Clas Malmeström

Neurofilament light protein and glial fibrillary acidic protein as biological markers in MS

Objective: To determine if CNS-derived proteins present in the CSF of multiple sclerosis (MS) patients reflect different pathologic processes of MS and if these proteins could be useful as biologic markers of disease activity. Methods: Concentrations of the neurofilament light protein (NFL), glial fibrillary acidic protein (GFAP), S100B, and the neuron-specific enolase protein (NSE) were determined in the CSF of 66 MS patients and 50 healthy control subjects with immunoassays. Results: The mean levels of the NFL were increased during all stages of MS compared with controls (p < 0.001), peaking almost 10 times higher during acute relapses. The highest levels of GFAP were found during the secondary progressive course (p < 0.001) with a strong correlation with neurologic deficits (Expanded Disability Status Scale score, r = 0.73, p < 0.001). No increase of S100B or NSE protein was found in the CSF of MS patients compared with control subjects. Conclusions: Increased level of NFL is a general feature of MS, indicating continuous axonal damage during the entire course of the disease with the most profound damage during acute relapses. GFAP may serve as a biomarker for disease progression, probably reflecting the increasing rate of astrogliosis.

Acyclovir Levels in Serum and Cerebrospinal Fluid after Oral Administration of Valacyclovir

ABSTRACT The possible involvement of herpesviruses in the pathogenesis of multiple sclerosis (MS) was recently investigated in a clinical trial of valacyclovir in patients with MS. As an important part of that survey we performed an independent pharmacokinetic study in order to determine the concentration of acyclovir in cerebrospinal fluid (CSF). The concentrations of acyclovir in serum and CSF were measured at steady state after 6 days of oral treatment with 1,000 mg of valacyclovir three times a day. Samples were obtained from 10 patients with MS. All patients had normal renal function, and none had signs of a damaged blood-CSF barrier. The maximum concentration of acyclovir in serum was reached after 1 to 3 h (mean ± standard deviation [SD], 27.1 ± 5.6 μM), and the minimum concentration in serum was 3.1 ± 1.1 μM (mean ± SD). The acyclovir concentrations in CSF at 2 and 8 h were essentially stable, with the mean ± SD levels being 2.5 ± 0.9 and 2.3 ± 0.7 μM, respectively. Similar levels were recorded in serum and CSF samples from five other MS patients after 6 months of oral treatment with valacyclovir at identical dosages. The area under the concentration-time curve (AUC) for acyclovir in CSF to the AUC for acyclovir in serum (CSF/serum AUC ratio) was approximately 20%. We conclude that the improved bioavailability previously reported for valacyclovir in plasma results in higher concentrations in CSF, while the CSF/serum AUC ratio remains constant.

Rituximab versus fingolimod after natalizumab in multiple sclerosis patients

Many JC virus antibody‐positive relapsing–remitting multiple sclerosis (RRMS) patients who are stable on natalizumab switch to other therapies to avoid progressive multifocal leukoencephalopathy.

Immunosuppressive therapy reduces axonal damage in progressive multiple sclerosis

Background: In progressive multiple sclerosis (PMS), disease-modifying therapies have not been shown to reduce disability progression. Objective: The impact from immunosuppressive therapy in PMS was explored by analyzing cerebrospinal fluid (CSF) biomarkers of axonal damage (neurofilament light protein, NFL), astrogliosis (glial fibrillary acidic protein, GFAP), and B-cell regulation (CXCL13). Methods: CSF was obtained from 35 patients with PMS before and after 12–24 months of mitoxantrone (n=30) or rituximab (n=5) treatment, and from 14 age-matched healthy control subjects. The levels of NFL, GFAP, and CXCL13 were determined by immunoassays. Results: The mean NFL level decreased by 51% (1781 ng/l, SD 2018 vs. 874 ng/l, SD 694, p=0.007), the mean CXCL13 reduction was 55% (9.71 pg/ml, SD 16.08, vs. 4.37 pg/ml, SD 1.94, p=0.008), while GFAP levels remained unaffected. Subgroup analysis showed that the NFL reduction was confined to previously untreated patients (n=20) and patients with Gd-enhancing lesions on magnetic resonance imaging (n=12) prior to study baseline. Conclusions: Our data imply that 12–24 months of immunosuppressive therapy reduces axonal damage in PMS, particularly in patients with ongoing disease activity. Determination of NFL levels in CSF is a potential surrogate marker for treatment efficacy and as endpoint in phase II trials of MS.

Reduced cerebrospinal fluid BACE1 activity in multiple sclerosis.

Background Cell and animal experiments have shown that β-site APP-cleaving enzyme 1 (BACE1) may be involved in myelination. Objective Here, we assess the association of cerebrospinal fluid (CSF) BACE1 activity with multiple sclerosis (MS). Methods BACE1 activity and levels of secreted amyloid precursor protein (APP) and amyloid-β (Aβ) isoforms were analyzed in CSF from 100 patients with MS and 114 neurologically healthy controls. Patients with systemic lupus erythematosus (SLE), 26 with and 41 without cerebral engagement, were also included to enable comparisons with regards to another autoimmune disease. A subset of patients with MS and controls underwent a second lumbar puncture after 10 years. Results MS patients had lower CSF BACE1 activity than controls (P = 0.03) and patients with cerebral SLE (P < 0.001). Patients with cerebral SLE had higher BACE1 activity than any other group (P < 0.05 for all comparisons). BACE1 activity correlated with the different amyloid markers in all study groups. BACE1 activity decreased over 10 years in the MS group (P = 0.039) and correlated weakly with clinical disease severity scores in an inverse manner. Conclusions These results suggest an involvement of BACE1 in the MS disease process.

Rituximab in multiple sclerosis: A retrospective observational study on safety and efficacy

Objective: To investigate the safety and efficacy of rituximab in multiple sclerosis (MS). Methods: In this retrospective uncontrolled observational multicenter study, off-label rituximab-treated patients with MS were identified through the Swedish MS register. Outcome data were collected from the MS register and medical charts. Adverse events (AEs) grades 2–5 according to the Common Terminology Criteria for Adverse Events were recorded. Results: A total of 822 rituximab-treated patients with MS were identified: 557 relapsing-remitting MS (RRMS), 198 secondary progressive MS (SPMS), and 67 primary progressive MS (PPMS). At baseline, 26.2% had contrast-enhancing lesions (CELs). Patients were treated with 500 or 1,000 mg rituximab IV every 6–12 months, during a mean 21.8 (SD 14.3) months. During treatment, the annualized relapse rates were 0.044 (RRMS), 0.038 (SPMS), and 0.015 (PPMS), and 4.6% of patients displayed CELs. Median Expanded Disability Status Scale remained unchanged in RRMS (p = 0.42) and increased by 0.5 and 1.0 in SPMS and PPMS, respectively (p = 0.10 and 0.25). Infusion-related AEs occurred during 7.8% of infusions and most were mild. A total of 89 AEs grades ≥2 (of which 76 infections) were recorded in 72 patients. No case of progressive multifocal leukoencephalopathy was detected. Conclusions: This is the largest cohort of patients with MS treated with rituximab reported so far. The safety, clinical, and MRI findings in this heterogeneous real-world cohort treated with different doses of rituximab were similar to those reported in previous randomized controlled trials on B-cell depletion therapy in MS. Classification of evidence: This study provides Class IV evidence that for patients with MS, rituximab is safe and effective.

MicroRNA regulate immune pathways in T-cells in multiple sclerosis (MS)

BackgroundMicroRNA are small noncoding RNA molecules that are involved in the control of gene expression. To investigate the role of microRNA in multiple sclerosis (MS), we performed genome-wide expression analyses of mRNA and microRNA in T-cells from MS patients and controls.MethodsHeparin-anticoagulated peripheral blood was collected from MS-patients and healthy controls followed by isolation of T-cells. MicroRNA and RNA from T-cells was prepared and hybridized to Affymetrix miR 2.0 array and Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA), respectively. Verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsWe identified 2,452 differentially expressed genes and 21 differentially expressed microRNA between MS patients and controls. By Kolmogorov-Smirnov test, 20 of 21 differentially expressed microRNA were shown to affect the expression of their target genes, many of which were involved in the immune system. Tumor necrosis factor ligand superfamily member 14 (TNFSF14) was a microRNA target gene significantly decreased in MS. The differential expression of mir-494, mir-197 and the predicted microRNA target gene TNFSF14 was verified by real-time PCR and ELISA.ConclusionThese findings indicate that microRNA may be important regulatory molecules in T-cells in MS.

Cerebrospinal fluid biomarkers as a measure of disease activity and treatment efficacy in relapsing-remitting multiple sclerosis

Cerebrospinal fluid (CSF) biomarkers can reflect different aspects of the pathophysiology of relapsing‐remitting multiple sclerosis (RRMS). Understanding the impact of different disease modifying therapies on the CSF biomarker profile may increase their implementation in clinical practice and their appropriateness for monitoring treatment efficacy. This study investigated the influence of first‐line (interferon beta) and second‐line (natalizumab) therapies on seven CSF biomarkers in RRMS and their correlation with clinical and radiological outcomes. We included 59 RRMS patients and 39 healthy controls. The concentrations of C‐X‐C motif chemokine 13 (CXCL13), C‐C motif chemokine ligand 2 (CCL2), chitinase‐3‐like protein 1 (CHI3L1), glial fibrillary acidic protein, neurofilament light protein (NFL), and neurogranin were determined by ELISA, and chitotriosidase (CHIT1) was analyzed by spectrofluorometry. RRMS patients had higher levels of NFL, CXCL13, CHI3L1, and CHIT1 than controls (p < 0.001). Subgroup analysis revealed higher NFL, CXCL13 and CHIT1 levels in patients treated with first‐line therapy compared to second‐line therapy (p = 0.008, p = 0.001 and p = 0.026, respectively). NFL and CHIT1 levels correlated with relapse status, and NFL and CXCL13 levels correlated with the formation of new magnetic resonance imaging lesions. Furthermore, we found an association between inflammatory and degenerative biomarkers. The results indicate that CSF levels of NFL, CXCL13, CHI3L1, and CHIT1 correlate with the clinical and/or radiological disease activity, providing additional dimensions in the assessment of treatment efficacy.

Cerebrospinal fluid biomarkers of β-amyloid metabolism in multiple sclerosis:

Background: Amyloid precursor protein (APP) and amyloid β (Aβ) peptides are intensely studied in neuroscience and their cerebrospinal fluid (CSF) measurements may be used to track the metabolic pathways of APP in vivo. Reduced CSF levels of Aβ and soluble APP (sAPP) fragments are reported in inflammatory diseases, including multiple sclerosis (MS); but in MS, the precise pathway of APP metabolism and whether it can be affected by disease-modifying treatments remains unclear. Objective: To characterize the CSF biomarkers of APP degradation in MS, including the effects of disease-modifying therapy. Methods: CSF samples from 87 MS patients (54 relapsing–remitting (RR) MS; 33 secondary progressive (SP) MS and 28 controls were analyzed for sAPP and Aβ peptides by immunoassays, plus a subset of samples was analyzed by immunoprecipitation and mass spectrometry (IP-MS). Patients treated with natalizumab or mitoxantrone were examined at baseline, and after 1–2 years of treatment. Results: CSF sAPP and Aβ peptide levels were reduced in MS patients; but they increased again towards normal, after natalizumab treatment. A multivariate model of IP-MS-measured Aβ species separated the SPMS patients from controls, with RRMS patients having intermediate levels. Conclusions: We confirmed and extended our previous observations of altered CSF sAPP and Aβ peptide levels in MS patients. We found that natalizumab therapy may be able to counteract the altered APP metabolism in MS. The CSF Aβ isoform distribution was found to be distinct in SPMS patients, as compared to the controls.

Soluble TREM-2 in cerebrospinal fluid from patients with multiple sclerosis treated with natalizumab or mitoxantrone.

Background: Microglia-mediated proteolysis of the triggering receptor expressed on myeloid cells-2 (TREM-2) produces soluble TREM-2 (sTREM-2) that can be measured in cerebrospinal fluid (CSF) samples. Loss-of-function mutations in TREM2 or in the gene encoding its adaptor protein cause the rare Nasu–Hakola disease (NHD). Multiple sclerosis (MS) is an autoimmune disease that in common with NHD is characterized by demyelination and microglial activation. Objective: To investigate the potential utility of sTREM-2 as a biomarker for MS and to follow treatment effects. Methods: sTREM-2 was analyzed in CSF samples from subjects with MS (N = 59); relapsing-remitting MS (RRMS) (N = 36), secondary progressive MS (SPMS) (N = 20) and primary progressive MS (PPMS) (N = 3), and controls (N = 27). CSF levels of sTREM-2 were also assessed before and after treatment of patients with natalizumab or mitoxantrone. Results: CSF levels of sTREM-2 were significantly increased in patients with RRMS, SPMS, and PPMS compared with controls. After natalizumab treatment, the levels of sTREM-2 were normalized to control levels. The levels of sTREM-2 were also reduced after mitoxantrone treatment. Conclusion: Increased CSF levels of sTREM-2, a new marker of microglial activation, in MS and normalization upon treatment with either natalizumab or mitoxantrone support a role for microglial activation in active MS.