Jens Kuhle

A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in CSF are being used in clinical practice. One of the most critical factors in CSF biomarker research is the inadequate powering of studies because of the lack of sufficient samples that can be obtained in single-center studies. Therefore, collaboration between investigators is needed to establish large biobanks of well-defined samples. Standardized protocols for biobanking are a prerequisite to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by preanalytical factors. Here, a consensus report on recommendations for CSF collection and biobanking is presented, formed by the BioMS-eu network for CSF biomarker research in multiple sclerosis. We focus on CSF collection procedures, preanalytical factors, and high-quality clinical and paraclinical information. The biobanking protocols are applicable for CSF biobanks for research targeting any neurologic disease.

FTY720 therapy exerts differential effects on T cell subsets in multiple sclerosis

Background: The oral immunomodulator FTY720 has shown efficacy in patients with relapsing multiple sclerosis (MS). FTY720 functionally antagonizes sphingosine 1-phosphate receptor-1 (S1P1) on T cells and consequently inhibits S1P/S1P1-dependent lymphocyte egress from secondary lymphoid organs. Little is known about the phenotype and function of T cells remaining in peripheral blood during long-term FTY720 treatment. Methods: T cells from FTY720-treated, interferon-beta (IFNβ)-treated and untreated patients with MS, and healthy donors (HD) were analyzed with respect to T cell subpopulation composition, proliferation, and cytokine production. Results: In FTY720-treated patients (n = 16), peripheral blood CD4+ and CD8+ T cell counts were reduced by approximately 80% and 60% when compared to the other groups (IFNβ: n = 7; untreated: n = 5; HD: n = 10). This related to selective reduction of naïve (CCR7+CD45RA+) and central memory (CCR7+CD45RA−) T cells (TCM), and resulted in a relative increase of peripheral effector memory (CCR7−CD45RA− [TEM] and CCR7−CD45RA+ [TEMRA]) T cells. The remaining blood T cell populations displayed a reduced potential to secrete IL-2 and to proliferate in vitro, but rapidly produced interferon-gamma upon reactivation, confirming a functional TEM/TEMRA phenotype. Neither FTY720 nor FTY720-P directly suppressed proliferation or cytokine production by T cells. Conclusion: Therapeutic dosing of FTY720 reduces naïve T cells and TCM, but not TEM, in blood, without affecting T cell function. This is presumably because naïve T cells and TCM express the homing receptor CCR7, allowing recirculation to secondary lymphoid tissues on a regular basis and, thus, trapping of the cells by FTY720 in lymph nodes.

Th17 central memory T cells are reduced by FTY720 in patients with multiple sclerosis

Objective: FTY720 is a sphingosine 1-phosphate (S1P) receptor modulator that showed efficacy in phase II and III clinical trials in patients with multiple sclerosis (MS). FTY720 inhibits lymphocyte egress from secondary lymphoid organs into the peripheral circulation, thereby reducing the number of circulating naïve and central memory T cells, but not effector memory T cells in blood. Little is known to which of these memory T-cell subsets interleukin 17 (IL-17)-producing T cells (Th17 cells) belong, which are considered to be key mediators of inflammation in MS, and how they are affected by treatment with FTY720. In this study, we determined the phenotype and frequency of Th17 cells in blood of untreated, FTY720-treated, and interferon-β (IFNβ)-treated patients with MS and healthy donors. Methods: In a prospective observational study, circulating T cells were phenotypically characterized and Th17 cells enumerated in T-cell subsets ex vivo. Production of IL-17 upon activation and expression of the Th17-specific transcription factor RORC2 was assessed in vitro. Results: Th17 cells were found primarily within central memory T cells in all study populations. FTY720 treatment reduced blood central memory T cells, including RORC2+ and IL-17-producing T cells, by >90%. FTY720 did not per se affect IL-17 production when added to activated T cells in vitro. Conclusion: Phenotypic Th17 cells are defined by a central memory T-cell phenotype. FTY720 reduces these Th17 cells in blood. This is presumably because central memory T cells are retained by FTY720 in secondary lymphoid organs.

Altered expression of miR-17-5p in CD4+ lymphocytes of relapsing-remitting multiple sclerosis patients

MicroRNA (miRNA) are a class of post‐transcriptional regulators of gene expression targeting mRNA for translational repression and/or degradation. We analyzed the expression of 365 miRNA in lymphocytes in relapsing–remitting MS patients, and show the first evidence for distinct miRNA expression profiles in CD4+, CD8+ and B cells in MS when compared with those in healthy volunteers. MiR‐17‐5p, which is involved in autoimmunity, was up‐regulated in CD4+ cells from MS patients. This was correlated with alterations in the expression of potential target genes of miR‐17‐5p, i.e. phosphatase and tensin homology and phosphatidyl‐inositol‐3‐kinase regulatory subunit 1, which were down‐regulated upon stimulation of CD4+ cells with anti‐CD3/CD28 in vitro. Functional experiments with a synthetic inhibitor of miR‐17 supported the link between miRNA expression and the altered target gene expression. Moreover, we found distinct responses of deregulated miRNA to stimulation, i.e. miR‐17‐5p and miR‐193a were strongly up‐regulated, in contrast to the down‐regulation of miR‐497, miR‐1 and miR‐126. Other deregulated miRNA did not respond to the stimulation probably due to other, non‐T‐cell activation related, mechanisms in their mode of action. Our findings support the role of miRNA‐dependent regulatory mechanisms in the immunopathogenesis of MS.

Age-Dependent B Cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis

Multiple sclerosis (MS) typically manifests in early to mid adulthood, but there is increasing recognition of pediatric-onset MS, aided by improvements in imaging techniques. The immunological mechanisms of disease are largely unexplored in pediatric-onset MS, in part because studies have historically focused on adult-onset disease. We investigated autoantibodies to myelin surface Ags in a large cohort of pediatric MS cases by flow cytometric labeling of transfectants that expressed different myelin proteins. Although Abs to native myelin oligodendrocyte glycoprotein (MOG) were uncommon among adult-onset patients, a subset of pediatric patients had serum Abs that brightly labeled the MOG transfectant. Abs to two other myelin surface Ags were largely absent. Affinity purification of MOG Abs as well as competition of binding with soluble MOG documented their binding specificity. Such affinity purified Abs labeled myelin and glial cells in human CNS white matter as well as myelinated axons in gray matter. The prevalence of such autoantibodies was highest among patients with a very early onset of MS: 38.7% of patients less than 10 years of age at disease onset had MOG Abs, compared with 14.7% of patients in the 10- to 18-year age group. B cell autoimmunity to this myelin surface Ag is therefore most common in patients with a very early onset of MS.

Increased neurofilament light chain blood levels in neurodegenerative neurological diseases

Objective Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are cytoskeletal proteins of neurons and their release into cerebrospinal fluid has shown encouraging results as a biomarker for neurodegeneration. This study aimed to validate the quantification of the Nf light chain (NfL) in blood samples, as a biofluid source easily accessible for longitudinal studies. Methods We developed and applied a highly sensitive electrochemiluminescence (ECL) based immunoassay for quantification of NfL in blood and CSF. Results Patients with Alzheimer’s disease (AD) (30.8 pg/ml, n=20), Guillain-Barré-syndrome (GBS) (79.4 pg/ml, n=19) or amyotrophic lateral sclerosis (ALS) (95.4 pg/ml, n=46) had higher serum NfL values than a control group of neurological patients without evidence of structural CNS damage (control patients, CP) (4.4 pg/ml, n=68, p<0.0001 for each comparison, p=0.002 for AD patients) and healthy controls (HC) (3.3 pg/ml, n=67, p<0.0001). Similar differences were seen in corresponding CSF samples. CSF and serum levels correlated in AD (r=0.48, p=0.033), GBS (r=0.79, p<0.0001) and ALS (r=0.70, p<0.0001), but not in CP (r=0.11, p=0.3739). The sensitivity and specificity of serum NfL for separating ALS from healthy controls was 91.3% and 91.0%. Conclusions We developed and validated a novel ECL based sandwich immunoassay for the NfL protein in serum (NfLUmea47:3); levels in ALS were more than 20-fold higher than in controls. Our data supports further longitudinal studies of serum NfL in neurodegenerative diseases as a potential biomarker of on-going disease progression, and as a potential surrogate to quantify effects of neuroprotective drugs in clinical trials.

Increased levels of inflammatory chemokines in amyotrophic lateral sclerosis

Background and purpose:  Amyotrophic lateral sclerosis (ALS) is classically assumed to be a neurodegenerative disorder. Inflammation has been observed in CNS tissue in ALS patients. We investigated the expression and prognostic relevance of proinflammatory chemokines in ALS.

Neurofilament light chain: A prognostic biomarker in amyotrophic lateral sclerosis.

Objective: To test blood and CSF neurofilament light chain (NfL) levels in relation to disease progression and survival in amyotrophic lateral sclerosis (ALS). Methods: Using an electrochemiluminescence immunoassay, NfL levels were measured in samples from 2 cohorts of patients with sporadic ALS and healthy controls, recruited in London (ALS/control, plasma: n = 103/42) and Oxford (ALS/control, serum: n = 64/36; paired CSF: n = 38/20). NfL levels in patients were measured at regular intervals for up to 3 years. Change in ALS Functional Rating Scale–Revised score was used to assess disease progression. Survival was evaluated using Cox regression and Kaplan–Meier analysis. Results: CSF, serum, and plasma NfL discriminated patients with ALS from healthy controls with high sensitivity (97%, 89%, 90%, respectively) and specificity (95%, 75%, 71%, respectively). CSF NfL was highly correlated with serum levels (r = 0.78, p < 0.0001). Blood NfL levels were approximately 4 times as high in patients with ALS compared with controls in both cohorts, and maintained a relatively constant expression during follow-up. Blood NfL levels at recruitment were strong, independent predictors of survival. The highest tertile of blood NfL at baseline had a mortality hazard ratio of 3.91 (95% confidence interval 1.98–7.94, p < 0.001). Conclusion: Blood-derived NfL level is an easily accessible biomarker with prognostic value in ALS. The individually relatively stable levels longitudinally offer potential for NfL as a pharmacodynamic biomarker in future therapeutic trials. Classification of evidence: This report provides Class III evidence that the NfL electrochemiluminescence immunoassay accurately distinguishes patients with sporadic ALS from healthy controls.

Natalizumab alters transcriptional expression profiles of blood cell subpopulations of multiple sclerosis patients

Natalizumab, the most recently approved treatment for relapsing multiple sclerosis (MS) exerts its action through binding to alpha4 integrins. We studied longitudinally gene expression profiles in peripheral blood of MS patients, treated with natalizumab for more than 2 years. The majority of altered genes relates to immune response, signal transduction, adhesion and metabolism. Not only gene expression relevant for T lymphocytes was altered, but also genes regulating B-lymphocyte, neutrophil and erythrocyte functions. Understanding these different gene effects and their interrelationships will provide more insights into additional mechanisms of action of natalizumab and possibly allow better prediction of adverse events.

Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa

Abstract Background: Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are specific cytoskeletal proteins of neurons and their quantification has shown encouraging results as a biomarker for axonal injury. Methods: We aimed at comparing a widely used conventional ELISA for Nf light chain (NfL) with an electrochemiluminescence-based method (ECL assay) and a newly developed single-molecule array (Simoa) method in clinically relevant cerebrospinal fluid (CSF) and serum samples. Results: Analytical sensitivity was 0.62 pg/mL for Simoa, 15.6 pg/mL for the ECL assay, and 78.0 pg/mL for the ELISA. Correlations between paired CSF and serum samples were strongest for Simoa (r=0.88, p<0.001) and the ECL assay (r=0.78, p<0.001) and weaker for ELISA measurements (r=0.38, p=0.030). CSF NfL measurements between the platforms were highly correlated (r=1.0, p<0.001). Serum NfL levels were highly related between ECL assay and Simoa (r=0.86, p<0.001), and this was less visible between ELISA-ECL assay (r=0.41, p=0.018) and ELISA-Simoa (r=0.43, p=0.013). Multiple sclerosis (MS) patients had significantly higher serum NfL levels than controls when measured with Simoa (p=0.001) but not with the other platforms. Conclusions: We found Simoa to be more sensitive than ELISA or the ECL assay. Our results support the feasibility of quantifying NfL in serum; the results correlate with the more-established CSF NfL test. The highly sensitive Simoa technology deserves further studies in larger patient cohorts to clarify whether serum NfL could be used in the future to measure disease severity and determine prognosis or response to treatment interventions in neurological diseases.