted age difference (brain-PAD) was sensitive to the presence of MS, clinical progression and future outcomes. METHODS: In a longitudinal, multi-centre sample of 3,565 MRI scans, in 1,204 MS and clinically-isolated syndrome (CIS) patients and 150 healthy controls (mean follow-up time: patients 3.41 years, healthy controls 1.97 years), we measured 'brain-predicted age' using T1-weighted MRI. We compared brain-PAD between MS and CIS patients and healthy controls, and between disease subtypes. Relationships between brain-PAD and Expanded Disability Status Scale (EDSS) were explored. RESULTS: MS patients had markedly higher brain-PAD than healthy controls (mean brain-PAD +10.3 years [95% CI 8.5, 12.1] versus 4.3 years [-2.1, 6.4], p < 0.001). The highest brain-PADs were in secondary-progressive MS (+19.4 years [17.1, 21.9]). Brain-PAD at study entry predicted time-to-disability progression (hazard ratio 1.02 [1.01, 1.03], p < 0.001); though normalised brain volume was a stronger predictor. Greater annualised brain-PAD increases were associated with greater annualised EDSS score (r = 0.26, p < 0.001). INTERPRETATION: The brain-age paradigm is sensitive to MS-related atrophy and clinical progression. A higher brain-PAD at baseline was associated with more rapid disability progression and the rate of change in brain-PAD related to worsening disability. Potentially, 'brain-age' could be used as a prognostic biomarker in early-stage MS, to track disease progression or stratify patients for clinical trial enrolment. This article is protected by copyright. All rights reserved

A search for new MRI criteria for dissemination in space in subjects with a clinically isolated syndrome

The International Panel on the Diagnosis of Multiple Sclerosis (MS) incorporated the Barkhof/Tintoré (B/T) magnetic resonance criteria into their diagnostic scheme to provide evidence of dissemination in space of central nervous system lesions, a prerequisite for diagnosing MS in patients who present with clinically isolated syndromes (CIS). Although specific for MS, the B/T criteria were criticised for their low sensitivity and relative complexity in clinical use. We used lesion characteristics at onset from 349 CIS patients in logistic regression and recursive partitioning modelling in a search for simpler and more sensitive criteria, while maintaining current specificity. The resulting models, all based on the presence of periventricular and deep white matter lesions, performed roughly in agreement with the B/T criteria, but were unable to provide higher diagnostic accuracy based on information from a single scan. Apparently, findings from contrast-enhanced and follow-up magnetic resonance scans are needed to improve the diagnostic algorithm

Manual and automated tissue segmentation confirm the impact of thalamus atrophy on cognition in multiple sclerosis: A multicenter study

Background and rationale: Thalamus atrophy has been linked to cognitive decline in multiple sclerosis (MS) using various segmentation methods. We investigated the consistency of the association between thalamus volume and cognition in MS for two common automated segmentation approaches, as well as fully manual outlining. Methods: Standardized neuropsychological assessment and 3-Tesla 3D-T1-weighted brain MRI were collected (multi-center) from 57 MS patients and 17 healthy controls. Thalamus segmentations were generated manually and using five automated methods. Agreement between the algorithms and manual outlines was assessed with Bland-Altman plots; linear regression assessed the presence of proportional bias. The effect of segmentation method on the separation of cognitively impaired (CI) and preserved (CP) patients was investigated through Generalized Estimating Equations; associations with cognitive measures were investigated using linear mixed models, for each method and vendor. Results: In smaller thalami, automated methods systematically overestimated volumes compared to manual segmentations [ρ=(-0.42)-(-0.76); p-values < 0.001). All methods significantly distinguished CI from CP MS patients, except manual outlines of the left thalamus (p = 0.23). Poorer global neuropsychological test performance was significantly associated with smaller thalamus volumes bilaterally using all methods. Vendor significantly affected the findings. Conclusion: Automated and manual thalamus segmentation consistently demonstrated an association between thalamus atrophy and cognitive impairment in MS. However, a proportional bias in smaller thalami and choice of MRI acquisition system might impact the effect size of these findings

Cerebral atrophy as outcome measure in short-term phase 2 clinical trials in multiple sclerosis

Cerebral atrophy is a compound measure of the neurodegenerative component of multiple sclerosis (MS) and a conceivable outcome measure for clinical trials monitoring the effect of neuroprotective agents. In this study, we evaluate the rate of cerebral atrophy in a 6-month period, investigate the predictive and explanatory value of other magnetic resonance imaging (MRI) measures in relation to cerebral atrophy, and determine sample sizes for future short-term clinical trials using cerebral atrophy as primary outcome measure