Cecilia Rajda

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.

Catecholamine levels in peripheral blood lymphocytes from multiple sclerosis patients

Circumstantial evidence suggests the involvement of sympathoadrenergic mechanisms in the progress of multiple sclerosis (MS). We studied peripheral blood lymphocytes from MS patients. The levels of dopamine (DA), norepinephrine (NE), epinephrine (E) and their metabolites in extracts of lymphocytes from 58 MS patients and 19 healthy controls were measured by using capillary electrophoresis. The MS patients were divided into clinical subgroups: a laboratory-supported definitive (first-attack) MS group, and a relapsing-remitting (RR) group in remission. The peripheral blood lymphocyte level of epinephrine was significantly higher in the first-attack MS patients (p=0.028) than in the controls. However, the norepinephrine levels were significantly (p=0.027) lower in the RR patients in remission. The catecholamines are known to be able to affect the lymphocyte activity, both by stimulation and by immunosuppression. Our results suggest that the catecholamines are important regulators of lymphocyte activation in MS, and of potential importance as concerns new diagnostic and therapeutic methods.

Interferon-β affects the tryptophan metabolism in multiple sclerosis patients

Tryptophan and its metabolites are of great interest in understanding the pathogenesis of multiple sclerosis (MS). The total levels of tryptophan and its metabolites, kynurenine and kynurenic acid were determined in plasma by capillary liquid chromatography electrospray ionisation tandem mass spectrometry. This is the first report of the plasma levels of these analytes in healthy controls and relapsing–remitting MS patients receiving long‐term and acute interferon‐β (IFN‐β) treatment. Twenty‐four hours post‐administration increased kynurenine levels (first IFN MS versus healthy, P = 0.042) and kynurenine/tryptophan ratio (K/T; first IFN MS versus healthy, P =0.027; first IFN MS versus long‐term IFN MS, P = 0.036) were found. The long‐term IFN MS group had higher K/T ratios at 4 and 12 h post‐administration (P = 0.015 and 0.009, respectively). The increase of K/T ratio in the first IFN MS group indicate an induction of the enzyme indolamine‐2,3‐dioxygenase (IDO), as reported earlier in experimental allergic encephalomyelitis. As IDO is participating in both inflammatory and neurodegenerative processes, further knowledge of its involvement in the pathogenesis of MS is of great importance.

Consensus Guidelines for CSF and Blood Biobanking for CNS Biomarker Studies.

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in cerebrospinal fluid (CSF) are being used in clinical practice. Anti-aquaporin-4 antibodies in serum are currently useful for the diagnosis of neuromyelitis optica (NMO), but we could expect novel CSF biomarkers that help define prognosis and response to treatment for this disease. One of the most critical factors in biomarker research is the inadequate powering of studies performed by single centers. Collaboration between investigators is needed to establish large biobanks of well-defined samples. A key issue in collaboration is to establish standardized protocols for biobanking to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by pre-analytical factors. Here, consensus guidelines for CSF collection and biobanking are presented, based on the guidelines that have been published by the BioMS-eu network for CSF biomarker research. We focussed on CSF collection procedures, pre-analytical factors and high quality clinical and paraclinical information. Importantly, the biobanking protocols are applicable for CSF biobanks for research targeting any neurological disease.

A pilot study on the antibodies to HHV-6 variants and HHV-7 in CSF of MS patients

In the possible role for human herpesviruses (HHV) in the pathogenesis of multiple sclerosis (MS) neither clear distinction between the two variants of HHV-6, nor the involvement of HHV-7 have been described. Therefore, we quantitated HHV-6 variant specific and HHV-7 reacting antibodies in the CSF of 13 patients with MS or other neurological disorders by ELISA. Predominance in the positivity of IgG (67%) and IgM (44%) to HHV-6B over that of IgG (44%) with no detectable IgM to HHV-6A, and no antibodies to HHV-7 were found in the CSF of MS patients. None of these antibodies were found in the CSF of controls. This suggests that, intrathecal chronic active or primary HHV-6B infection might contribute to MS progression, while the local effects of HHV-6A and HHV-7 seem to be less important.

The effects of reward and punishment contingencies on decision-making in multiple sclerosis.

Many patients with multiple sclerosis (MS) show cognitive and emotional disorders. The purpose of this study is to evaluate the role of contingency learning in decision-making in young, non-depressed, highly functioning patients with MS (n=21) and in matched healthy controls (n=30). Executive functions, attention, short-term memory, speed of information processing, and selection and retrieval of linguistic material were also investigated. Contingency learning based on the cumulative effect of reward and punishment was assessed using the Iowa Gambling Test (IGT). In the classic ABCD version of the IGT, advantageous decks are characterized by immediate small reward but even smaller future punishment. In the modified EFGH version, advantageous decks are characterized by immediate large punishment but even larger future reward. Results revealed that patients with MS showed significant dysfunctions in both versions of the IGT. Performances on neuropsychological tests sensitive to dorsolateral prefrontal functions did not predict and did not correlate with the IGT scores. These results suggest that patients with MS show impaired performances on tasks designed to assess decision-making in a situation requiring the evaluation of long-term outcomes regardless of gain or loss, and that this deficit is not a pure consequence of executive dysfunctions.

The Prevalence of Multiple Sclerosis, Distribution of Clinical Forms of the Disease and Functional Status of Patients in Csongrád County, Hungary

Objective: The aim of this study was to determine the prevalence of multiple sclerosis (MS) in the population of Csongrád County, Hungary (400,128 inhabitants) and to determine the functional status (based on the Expanded Disability Status Scale; EDSS) of the patients according to the clinical forms of the disease. Methods: The diagnosis was established with the aid of the Poser diagnostic criteria, and the degree of physical disability was determined using the Kurtzke EDSS. Results: In Csongrád County, the prevalence of MS is 62/100,000. The distribution of patients according to the clinical forms of MS was as follows: 15% had the benign form, 54% had relapsing-remitting MS, 20% had secondary chronic progressive MS and 11% had the primary chronic progressive form of MS. Sixty percent of relapsing-remitting MS patients had an EDSS score of 0–4 points and 33% had an EDSS score of 4.5–6.5 points. Conclusion: The distribution of patients according to the clinical forms of the disease in this representative population is comparable to results in other regions of the world.

Linkage disequilibrium screening for multiple sclerosis implicates JAG1 and POU2AF1 as susceptibility genes in Europeans.

By combining all the data available from the Genetic Analysis of Multiple sclerosis in EuropeanS (GAMES) project, we have been able to identify 17 microsatellite markers showing consistent evidence for apparent association. As might be expected five of these markers map within the Major Histocompatibility Complex (MHC) and are in LD with HLA-DRB1. Individual genotyping of the 12 non-MHC markers confirmed association for three of them — D11S1986, D19S552 and D20S894. Association mapping across the candidate genes implicated by these markers in 937 UK trio families revealed modestly associated haplotypes in JAG1 (p=0.019) on chromosome 20p12.2 and POU2AF1 (p=0.003) on chromosome 11q23.1.

Proteomic Analysis of Cerebrospinal Fluid in a Fulminant Case of Multiple Sclerosis

Multiple Sclerosis (MS) is a chronic disease, but in rare fulminant cases rapid progression may lead to death shortly after diagnosis. Currently there is no diagnostic test to predict disease course. The aim of this study was to identify potential biomarkers/proteins related to rapid progression. We present the case history of a 15-year-old male MS patient. Cerebrospinal fluid (CSF) was taken at diagnosis and at the time of rapid progression leading to the patient’s death. Using isobaric tag labeling and nanoflow liquid chromatography in conjunction with matrix assisted laser desorption/ionization time of flight tandem mass spectrometry we quantitatively analyzed the protein content of two CSF samples from the patient with fulminant MS as well as one relapsing-remitting (RR) MS patient and one control headache patient, whose CSF analysis was normal. Seventy-eight proteins were identified and seven proteins were found to be more abundant in both fulminant MS samples but not in the RR MS sample compared to the control. These proteins are involved in the immune response, blood coagulation, cell proliferation and cell adhesion. In conclusion, in this pilot study we were able to show differences in the CSF proteome of a rapidly progressing MS patient compared to a more typical clinical form of MS and a control subject.

The norepinephrine level is decreased in the lymphocytes of long-term interferon-beta-treated multiple sclerosis patients

The mutual involvement of dopamine and its metabolites in the nervous and immune systems has the potential to provide information on the interaction of these two systems. During a 24-hour period, we used capillary electrophoresis with electrochemical detection to repeatedly measure the intracellular catecholamine concentrations in the peripheral blood lymphocytes of relapsing-remitting multiple sclerosis (RRMS) patients receiving interferon (IFN)-beta-1b (n = 13), and those of IFN-naïve RRMS patients receiving their first IFN-beta-1a injection (n = 19) during this study, and compared them with the levels in healthy controls (n = 12). At baseline, the norepinephrine level was significantly decreased (P = 0.003) in the long-term IFN MS patients compared with the controls. The Time × Group interactions for dopamine (P= 0.5854) and norepinephrine (P = 0.6192) were not significant. The group effects for the individual drugs were P = 0.3529 and 0.1282, respectively. The lower norepinephrine level at baseline in the long-term IFN MS group suggests an immunologically stable phase, in line with our previous findings. This is the first report of the effects of IFN-beta administration on intracellular catecholamines in MS patients. Further studies are necessary to elucidate the immune reactions affected by the catecholamines in MS and to evaluate the roles of these potential immunotransmitters.