Towards quantifying axonal damage in blood samples from patients with neurological diseases.

PhDReliable biomarkers of axonal damage are urgently needed in neurological diseases. Neurofilaments (Nf) are specific structural elements of neurons composed of at least three subunits: Nf light chain (NfL), Nf medium and Nf heavy chain (NfH). This PhD aimed to characterise NfL levels and their correlation with clinical features in patients with neurological diseases with a different rate of progression and following and under different treatment regimes. An important aim was also to develop a bioassay for NfL measurements in blood. Cerebrospinal fluid (CSF) NfL levels discriminated patients with a clinically isolated syndrome (CIS) (p=0.001) or multiple sclerosis (MS) (p=0.035) from healthy controls more efficiently, and was more sensitive to change after natalizumab therapy (p<0.0001) than CSF NfH (p=0.002). Further, CSF NfL levels decreased in fingolimodtreated MS patients (p=0.001), but not in those receiving placebo (p=0.433). Based on these findings, a sensitive method for the detection of NfL in serum was developed and validated. Patients with neurological diseases had higher serum NfL values than controls. In acute spinal cord injury (SCI), serum NfL levels correlated with injury severity and long-term motor outcome, and Minocycline treatment was associated with decreased NfL levels in complete SCI patients compared to placebo. Finally, I found that serum NfL levels were higher in CIS patients than in healthy controls but did not predict conversion to clinically definite MS (CDMS). Independent predictors of CDMS were instead oligoclonal bands, number of T2 lesions and age at CIS. Lower 25-OHvitamin D levels were associated with CDMS in univariate analysis, but this was attenuated in the multivariate model. In conclusion, NfL proved to be an analytically stable protein which is an important prerequisite for biomarkers. The role of NfL quantification as a surrogate measure of neuroaxonal damage is corroborated by my findings and further supports the usefulness of NfL as a putative biomarker of axonal damage in various neurological diseases

Validation of quantitative scores derived from motor evoked potentials in the assessment of primary progressive multiple sclerosis: a longitudinal study

Objective:; To evaluate the sensitivity to change of differently calculated quantitative scores from motor evoked potentials (MEP) in patients with primary progressive multiple sclerosis (PPMS).; Methods:; Twenty patients with PPMS had MEP to upper and lower limbs at baseline, years 1 and 2 measured in addition to clinical assessment [Expanded Disability Status Scale (EDSS), ambulation score]; a subsample (; n; = 9) had a nine-hole peg test (NHPT) and a timed 25-foot walk (T25FW). Quantitative MEP scores for upper limbs (qMEP-UL), lower limbs (qMEP-LL), and all limbs (qMEP) were calculated in three different ways, based on; z; -transformed central motor conduction time (CMCT), shortest corticomuscular latency (CxM-sh), and mean CxM (CxM-mn). Changes in clinical measures and qMEP metrics were analyzed by repeated-measures analysis of variance (rANOVA), and a factor analysis was performed on change in qMEP metrics.; Results:; Expanded Disability Status Scale and ambulation score progressed in the rANOVA model (; p; < 0.05;; post-hoc; comparison baseline-year 2,; p; < 0.1). Lower limb and combined qMEP scores showed significant deterioration of latency (; p; < 0.01, MEP-LL_CxM-sh:; p; < 0.05) and in; post-hoc; comparisons (baseline-year 2,; p; < 0.05), qMEP_CxM-mn even over 1 year (; p; < 0.05). Effect sizes were higher for qMEP scores than for clinical measures, and slightly but consistently higher when based on CxM-mn compared to CxM-sh or CMCT. Subgroup analysis yielded no indication of higher sensitivity of timed clinical measures over qMEP scores. Two independent factors were detected, the first mainly associated with qMEP-LL, the second with qMEP-UL, explaining 65 and 29% of total variability, respectively.; Conclusions:; Deterioration in qMEP scores occurs earlier than EDSS progression in patients with PPMS. Upper and lower limb qMEP scores contribute independently to measuring change, and qMEP scores based on mean CxM are advantageous. The capability to detect subclinical changes longitudinally is a unique property of EP and complementary to clinical assessment. These features underline the role of EP as candidate biomarkers to measure effects of therapeutic interventions in PPMS

Alternatives to current disease-modifying treatment in MS: what do we need and what can we expect in the future?

Abstract. : Disease-modifying treatments (DMTs) for multiple sclerosis (MS) are now widely available, and their beneficial effects on relapse rates, magnetic resonance imaging outcomes and, in some cases, relapse-related disability have been shown in numerous clinical studies. However, as these treatments are only partially effective in halting the MS disease process, the search for improved treatment regimens and novel therapies must continue. Strategies to improve our therapeutic armamentarium have to take into account the different phases or parts of the pathogenesis of the disease. Available treatments address systemic immune dysfunction, blood-brain barrier permeability and the inflammatory process in the central nervous system. Currently, patients who fail to respond adequately to first-line DMTs are often considered as candidates for intensive immunosuppression with cytostatic agents or even autologous stem cell transplantation.However, new approaches are being developed. Combination therapies offer an alternative approach that may have considerable potential to improve therapeutic yield and, although likely to present considerable challenges in terms of trial design, this certainly seems to be a logical step forward in view of the complex pathology of MS. Several new drugs are also being developed with the aim of providing more effective, convenient and/or specific modulation of the inflammatory component of the disease. These treatments include humanised monoclonal antibodies such as the anti-VLA-4 antibody natalizumab, inhibitors of intracellular activation, signalling pathways and T-cell proliferation, and oral immunomodulators such as sirolimus, teriflunomide or statins. There remains, however, an urgent need for treatments that protect against demyelination and axonal loss, or promote remyelination/regeneration. Due to the chronicity of MS, the therapeutic window for neuroprotective agents is wider than that following stroke or acute spinal cord injury, and may therefore allow the use of some drugs that have proven disappointing in other situations. Novel potential neuroprotective agents such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid antagonists and ion-channel blockers will be entering Phase II trials in MS in the near future, and it is hoped that these agents will mark the start of a new era for DMTs for M

Screening for balance disorders in mildly affected multiple sclerosis patients

Multiple sclerosis (MS) patients often complain about balance problems when Romberg's test and tandem gait are normal. The aim of the study was to determine if measures of trunk sway taken during a battery of stance and gait tasks could be used to detect subclinical balance disorders. We recorded trunk angular sway in the pitch and roll directions from 20 MS patients (EDSS 1.4±0.5) and 20 age- and gender-matched healthy controls (HCs), during 12 stance and gait tasks. We filmed 22 subjects simultaneously. Two neurologists assessed the videos, deciding whether task performance was pathological. Sway measures were significantly different between patients and HCs in eight out of 12 balance tasks. The most significant differences between MS patients and HCs were pitch angle range standing on one leg with eyes open on a firm surface (mean 3.13° vs. 2.09°, p=0.005), and on a foam support surface (mean 6.24° vs. 2.96°, p=0.006), pitch velocity range walking 8m with eyes closed (mean 75.5 vs. 50.2°/s, p<0.001) and pitch velocity range walking 3m on heels (mean 85.37 vs. 60.9°/s, p=0.002). Multivariate analysis revealed a model with three tasks which detected balance disorders in 84% of the MS patients and 90% of the HCs correctly. The neurologists achieved accuracies of 30% for the MS patients and 82% for the HCs. Using trunk sway measures during stance and gait tasks is a sensitive screening method for balance problems in MS patients, and is more accurate than assessment by trained neurologist

Placental α-microglobulin-1 to detect uncertain rupture of membranes in a European cohort of pregnancies

Purpose: We evaluated the performance of the placental alpha-microglobulin-1 immunoassay (AmniSure®, AT) in cervicovaginal secretions in patients with uncertain rupture of membranes (ROM) and investigated the influence of the examiners experience. Methods: This prospective cohort study was performed in pregnant women (17-42weeks of gestation) with signs of possible ROM. Evaluation included clinical assessment, examination for cervical leakage, Nitrazine test and measurement of the amniotic fluid index by ultrasound and AT. ROM occurrence was based on review of the medical records after delivery. Results: 199 women were included. AT had a sensitivity of 94.4%; specificity of 98.6%; positive predictive value, 96.2%; negative predictive value, 98.0%. Clinical assessment showed a sensitivity of 72.2%; specificity of 97.8%; positive predictive value, 92.9%; negative predictive value, 90.6%. AT was more sensitive for diagnosing ROM (p=0.00596) compared to clinical assessment, independent of the examiners experience. Furthermore, the sole use of AT reduced costs by 58.4% compared to clinical assessment. Conclusions: AT was more sensitive compared to clinical assessment, independent of the examiners experience and gestational age. Our data extend its use in patients with uncertain ROM. Moreover, AT seems to be a cost-effective approach in the assessment of these patient

Genomics and proteomics: role in the management of multiple sclerosis

Epidemiological studies and neuro-imaging have provided important insights into the natural course and prognostic factors of multiple sclerosis (MS), but our ability to predict different courses of the disease, and especially its response to treatment, is still very limited. Pharmacogenetic, pharmacogenomic and proteomic studies aim to assess gene and protein function in disease and promise to help to fill this important gap in our knowledge. Such studies may increase our understanding of disease mechanisms and responses to therapeutic compounds. Large-scale transcriptional expression profiling can be performed using gene chip microarrays; this technology allows screening for differentially expressed genes without having well-defined underlying hypotheses ("discovery-driven research”). To complement the technique, real time reverse transcription and polymerase chain reaction (RT-PCR) can be used for more targeted profiling and provides quantitative data on pre-selected genes. However, to maximise their clinical utility, expression profiling results need to be combined with well-documented clinical and imaging data. Two forthcoming studies will investigate the long-term effects of early treatment with interferon beta-1b (IFNβ) on the course of MS. The BENEFIT (BEtaseron®/Betaferon® in Newly Emerging MS for Initial Treatment) study will incorporate pharmacogenetic and pharmacogenomic analyses to determine the genetic elements controlling treatment response. BEST-PGx (Betaferon®/Betaseron® in Early relapsing-remitting MS Surveillance Trial—Pharmacogenomics) is an exploratory 2-year study that will investigate the value of RNA expression profiling and pharmacogenetics in predicting treatment response to IFNβ in patients with early relapsing MS. The main goal of BEST-PGx is the identification of differences in gene expression profiles of patients showing differential treatment responses. In addition, this study may reveal new information relevant to the mechanism of action of interferon treatment in MS and also to differences in the underlying pathology of the immune system. These data may help us approach the goal of a really "individualised therapy” with increased efficacy, reduced adverse drug reactions and more efficient use of healthcare resource

Adaptation of antiretroviral therapy in human immunodeficiency virus infection with central nervous system involvement

The authors describe a patient with known human immunodeficiency virus (HIV)-1 infection who presented with two generalized seizures and was found to have extensive white matter disease and a left/bilateral temporo-occipital focal slowing on electroencephalography (EEG). There were no magnetic resonance imaging (MRI) or cerebrospinal fluid (CSF) indications for opportunistic infection. Plasma viremia was controlled, whereas viral replication was uncontrolled in CSF. CSF-specific genotype-guided adaptation of the antiretroviral therapy in order to optimize central nervous system (CNS) penetration resulted in clinical improvement and normalization of MRI and EEG. Our case report illustrates the importance of individualized antiretroviral therapy in HIV infected patients with neurological complication

The CCR5 antagonist maraviroc exerts limited neuroprotection without improving neurofunctional outcome in experimental pneumococcal meningitis.

One-third of pneumococcal meningitis (PM) survivors suffer from neurological sequelae including learning disabilities and hearing loss due to excessive neuroinflammation. There is a lack of efficacious compounds for adjuvant therapy to control this long-term consequence of PM. One hallmark is the recruitment of leukocytes to the brain to combat the bacterial spread. However, this process induces excessive inflammation, causing neuronal injury. Maraviroc (MVC)-a CCR5 antagonist-was demonstrated to inhibit leukocyte recruitment and attenuate neuroinflammation in several inflammatory diseases. Here, we show that in vitro, MVC decreased nitric oxide production in astroglial cells upon pneumococcal stimulation. In vivo, infant Wistar rats were infected with 1 × 104 CFU/ml S. pneumoniae and randomized for treatment with ceftriaxone plus MVC (100 mg/kg) or ceftriaxone monotherapy. During the acute phase, neuroinflammation in the CSF was measured and histopathological analyses were performed to determine neuronal injury. Long-term neurofunctional outcome (learning/memory and hearing capacity) after PM was assessed. MVC treatment reduced hippocampal cell apoptosis but did not affect CSF neuroinflammation and the neurofunctional outcome after PM. We conclude that MVC treatment only exerted limited effect on the pathophysiology of PM and is, therefore, not sufficiently beneficial in this experimental paradigm of PM

Dynamics of Inflammatory and Neurodegenerative Biomarkers after Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis

Autologous hematopoietic stem cell transplantation (aHSCT) is a highly efficient treatment of multiple sclerosis (MS), and hence it likely normalizes pathological and/or enhances beneficial processes in MS. The disease pathomechanisms include neuroinflammation, glial cell activation and neuronal damage. We studied biomarkers that in part reflect these, like markers for neuroinflammation (C-X-C motif chemokine ligand (CXCL) 9, CXCL10, CXCL13, and chitinase 3-like 1 (CHI3L1)), glial perturbations (glial fibrillary acidic protein (GFAP) and in part CHI3L1), and neurodegeneration (neurofilament light chain (NfL)) by enzyme-linked immunosorbent assays (ELISA) and single-molecule array assay (SIMOA) in the serum and cerebrospinal fluid (CSF) of 32 MS patients that underwent aHSCT. We sampled before and at 1, 3, 6, 12, 24 and 36 months after aHSCT for serum, as well as before and 24 months after aHSCT for CSF. We found a strong increase of serum CXCL10, NfL and GFAP one month after the transplantation, which normalized one and two years post-aHSCT. CXCL10 was particularly increased in patients that experienced reactivation of cytomegalovirus (CMV) infection, but not those with Epstein-Barr virus (EBV) reactivation. Furthermore, patients with CMV reactivation showed increased Th1 phenotype in effector memory CD4+ T cells. Changes of the other serum markers were more subtle with a trend for an increase in serum CXCL9 early post-aHSCT. In CSF, GFAP levels were increased 24 months after aHSCT, which may indicate sustained astroglia activation 24 months post-aHSCT. Other CSF markers remained largely stable. We conclude that MS-related biomarkers indicate neurotoxicity early after aHSCT that normalizes after one year while astrocyte activation appears increased beyond that, and increased serum CXCL10 likely does not reflect inflammation within the central nervous system (CNS) but rather occurs in the context of CMV reactivation or other infections post-aHSCT

Antimyelin antibodies in clinically isolated syndromes correlate with inflammation in MRI and CSF

Objective: We investigated the correlation of anti-myelin oligodendrocyte glycoprotein- (anti-MOG) and anti-myelin basic protein antibodies (anti-MBP) in serum of CIS patients with inflammatory signs in MRI and in CSF and, as previously suggested, the incidence of more frequent and rapid progression to clinically definite MS (CDMS). Methods: 133 CIS patients were analysed for anti-MOG and anti-MBP (Western blot). Routine CSF and cranial MRI (quantitatively and qualitatively) measures were analyzed. 55 patients had a follow-up of at least 12 months or until conversion to CDMS. Results: Patients with anti-MOG and anti-MBP had an increased intrathecal IgG production and CSF white blood cell count (p = 0.048 and p = 0.036). When anti-MBP alone, or both antibodies were present the cranial MRI showed significantly more T2 lesions (p = 0.007 and p = 0.01, respectively). There was a trend for more lesion dissemination in anti-MBP positive patients (p = 0.076). Conversely, anti-MOG- and/or anti-MBP failed to predict conversion to CDMS in our follow-up group (n = 55). Only in female patients with at least one MRI lesion (n = 34) did the presence of anti-MOG correlate with more frequent (p = 0.028) and more rapid (p = 0.0209) transition to CDMS. Conclusions: Presence of anti-MOG or anti-MBP or both was not significantly associated with conversion to CDMS in our CIS cohort. However, patients with anti-MOG and anti-MBP had higher lesion load and more disseminated lesions in cranial MRI as well as higher values for CSF leucocytes and intrathecal IgG production. Our data support a correlation of anti-MOG and anti-MBP to inflammatory signs in MRI and CSF. The prognostic value of these antibodies for CDMS, however, seems to be less pronounced than previously reporte