Low-intensity repetitive transcranial magnetic stimulation is safe and well tolerated by people living with MS - outcomes of the phase I randomised controlled trial (TAURUS).

Background: Low-intensity repetitive transcranial magnetic stimulation (rTMS), delivered as a daily intermittent theta burst stimulation (iTBS) for four consecutive weeks, increased the number of new oligodendrocytes in the adult mouse brain. Therefore, rTMS holds potential as a remyelinating intervention for people with multiple sclerosis (MS). Objective: Primarily to determine the safety and tolerability of our rTMS protocol in people with MS. Secondary objectives include feasibility, blinding and an exploration of changes in magnetic resonance imaging (MRI) metrics, patient-reported outcome measures (PROMs) and cognitive or motor performance. Methods: A randomised (2:1), placebo controlled, single blind, parallel group, phase 1 trial of 20 rTMS sessions (600 iTBS pulses per hemisphere; 25% maximum stimulator output), delivered over 4-5 weeks. Twenty participants were randomly assigned to 'sham' (n = 7) or active rTMS (n = 13), with the coil positioned at 90° or 0°, respectively. Results: Five adverse events (AEs) including one serious AE reported. None were related to treatment. Protocol compliance was high (85%) and blinding successful. Within participant MRI metrics, PROMs and cognitive or motor performance were unchanged over time. Conclusion: Twenty sessions of rTMS is safe and well tolerated in a small group of people with MS. The study protocol and procedures are feasible. Improvement of sham is warranted before further investigating safety and efficacy.

Improving multiple sclerosis lesion segmentation across clinical sites: A federated learning approach with noise-resilient training.

Accurately measuring the evolution of Multiple Sclerosis (MS) with magnetic resonance imaging (MRI) critically informs understanding of disease progression and helps to direct therapeutic strategy. Deep learning models have shown promise for automatically segmenting MS lesions, but the scarcity of accurately annotated data hinders progress in this area. Obtaining sufficient data from a single clinical site is challenging and does not address the heterogeneous need for model robustness. Conversely, the collection of data from multiple sites introduces data privacy concerns and potential label noise due to varying annotation standards. To address this dilemma, we explore the use of the federated learning framework while considering label noise. Our approach enables collaboration among multiple clinical sites without compromising data privacy under a federated learning paradigm that incorporates a noise-robust training strategy based on label correction. Specifically, we introduce a Decoupled Hard Label Correction (DHLC) strategy that considers the imbalanced distribution and fuzzy boundaries of MS lesions, enabling the correction of false annotations based on prediction confidence. We also introduce a Centrally Enhanced Label Correction (CELC) strategy, which leverages the aggregated central model as a correction teacher for all sites, enhancing the reliability of the correction process. Extensive experiments conducted on two multi-site datasets demonstrate the effectiveness and robustness of our proposed methods, indicating their potential for clinical applications in multi-site collaborations to train better deep learning models with lower cost in data collection and annotation.

A real-world clinical validation for AI-based MRI monitoring in multiple sclerosis.

Modern management of MS targets No Evidence of Disease Activity (NEDA): no clinical relapses, no magnetic resonance imaging (MRI) disease activity and no disability worsening. While MRI is the principal tool available to neurologists for monitoring clinically silent MS disease activity and, where appropriate, escalating treatment, standard radiology reports are qualitative and may be insensitive to the development of new or enlarging lesions. Existing quantitative neuroimaging tools lack adequate clinical validation. In 397 multi-center MRI scan pairs acquired in routine practice, we demonstrate superior case-level sensitivity of a clinically integrated AI-based tool over standard radiology reports (93.3% vs 58.3%), relative to a consensus ground truth, with minimal loss of specificity. We also demonstrate equivalence of the AI-tool with a core clinical trial imaging lab for lesion activity and quantitative brain volumetric measures, including percentage brain volume loss (PBVC), an accepted biomarker of neurodegeneration in MS (mean PBVC -0.32% vs -0.36%, respectively), whereas even severe atrophy (>0.8% loss) was not appreciated in radiology reports. Finally, the AI-tool additionally embeds a clinically meaningful, experiential comparator that returns a relevant MS patient centile for lesion burden, revealing, in our cohort, inconsistencies in qualitative descriptors used in radiology reports. AI-based image quantitation enhances the accuracy of, and value-adds to, qualitative radiology reporting. Scaled deployment of these tools will open a path to precision management for patients with MS.

Multiple sclerosis lesion segmentation: revisiting weighting mechanisms for federated learning.

Background and introduction: Federated learning (FL) has been widely employed for medical image analysis to facilitate multi-client collaborative learning without sharing raw data. Despite great success, FL's applications remain suboptimal in neuroimage analysis tasks such as lesion segmentation in multiple sclerosis (MS), due to variance in lesion characteristics imparted by different scanners and acquisition parameters. Methods: In this work, we propose the first FL MS lesion segmentation framework via two effective re-weighting mechanisms. Specifically, a learnable weight is assigned to each local node during the aggregation process, based on its segmentation performance. In addition, the segmentation loss function in each client is also re-weighted according to the lesion volume for the data during training. Results: The proposed method has been validated on two FL MS segmentation scenarios using public and clinical datasets. Specifically, the case-wise and voxel-wise Dice score of the proposed method under the first public dataset is 65.20 and 74.30, respectively. On the second in-house dataset, the case-wise and voxel-wise Dice score is 53.66, and 62.31, respectively. Discussions and conclusions: The Comparison experiments on two FL MS segmentation scenarios using public and clinical datasets have demonstrated the effectiveness of the proposed method by significantly outperforming other FL methods. Furthermore, the segmentation performance of FL incorporating our proposed aggregation mechanism can achieve comparable performance to that from centralized training with all the raw data.

Multiple sclerosis: structural and functional integrity of the visual system following alemtuzumab therapy.

OBJECTIVE: To investigate potential neuroprotective and pro-remyelinating effects of alemtuzumab in multiple sclerosis (MS), using the visual pathway as a model. METHODS: We monitored clinical, multifocal visual evoked potential (mfVEP) and MRI outcomes in 30 patients commencing alemtuzumab for relapsing MS, and a reference group of 20 healthy controls (HCs), over 24 months. Change in mfVEP latency was the primary endpoint; change in optic radiation (OR) lesion diffusion metrics and Mars letter contrast sensitivity over the course of the study were secondary endpoints. RESULTS: In patients, we observed a mean shortening of mfVEP latency of 1.21 ms over the course of the study (95% CI 0.21 to 2.21, p=0.013), not altered by correction for age, gender, disease duration or change in OR T2 lesion volume. Mean mfVEP latency in the HC group increased over the course of the study by 0.72 ms (not significant). Analysis of chronic OR T2 lesions (patients) showed an increase in normalised fractional anisotropy and axial diffusivity between baseline and 24 months (both p<0.01). Mean Mars letter contrast sensitivity was improved at 24 months vs baseline (p<0.001), and driven by an early improvement, in both patients and HC. CONCLUSION: We found evidence of partial lesion remyelination after alemtuzumab therapy, indicating either natural restoration in the context of a 'permissive' local milieu; or potentially an independent, pro-reparative mechanism of action. The visual system presents a unique opportunity to study function-structure specific effects of therapy and inform the design of future phase 2 MS remyelination trials.

White matter tract-specific quantitative analysis in multiple sclerosis: Comparison of optic radiation reconstruction techniques.

The posterior visual pathway is commonly affected by multiple sclerosis (MS) pathology that results in measurable clinical and electrophysiological impairment. Due to its highly structured retinotopic mapping, the visual pathway represents an ideal substrate for investigating patho-mechanisms in MS. Therefore, a reliable and robust imaging segmentation method for in-vivo delineation of the optic radiations (OR) is needed. However, diffusion-based tractography approaches, which are typically used for OR segmentation are confounded by the presence of focal white matter lesions. Current solutions require complex acquisition paradigms and demand expert image analysis, limiting application in both clinical trials and clinical practice. In the current study, using data acquired in a clinical setting on a 3T scanner, we optimised and compared two approaches for optic radiation (OR) reconstruction: individual probabilistic tractography-based and template-based methods. OR segmentation results were applied to subjects with MS and volumetric and diffusivity parameters were compared between OR segmentation techniques. Despite differences in reconstructed OR volumes, both OR lesion volume and OR diffusivity measurements in MS subjects were highly comparable using optimised probabilistic tractography-based, and template-based, methods. The choice of OR reconstruction technique should be determined primarily by the research question and the nature of the available dataset. Template-based approaches are particularly suited to the semi-automated analysis of large image datasets and have utility even in the absence of dMRI acquisitions. Individual tractography methods, while more complex than template based OR reconstruction, permit measurement of diffusivity changes along fibre bundles that are affected by specific MS lesions or other focal pathologies.

Spectral Analysis Methods Based on Background Subtraction and Curvature Calculation Used in the Detection or Quantification of Hemolysis and Icterus in Blood-derived Clinical Samples.

Objective We aimed to find new methods to detect and quantify hemolysis and icterus which may cause assay biases. These methods need to determine each of these interferents in the presence of various other interferents. They also need to have less stringent requirements in development and implementation than those conventional analyzers currently must satisfy. Design and methods We developed two spectral analysis methods that obtain absorption signals of interest by background subtraction or by calculating the spectral curvatures near the peaks of interest. We optimized and tested the performance of these methods using a plasma sample set with permutations of the levels of hemolysis, icterus, and lipemia (using 510 samples in total). Results The processed signals correlated well with concentrations of hemoglobin and bilirubin, indicators of hemolysis and icterus, respectively. Through iterations of randomly splitting the samples for calibration and testing, the two new methods performed as well as those used on conventional analyzers. We demonstrated that the two methods can lessen the application requirements of 1) prior knowledge of the absorption spectra of individual interferents, 2) calibration over a wide concentration range for each interferent, and 3) the need for full-range spectrophotometers spanning most of the ultraviolet/visible spectrum. We also proposed a hardware setup to detect and quantify hemolysis or icterus with a camera and two optical filters. Conclusions This work indicates that new methods of spectral analysis can reduce practical constraints in the development of interference screening systems. These methods could also benefit other assays that rely on reading spectral signals.

Comprehensive proteome profiling of glioblastoma-derived extracellular vesicles identifies markers for more aggressive disease.

Extracellular vesicles (EVs) play key roles in glioblastoma (GBM) biology and represent novel sources of biomarkers that are detectable in the peripheral circulation. Despite this notionally non-invasive approach to assess GBM tumours in situ, a comprehensive GBM EV protein signature has not been described. Here, EVs secreted by six GBM cell lines were isolated and analysed by quantitative high-resolution mass spectrometry. Overall, 844 proteins were identified in the GBM EV proteome, of which 145 proteins were common to EVs secreted by all cell lines examined; included in the curated EV compendium (Vesiclepedia_559; http://microvesicles.org ). Levels of 14 EV proteins significantly correlated with cell invasion (invadopodia production; r2 > 0.5, p < 0.05), including several proteins that interact with molecules responsible for regulating invadopodia formation. Invadopodia, actin-rich membrane protrusions with proteolytic activity, are associated with more aggressive disease and are sites of EV release. Gene levels corresponding to invasion-related EV proteins showed that five genes (annexin A1, actin-related protein 3, integrin-ß1, insulin-like growth factor 2 receptor and programmed cell death 6-interacting protein) were significantly higher in GBM tumours compared to normal brain in silico, with common functions relating to actin polymerisation and endosomal sorting. We also show that Cavitron Ultrasonic Surgical Aspirator (CUSA) washings are a novel source of brain tumour-derived EVs, demonstrated by particle tracking analysis, TEM and proteome profiling. Quantitative proteomics corroborated the high levels of proposed invasion-related proteins in EVs enriched from a GBM compared to low-grade astrocytoma tumour. Large-scale clinical follow-up of putative biomarkers, particularly the proposed survival marker annexin A1, is warranted.

Membrane proteome analysis of glioblastoma cell invasion.

Glioblastoma multiforme (GBM) tumor invasion is facilitated by cell migration and degradation of the extracellular matrix. Invadopodia are actin-rich structures that protrude from the plasma membrane in direct contact with the extracellular matrix and are proposed to participate in epithelial-mesenchymal transition. We characterized the invasiveness of 9 established GBM cell lines using an invadopodia assay and performed quantitative mass spectrometry-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia, with a 65% difference between the most invasive cell line (U87MG) and the least invasive cell line (LN229) (p = 0.0001). Overall, 1,141 proteins were identified in the GBM membrane proteome; the levels of 49 proteins correlated with cell invasiveness. Ingenuity Pathway Analysis predicted activation "cell movement" (z-score = 2.608, p = 3.94E(-04)) in more invasive cells and generated a network of invasion-associated proteins with direct links to key regulators of invadopodia formation. Gene expression data relating to the invasion-associated proteins ITGA5 (integrin α5), CD97, and ANXA1 (annexin A1) showed prognostic significance in independent GBM cohorts. Fluorescence microscopy demonstrated ITGA5, CD97, and ANXA1 localization in invadopodia assays, and small interfering RNA knockdown of ITGA5 reduced invadopodia formation in U87MG cells. Thus, invasion-associated proteins, including ITGA5, may prove to be useful anti-invasive targets; volociximab, a therapeutic antibody against integrin α5ß1, may be useful for treatment of patients with GBM.

Muscle expression of mutant androgen receptor accounts for systemic and motor neuron disease phenotypes in spinal and bulbar muscular atrophy.

X-linked spinal and bulbar muscular atrophy (SBMA) is characterized by adult-onset muscle weakness and lower motor neuron degeneration. SBMA is caused by CAG-polyglutamine (polyQ) repeat expansions in the androgen receptor (AR) gene. Pathological findings include motor neuron loss, with polyQ-AR accumulation in intranuclear inclusions. SBMA patients exhibit myopathic features, suggesting a role for muscle in disease pathogenesis. To determine the contribution of muscle, we developed a BAC mouse model featuring a floxed first exon to permit cell-type-specific excision of human AR121Q. BAC fxAR121 mice develop systemic and neuromuscular phenotypes, including shortened survival. After validating termination of AR121 expression and full rescue with ubiquitous Cre, we crossed BAC fxAR121 mice with Human Skeletal Actin-Cre mice. Muscle-specific excision prevented weight loss, motor phenotypes, muscle pathology, and motor neuronopathy and dramatically extended survival. Our results reveal a crucial role for muscle expression of polyQ-AR in SBMA and suggest muscle-directed therapies as effective treatments.

Comprehensive tissue processing strategy for quantitative proteomics of formalin-fixed multiple sclerosis lesions.

Formalin-fixed (FF) autopsy tissue comprises the bulk of existing Multiple Sclerosis (MSc) pathology archives, providing a rich pool of material for biomarker discovery and disease characterization. Here, we present the development of a heat-induced extraction protocol for the proteomic analysis of FF brain tissue, its application to the study of lesion remyelination and its failure in MSc. A 4-round extraction strategy was optimized using FF tissue leading to a 35% increase in the number of proteins identified compared to a single extraction; and a 65% increase in proteins identified with ≥4 peptides. Histological staining of sections with oil red O and luxol fast blue-periodic acid Schiff, required to characterize MSc lesions was found to have minimal effect on LC-MS/MS. The application of the optimized protocol to chronic demyelinated and remyelinated FF MSc lesions and the adjacent periplaque white matter, isolated through laser guided manual dissection from 3 patients, identified 428 unique proteins (0.2% FDR) using LC-MS/MS. Comparison of the lesion types using iTRAQ and 2-D LC-MS/MS revealed 82 differentially expressed proteins. Protein quantitation by iTRAQ and spectral counting was well-correlated (r(s)= 0.7653; p < 10(-30)). The data generated from this work illustrates the scope of the methodology and provides insights into the pathogenesis of MSc and remyelination.

Protein and peptide fractionation, enrichment and depletion: tools for the complex proteome.

The identification, quantitation and global characterisation of all proteins within a given proteome are extremely challenging. This is due to the absolute detection limits of technology as well as the dynamic range in expression of proteins; and the extreme diversity and heterogeneity of the proteome. To overcome such issues, the use of separation technologies has played a critical role in reducing sample complexity. To date, a plethora of chromatographic and electrophoretic fractionation tools have evolved over the years assisting in simplifying complex protein and peptide mixtures. Here, we review a range of these technologies highlighting the challenges of protein and peptide analysis in the context of proteome research and some of the advantages and disadvantages of present techniques.

Mass and charge selective protein fractionation for the differential analysis of T-cell and CD34+ stem cell proteins from cord blood.

Studying biological processes through protein expression is difficult due to the dynamic flux of the proteome, the extreme diversity and heterogeneity, the wide range of cellular protein expression, and the limited detection range of technology. Fractionating complex biological mixtures can help overcome these issues but it can be a challenging process particularly when fractionating rare samples (

Peptide enrichment and protein fractionation using selective electrophoresis.

In recent times, the analysis of the peptidome has become increasingly valuable to gain a better understanding of the critical roles native peptides play in biological processes. Here, we show a technique using a novel electrophoretic device named MF10, for the fractionation of proteins and peptides based on size and also pH in low volume liquid phase under an electric field. A 1 microM, 7-protein and peptide standard mix ranging from 1 to 25 kDa has been used to show peptide migration into a fraction contained by 1-5 kDa membranes. Simultaneous fractionation of the higher mass protein standards to the correct fraction also occurred. To assess the MF10's ability to fractionate more complex samples, human plasma was used to enrich for the peptidome below 5 kDa in the presence of the proteome. Peptide enrichment was achieved while simultaneously fractionating higher mass proteins to three other mass restricted fractions. The utility of this approach is demonstrated with the identification (with at least 2 ppm mass accuracy) of 76 unique peptides, equating to 22 proteins enriched to the 1-5 kDa fraction of the MF10.

Dynamic assessment of fibroblast mechanical activity during Rac-induced cell spreading in 3-D culture.

The goal of this study was to determine the morphological and sub-cellular mechanical effects of Rac activation on fibroblasts within 3-D collagen matrices. Corneal fibroblasts were plated at low density inside 100 microm thick fibrillar collagen matrices and cultured for 1-2 days in serum-free media. Time-lapse imaging was then performed using Nomarski DIC. After an acclimation period, perfusion was switched to media containing PDGF. In some experiments, Y-27632 or blebbistatin were used to inhibit Rho-kinase (ROCK) or myosin II, respectively. PDGF activated Rac and induced cell spreading, which resulted in an increase in cell length, cell area, and the number of pseudopodial processes. Tractional forces were generated by extending pseudopodia, as indicated by centripetal displacement and realignment of collagen fibrils. Interestingly, the pattern of pseudopodial extension and local collagen fibril realignment was highly dependent upon the initial orientation of fibrils at the leading edge. Following ROCK or myosin II inhibition, significant ECM relaxation was observed, but small displacements of collagen fibrils continued to be detected at the tips of pseudopodia. Taken together, the data suggests that during Rac-induced cell spreading within 3-D matrices, there is a shift in the distribution of forces from the center to the periphery of corneal fibroblasts. ROCK mediates the generation of large myosin II-based tractional forces during cell spreading within 3-D collagen matrices, however residual forces can be generated at the tips of extending pseudopodia that are both ROCK and myosin II-independent.

Prefractionation, enrichment, desalting and depleting of low volume and low abundance proteins and peptides using the MF10.

The success of proteomics relies heavily in the ability to characterize very diverse species of proteins. This diversity stems from a proteins physicochemical properties, its copy number and abundance and its association with other proteins. Prefractionation and simplification of biological samples prior to downstream MS analysis is showing some virtue in obtaining greater depth of protein analysis. The MicroFlow MF10 is a prefractionation device for low volume, low abundance complex samples that can also enrich for very specific species of proteins based on charge and/or size either in native or denaturing format. It has also been used to desalt and deplete samples of contaminating ions or proteins. Although this instrument is only in its infancy in terms of exploring its capabilities, the technology has been used successfully for the fractionation of plasma proteins Wasinger(1), Omenn GS(2), as well as purification of human growth hormone Catzel D(3) antibodies Cheung(4) and IgY Gee(5) and other complex samples.

Analysis of the pattern of subcellular force generation by corneal fibroblasts after Rho activation.

PURPOSE: To determine the structural and subcellular mechanical effects of Rho activation on corneal fibroblasts in three-dimensional collagen matrices. METHODS: Human corneal fibroblasts were plated at low density in 100-microm thick fibrillar collagen matrices and cultured for 1 or 2 days in serum-free media. Time-lapse imaging was then performed at 1- to 2-minute intervals with Nomarski differential interference contrast. After 1 hour, perfusion was switched to serum-free media containing 1 micromol/L lysophosphatidic acid (LPA). After an additional 30 to 60 minutes, the Rho kinase (ROCK) inhibitor Y-27632 was added to the perfusion media. Changes in cell structure and extracellular matrix deformation were measured with MetaMorph. RESULTS: Addition of LPA activated Rho and induced retraction of cell processes and cellular contraction, as indicated by decreases in cell length (-12.1%+/-7.0%; P<0.05) and cell area (-13.1%+/-13.5%; P=0.06). Force generation was greatest along the cell body in all cases, as indicated by the location of maximum extracellular matrix compression. Subsequent addition of Y-27632 resulted in relaxation of extracellular matrix stress, and reextension of cellular processes. CONCLUSIONS: The data show that Rho induces rapid contraction of corneal fibroblasts in three-dimensional collagen matrices. Forces are generated primarily along the cell body through a ROCK-dependent mechanism.

Confocal assessment of the effects of fourth-generation fluoroquinolones on the cornea.

PURPOSE: To evaluate the toxicity of fourth-generation fluoroquinolone antibiotic solutions on the rabbit corneal epithelium. METHODS: In vivo confocal microscopy was used to assess epithelial structure in 18 rabbits, and tight junction integrity of superficial epithelial cells was evaluated with ZO-1 labeling in 10 rabbits. Eyes were bathed with commercial solutions of moxifloxacin (Vigamox) or gatifloxacin (Zymar) solution for 3 minutes, rinsed with balanced salt solution, and immediately examined. Balanced salt solution rinsing alone served as the control. RESULTS: A decrease in epithelial cell size was observed after treatment with Zymar (P < 0.05, two-way repeated-measures analysis of variance), but not with Vigamox or the control. Normal ZO-1 organization was observed in controls and eyes treated with Vigamox. ZO-1 staining in eyes treated with Zymar was disrupted, patchy, and generally weaker than that in control eyes. CONCLUSIONS: After short-term, intensive exposure to Vigamox, corneal epithelial integrity and tight junction organization are maintained. Zymar induces a loss of superficial epithelial cells and breakdown of tight junctions under similar conditions.

Evaluation of intrastromal lipid deposits after intacs implantation using in vivo confocal microscopy.

PURPOSE: To assess the structure and location of intrastromal lipid deposits after implantation of Intacs by using in vivo confocal microscopy. METHODS: Seven eyes of six patients were examined by in vivo confocal microscopy 5 years (n = 6) or 2 months (n = 1) after uncomplicated implantation of Intacs for the correction of mild myopia. Selected images from all corneal layers were qualitatively evaluated for structural changes, with special attention paid to areas surrounding the Intacs implants. RESULTS: In the peripheral cornea of eyes examined 5 years after surgery, epithelial and endothelial cell layers appeared normal. Tandem scanning confocal microscopy showed stromal haze surrounding the implants in all eyes examined, but no keratocyte activation was seen. Reflective amorphous or crystalline structures consistent with lipid deposition were detected in all eyes with long-term implantation of Intacs. Deposits were localized to the inner and outer edges of Intacs segments and to the region anterior to the implant. Confocal microscopy did not show any deposits in the eye examined 2 months after surgery, although the region anterior to the implant appeared hazy and edematous. Areas central to the implant appeared normal in all eyes. CONCLUSIONS: The mechanical and physiologic stresses introduced by the implantation of Intacs lead to the accumulation of lipid deposits in the extracellular matrix. By using in vivo confocal microscopy, the location and structure of these deposits can be determined.

RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins.

We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding exosite-2 (exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite-2. We conclude that a RNA aptamer to thrombin exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action.