Natalizumab exerts a suppressive effect on surrogates of B cell function in blood and CSF.

BACKGROUND: Natalizumab for multiple sclerosis (MS) increases the risk of progressive multifocal leukoencephalopathy (PML). OBJECTIVE: We aimed to assess the effect of natalizumab on cellular composition and functional B cell parameters including patients with natalizumab-associated PML (n=37). METHODS: Cellular composition by flow cytometry, levels of immunoglobulin (Ig)G/IgM by immunonephelometry, and oligoclonal bands by isoelectric focusing were studied in blood and cerebrospinal fluid. RESULTS: In MS patients treated with natalizumab without PML (n=59) the proportion of CD19+ B cells was higher in blood, but lower in cerebrospinal fluid compared with MS patients not treated with natalizumab (n=17). The CD4/CD8-ratio in cerebrospinal fluid was lower, and IgG and IgM levels as well as the IgG index dropped in longitudinal samples during natalizumab therapy. Oligoclonal bands persisted, but the total amount of the intrathecally produced IgG fraction, and the polyclonal intrathecal IgG reactivity to measles, rubella, and zoster declined. At the time of diagnosis of PML patients with natalizumab-associated PML had low total IgG levels in blood and cerebrospinal fluid. CONCLUSIONS: Natalizumab impacts B and T cell distribution and exerts an inhibitory effect on surrogates of B cell function in periphery and in cerebrospinal fluid, potentially contributing to the increased risk of developing PML.

Patterns of TH1/TH2 cytokines predict clinical response in multiple sclerosis patients treated with glatiramer acetate.

The patterns of Th1/Th2 cytokines in relapsing-remitting multiple sclerosis were analyzed to evaluate their relevance as biomarkers of therapy response to glatiramer acetate (GA). Serum interferon-γ (IFN-γ), osteopontin and interleukin (IL)-2, IL-4, and IL-10 were measured in 19 relapsing-remitting multiple sclerosis patients treated with GA in a prospective study over 3 years. The quotient (IL-2 + IFN-γ)/(IL-4 + IL-10) was elevated in patients with relapses as compared to relapse-free patients after 12 (p = 0.04), 24 (p = 0.02) and 36 months (p = 0.04). Our study indicates that specific patterns of Th1/Th2 cytokines predict the response to GA therapy.

Cerebrospinal fluid markers of idiopathic intracranial hypertension: is the renin-angiotensinogen system involved?

BACKGROUND: The causes underlying idiopathic intracranial hypertension (IIH) are poorly understood. METHODS: To identify disease-related biomarkers that could offer a new insight into IIH pathology, we analyzed the cerebrospinal fluid (CSF) of 18 patients with IIH and 18 controls using two-dimensional fluorescence differential in-gel electrophoresis (2-D DIGE). RESULTS: We found six proteins that were upregulated in IIH (sterol regulatory element-binding protein 1, zinc-alpha-2-glycoprotein, immunoglobulin heavy constant alpha 1 [IGHA1], alpha-1-antitrypsin [SERPINA1], serotransferrin, haptoglobin) and four proteins that were downregulated (hemopexin, angiotensinogen, vitamin-D-binding protein, transthyretin). The validity of our approach was confirmed for one candidate protein (angiotensinogen). To account for a dependency from blood-CSF barrier function, the ratio of angiotensinogen and albumin CSF-to-serum quotients (Qang/Qalb) was determined, which confirmed the downregulation of angiotensinogen in IIH (p = .04). CONCLUSION: Previous studies showed the intrinsic renin-angiotensin system (RAS) to regulate choroid plexus blood flow and CSF production. Altered levels of angiotensinogen could indicate an imbalance of the RAS in IIH that may provide new targets for therapeutic intervention.

The chemokine CXCL13 is a prognostic marker in clinically isolated syndrome (CIS).

BACKGROUND: There is increasing recognition of the importance of B lymphocytes in the immunopathogenesis of multiple sclerosis (MS), encouraging the evaluation of B cell-associated biomarkers in the cerebrospinal fluid (CSF). We aimed to evaluate the relevance of the B cell chemoattractant CXCL13 as a prognostic marker in patients with clinically isolated syndrome (CIS) regarding conversion to MS, and to compare it to Barkhof criteria in magnetic resonance imaging (MRI), oligoclonal bands (OCB) and the polyspecific intrathecal B cell response against measles, rubella and varicella zoster virus (MRZR). METHODOLOGY/PRINCIPAL FINDINGS: CXCL13 was determined in a prospective study over 2 years including 46 patients that remained CIS over follow-up (CIS-CIS), 45 patients that developed MS (CIS-RRMS), and 30 controls using ELISA. CSF CXCL13 was significantly elevated in CIS-RRMS as compared to CIS-CIS and controls (p<0.001). It was significantly elevated in CIS with OCB (p<0.001), positive MRZR (p=0.04), and gadolinium enhancement in MRI (p=0.02) and showed a significant correlation with CSF leukocyte count (p<0.001) and QIgG (p<0.001). CXCL13 showed the best positive predictive value (PPV) of all parameters investigated (70%, 95%-CI: 53-84%), which could be further increased by combination with Barkhof criteria in MRI (80%). CONCLUSIONS/SIGNIFICANCE: Our data indicate the relevance of CXCL13 in CIS to predict conversion to MS. It furthermore shows CXCL13 to be an important mediator in the inflammatory cascade associated with the polyspecific intrathecal B cell response that manifests itself in OCB and MRZR.

2D DIGE of the cerebrospinal fluid proteome in neurological diseases.

2D DIGE is a promising approach to comparative proteome analysis known for a high sensitivity and high reproducibility compared with classical 2DE techniques. It offers new possibilities for the detection of cerebrospinal fluid (CSF) biomarkers in neurological diseases, such as dementia, amyotrophic lateral sclerosis or multiple sclerosis. We review the first studies using 2D DIGE for analysis of the CSF proteome in neurological diseases and discuss advantages, as well as drawbacks, of the methodological approach with special emphasis on CSF-related aspects.

Proteome analysis reveals candidate markers of disease progression in amyotrophic lateral sclerosis (ALS).

OBJECTIVES: In amyotrophic lateral sclerosis (ALS) the pathological determinants of disease progression remain poorly understood. We aimed to identify a characteristic CSF protein pattern that could provide new candidate biomarkers of disease progression in ALS. METHODS: Using the two-dimensional difference in gel electrophoresis (2-D-DIGE), we compared CSF samples from patients with ALS that showed a rapid progression of disease (ALS-rp, n=9) over a follow-up time of 2 years and from patients with ALS that showed a slow progression of disease over follow-up (ALS-sl, n=9) over the same period. Protein spots that showed significant differences between patients and controls were selected for further analysis by MALDI-TOF mass spectrometry. For validation of identified spots ELISA and nephelometry were performed for two candidate proteins on a second cohort of patients (n=40). RESULTS: We identified 6 different proteins and their isoforms which were all upregulated in ALS-rp as compared to ALS-sl (heat shock protein1, alpha-1 antitrypsin, fetuin-A precursor, transferrin, transthyretin (TTR), nebulin-related anchoring protein). For Fetuin-A and TTR, our findings could be confirmed by quantitative assay. CONCLUSIONS: Fetuin-A and TTR are promising candidate markers for disease progression in ALS that warrant further evaluation on a larger cohort of patients.

Neurofilament ELISA validation.

BACKGROUND: Neurofilament proteins (Nf) are highly specific biomarkers for neuronal death and axonal degeneration. As these markers become more widely used, an inter-laboratory validation study is required to identify assay criteria for high quality performance. METHODS: The UmanDiagnostics NF-light (R)enzyme-linked immunoabsorbent assays (ELISA) for the neurofilament light chain (NfL, 68kDa) was used to test the intra-assay and inter-laboratory coefficient of variation (CV) between 35 laboratories worldwide on 15 cerebrospinal fluid (CSF) samples. Critical factors, such as sample transport and storage, analytical delays, reaction temperature and time, the laboratories' accuracy and preparation of standards were documented and used for the statistical analyses. RESULTS: The intra-laboratory CV averaged 3.3% and the inter-laboratory CV 59%. The results from the test laboratories correlated with those from the reference laboratory (R=0.60, p<0.0001). Correcting for critical factors improved the strength of the correlation. Differences in the accuracy of standard preparation were identified as the most critical factor. Correcting for the error introduced by variation in the protein standards improved the correlation to R=0.98, p<0.0001 with an averaged inter-laboratory CV of 14%. The corrected overall inter-rater agreement was subtantial (0.6) according to Fleiss' multi-rater kappa and Gwet's AC1 statistics. CONCLUSION: This multi-center validation study identified the lack of preparation of accurate and consistent protein standards as the main reason for a poor inter-laboratory CV. This issue is also relevant to other protein biomarkers based on this type of assay and will need to be solved in order to achieve an acceptable level of analytical accuracy. The raw data of this study is available online.

IgG antibodies against measles, rubella, and varicella zoster virus predict conversion to multiple sclerosis in clinically isolated syndrome.

BACKGROUND: Multiple sclerosis (MS) is characterized by a polyspecific B-cell response to neurotropic viruses such as measles, rubella and varicella zoster, with the corresponding antibodies measurable in CSF as the so-called "MRZ reaction" (MRZR). We aimed to evaluate the relevance of MRZR to predict conversion of patients with clinically isolated syndrome (CIS) to MS, and to compare it to oligoclonal bands (OCB) and MRI. METHODOLOGY/PRINCIPAL FINDINGS: MRZR was determined in a prospective study over 2 years including 40 patients that remained CIS over follow-up (CIS-CIS) and 49 patients that developed MS (CIS-RRMS) using ELISA. Using logistic regression, a score (MRZS) balancing the predictive value of the antibody indices included in MRZR was defined (9 points measles, 8 points rubella, 1 point varicella zoster, cutpoint: sum of scores greater 10). MRZR and MRZS were significantly more frequent in CIS-RRMS as compared to CIS-CIS (p=0.04 and p=0.02). MRZS showed the best positive predictive value (PPV) of all parameters investigated (79%, 95%-CI: 54-94%), which could be further increased by combination with MRI (91%, 95%-CI: 59-99%). CONCLUSIONS/SIGNIFICANCE: Our data indicate the relevance of MRZR to predict conversion to MS. It furthermore shows the importance of weighting the different antibody indices included in MRZR and suggest that patients with positive MRZR are candidates for an early begin of immunomodulatory therapy.

Candidate biomarkers of chronic inflammatory demyelinating polyneuropathy (CIDP): proteome analysis of cerebrospinal fluid.

We aimed to identify disease-related biomarkers in CIDP. Using the two-dimensional difference in gel electrophoresis (2-D-DIGE), we compared CSF from patients with CIDP (n = 11) and controls (n = 11). Protein spots that showed a significant difference were further analyzed by MALDI-TOF mass spectrometry. We identified 10 proteins that were upregulated in CIDP (two transferrin isoforms, alpha-1 acid glycoprotein 1 precursor, apolipoprotein A IV, two haptoglobin isoforms, transthyretin (TTR), retinol binding protein and two isoforms of proapolipoprotein) and 1 protein that was downregulated (integrin beta 8). The pathophysiological role of these proteins remains to be clarified by further studies.

Stage-dependent vulnerability of fetal mesencephalic neuroprogenitors towards dopaminergic neurotoxins.

Although extensive knowledge exists on selective vulnerability of dopaminergic neurons against parkinsonism-inducing neurotoxins, there is a complete lack of such data on immature neuroprogenitors. Here we investigated the toxicity of 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine (6-OHDA) and the free radical generator H2O2 on various developmental stages of predopaminergic mesencephalic neuroprogenitors (mNPCs) to evaluate stage-dependency of selective dopaminergic neurotoxicity. Striatal NPCs (sNPCs) without dopaminergic differentiation potential served as controls. Exposure of both undifferentiated NPCs to MPP+ resulted in concentration-dependent cell death at concentrations of >10 microM after 72 h without differences between both cell types, while 6-OHDA led to relevant cell death at 1000 microM after 24h with significant higher sensitivity of mNPCs compared to sNPCs. H2O2 did not induce relevant cell death in all cell types. In NPC cultures differentiated for 14 days, MPP+ showed enhanced toxicity compared to the undifferentiated counterparts, but no significant differences between both NPC type and differentiation conditions. 6-OHDA showed similar toxicity pattern in differentiated compared to undifferentiated NPCs. By evaluating the toxicity of MPP+ on MAP2ab+ neurons derived from both mNPCs and sNPCs as well as tyrosine hydroxylase (TH)+ dopaminergic cells from mNPCs, we found concentration-dependent cell death of all cell types with no increased vulnerability of TH+ cells. Primary TH+ neurons showed significantly higher vulnerability to MPP+. Together, we demonstrated stage-dependent vulnerability of NPCs towards dopaminergic neurotoxins, but no selective vulnerability of NPC-derived TH+ dopaminergic cells towards MPP+. This cell system seems not suitable as a screening tool for selective dopaminergic toxicity.

Proteome analysis of cerebrospinal fluid in amyotrophic lateral sclerosis (ALS).

Cerebrospinal fluid (CSF) is a promising source of biomarkers in amyotrophic lateral sclerosis (ALS). Using the two-dimensional difference in gel electrophoresis (2-D-DIGE), we compared CSF samples from patients with ALS (n = 14) with those from normal controls (n = 14). Protein spots that showed significant differences between patients and controls were selected for further analysis by MALDI-TOF mass spectrometry. For validation of identified spots western blot analysis and ELISA was performed. We identified 2 proteins that were upregulated and 3 proteins that were down-regulated in CSF in ALS. Of these, two proteins (Zn-alpha-2-glycoprotein and ceruloplasmin precursor protein) have not been reported in CSF of patients with ALS so far. In contrast, several other proteins (transferrin, alpha-1-antitrypsin precursor and beta-2-microglobulin) seem to be unspecifically affected in different neurological diseases and may therefore be of limited value as disease-related biochemical markers in ALS. Further evaluation of the candidate proteins identified here is necessary.

Dopamine transporter-mediated cytotoxicity of 6-hydroxydopamine in vitro depends on expression of mutant alpha-synucleins related to Parkinson's disease.

6-Hydroxydopamine (6-OHDA) is widely used to produce animal models of Parkinson's disease (PD) by selectively destroying the nigro-striatal dopaminergic systems, but selective toxicity of 6-OHDA towards dopaminergic cells in vitro remains controversial. Mutant (A30P and A53T) alpha-synuclein isoforms cause increased vulnerability of cells towards various toxic insults and enhance dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin and mitochondrial complex I inhibitor MPP(+) in vitro. Here we extend our recent studies on DAT-mediated toxicity to elucidate the mechanisms involved in selective dopaminergic toxicity of 6-OHDA. We studied the cytotoxicity as well as the toxic mechanisms of 6-OHDA in human embryonic kidney HEK-293 cells ectopically co-expressing mutant alpha-synucleins and the human DAT protein. 6-OHDA showed half-maximal toxic concentration (TC(50)) of 88 microM in HEK-hDAT cells without alpha-synuclein expression after 24 h, whereas the TC(50) values significantly decreased to 58 and 39 microM by expression of A30P and A53T alpha-synuclein, respectively. alpha-Synuclein expression did not affect 6-OHDA toxicity in HEK-293 cells not expressing the DAT. Analysis of intracellular parameters of cellular energy metabolism revealed that the co-expression of mutant alpha-synucleins in HEK-hDAT cells accelerates the reduction of intracellular net ATP levels and ATP/ADP ratios induced by 6-OHDA. Uptake function of the DAT was not altered by expression of alpha-synuclein isoforms. Our data suggest a mechanism of 6-OHDA-induced dopaminergic toxicity involving an interaction of mutant alpha-synucleins with the DAT molecule and subsequent acceleration of cellular energy depletion that might be relevant for the pathogenesis of PD.

Expression of mutant alpha-synucleins enhances dopamine transporter-mediated MPP+ toxicity in vitro.

Mutations in the alpha-synuclein gene (A30P and A53T) are reported to cause familial Parkinson's disease (PD), but it is not known how they result in selective dopaminergic cell death. Here we report on effects of mutant alpha-synucleins on dopamine transporter (DAT)-mediated toxicity of the selective dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+) in vitro. We established human embryonic kidney HEK-293 cell lines stably co-expressing each alpha-synuclein isoform and the human DAT. We demonstrate that expression of all alpha-synuclein isoforms enhances toxicity of general complex I inhibition (rotenone), but only the expression of mutant alpha-synucleins induces significant increased DAT-dependent toxicity of very low concentrations of MPP+ compared to wild-type protein. Proteasomal inhibition by lactacystin does not alter MPP+-toxicity in all cell lines. Our data suggest a new mechanism of MPP+-induced dopaminergic toxicity by an interaction between mutant alpha-synucleins and the DAT, which is independent of the function of the proteasome.