Search

Background: Adult opsoclonus-myoclonus (OM), a disorder of eye movements accompanied by myoclonus affecting the trunk, limbs, or head, is commonly associated with an underlying malignancy or precipitated by viral infection. Methods: We present the first two reports of post-streptococcal OM associated with antibodies against a 56 kDa protein. Two young girls presented with opsoclonus and myoclonus following a febrile illness and pharyngitis. Protein purification techniques were employed. Amino acid sequences of human neuroleukin (NLK) and streptococcal proteins were compared using the protein-protein BLAST application. Results: The antigen was identified as NLK (glucose-6-phosphate isomerase, GPI). GPI is present on the cell surface of streptococcus making the protein a candidate target for molecular mimicry. Conclusions: We have identified NLK as an antigenic target in two patients with post-streptococcal OM. The pathogenicity of the antibodies is uncertain. The potential role of anti-neuroleukin antibodies in the pathogenesis of OM is discussed. We propose that OM may represent a further syndrome in the growing spectrum of post-streptococcal neurological disorders. The role of streptococcus in OM and the frequency with which anti-NLK responses occur in both post-infectious and paraneoplastic OM should be investigated further.

OBJECTIVE: To determine whether concentrations of soluble E-selectin (sE-selectin), an immunological marker of endothelial activation, were correlated with gadolinium-DPTA enhancement on MRI in patients with multiple sclerosis. METHODS: Serial sE-selectin concentrations were measured in 28 patients with multiple sclerosis undergoing monthly gadolinium (Gd) enhanced MRI of the brain and spinal cord, and in 10 control subjects. C reactive protein (CRP), von Willebrand factor (vWF), and tumour necrosis factor-alpha (TNF alpha) were also determined. RESULTS: Primary progressive patients had significantly increased sE-selectin concentrations compared with the relapsing remitting and secondary progressive patients who had normal sE-selectin concentrations (22.2 (SD1 6.1) ng/ml v 9.8 (SD2.1) ng/ml and 7.7 (SD2.7) ng/ml, respectively, P = 0.03). This difference was attributable to five of the 10 primary progressive patients who had persistently raised sE-selectin concentrations, with relatively inactive MRI studies. No correlation could be found between sE-selectin concentrations and Gd enhancement on MRI, but a close correlation existed between mean concentrations of sE-selectin and TNF alpha (r = 0.71, P < 0.001). Despite raised sE-selectin and TNF alpha concentrations, primary progressive patients had normal CRP concentrations (1.03 (SD1.14) mg/l), which were significantly lower than the relapsing remitting (3.16 (SD2.54) mg/l) and secondary progressive patients (2.28 (SD2.1) mg/l, P = 0.03). Raised CRP concentrations did correlate with infectious episodes, clinical relapse, and Gd enhancement, and were significantly raised when no MRI activity was found. Concentrations of vWF were normal in all patient groups. CONCLUSIONS: The results further high-light the differences between patients with primary progressive and those with relapsing remitting/secondary progressive multiple sclerosis.

Background and purpose It has previously been reported that serum levels of vascular endothelial growth factor are raised after acute ischemic stroke compared to healthy controls. The aim of this prospective study was to ascertain whether serum vascular endothelial growth factor measurements could be used to distinguish between acute ischemic stroke and common stroke mimics in the emergency room. Methods Blood samples were taken on arrival to hospital and daily for six-days, in 44 patients with suspected ischemic stroke (29 acute infarcts and 15 stroke mimics), arriving within 24 h of symptom onset. Vascular endothelial growth factor levels were measured by enzyme-linked immunoassay. The neurological deficit was recorded daily using the National Institute of Health Stroke Scale. Evaluation of infarct volumes was based on diffusion-weighted magnetic resonance imaging. Results Serum vascular endothelial growth factor levels were significantly raised in acute ischemic stroke patients on the day of symptom onset and at all other time points, compared to healthy controls ( P < 0.01). The sensitivity and specificity of vascular endothelial growth factor for diagnosing acute ischemic stroke on admission to hospital were only 69% and 73%, respectively. Vascular endothelial growth factor levels were also elevated in four out of 15 stroke mimics, including three patients presenting with postictal paresis. Conclusions Vascular endothelial growth factor has limited clinical utility in the diagnosis of acute ischemic stroke in the emergency room because levels are also raised in common stroke mimics. Further studies are required to establish the mechanism of vascular endothelial growth factor elevation in postictal paresis.

Background: External lumbar drainage (ELD) is known as a good predictor of favourable outcome in shunting patients suffering from idiopathic normal pressure hydrocephalus (iNPH). Methods: Eleven patients suffering from iNPH had a lumbar drain (LD) inserted for 72 h and participated in a research study to quantify any improvement in their clinical symptoms. The lumbar cerebrospinal fluid (CSF) levels of lactate, 8-isoprostane, vascular endothelial growth factor (VEGF), glial fibrillar acidic protein (GFAP), neurofilament (heavy chain) protein (NF (h)), Aβ 1–42 (β-amyloid) and total tau were assayed samples from all three time points. Results: The concentrations of lactate, VEGF, GFAP and tau increased significantly during the 72 h of drainage. There were also increases in 8-isoprostane and Aβ 1–42 (non significant). The concentration of NF (h) was reduced significantly following 72 h of drainage. There was a significant positive correlation between Aβ 1–42 and total tau in the first sample. GFAP was negatively correlated in a significant fashion with both Aβ 1–42 and total tau. NF (h) was negatively correlated with VEGF. Conclusion: Evidence is provided that ELD is producing measurable changes in the CSF composition of patients with iNPH. The present paper discusses how such changes may be implicated in the pathophysiology of the condition.

Early predictors of prognosis in Guillain-Barre syndrome (GBS) are needed to identify patients who are likely to make a poor recovery and to guide therapeutic decision-making in the acute phase. Here we investigate whether axonal protein biomarkers released into the cerebrospinal fluid (CSF) following proximal axonal damage improve the early prognostic accuracy in GBS. A prospective multicenter study including 132 patients (38 GBS, 38 neurological controls, 42 headaches, 14 chronic inflammatory demyelinating neuropathy). CSF levels of axonal [neurofilament (NfH) and tau] and glial (S100B and glial fibrillary acidic protein) protein biomarkers were measured on admission. Nerve conduction studies were performed at the time of lumbar puncture and patients were classified according to neurophysiological criteria. Outcome was assessed on the Hughes functional score (F-score). Poor outcome was defined as the inability to walk independently (F-score > or = 3). High NfH levels (>0.73 ng/ml) predicted poor outcome (P = 0.01) with an odds ratio of 7.3 and correlated with the outcome F-score (R = 0.51, P < 0.01), as did hTau levels (R = 0.47, P < 0.01). Patients with poor outcome had significantly higher CSF NfH (median 1.78 ng/ml) when compared to those with good outcome (0.03 ng/ml) or all of the control groups (neurological controls 0.18 ng/ml, headaches 0.06 ng/ml, chronic inflammatory demyelinating neuropathy 0.05 ng/ml). Except for age (P < 0.05) and need for ventilatory support (P < 0.05), none of the other features reliably predicted outcome. Improved prognostic accuracy in the acute phase of GBS seems possible using CSF NfH levels.

Background Two cerebrospinal fluid (CSF) biomarkers specific for neurodegeneration have recently emerged — the neurofilament light (NfL, 68 kDa) and heavy (NfH, 190–210 kDa) chains. This study investigated whether the CSF NfH and NfL levels or their stoichiometric relationship changed over time in a neuroprotective treatment trial. Methods Serial CSF samples (n = 95) from 42 patients with multiple system atrophy (MSA), half randomized to treatment with recombinant human growth hormone (r-hGH) and the other half to placebo, were collected at baseline, 6 and 12 months. The concentration of CSF NfL and NfH was determined using standard ELISAs. Results There was no consistent change in the levels of either protein over the 12 month period, or between treatment with active r-hGH versus placebo. The molar stoichiometry of CSF NfL:NfH was 4:1 (R = 0.37, p = 0.0002) and increased following treatment with r-hGH (p = 0.03). Conclusion These results indicate that CSF levels of both NfL and NfH on their own are not useful markers of disease progression in MSA, at least over a 12-month period. Future work is needed to elucidate whether the CSF stoichiometry and dynamics of Nf subunits in individual patients are a feature of the underlying pathology and of diagnostic or prognostic value.

Background and Purpose: Serum S100B has been widely studied as a biomarker in acute ischaemic stroke. The main objective of this review was to appraise the published literature on S100B and determine its clinical applicability. Methods: Medline was searched to identify studies on S100B (or S-100B) in acute ischaemic stroke. The authors have proposed the criteria for a clinically informative serum biomarker for acute ischaemic stroke, and relevant articles relating to these criteria were then selected. Results: Studies have shown that S100B has a low specificity for acute ischaemic stroke because of its tendency to be raised from extracranial sources. Data regarding S100B kinetics compiled from 6 longitudinal studies show that serum levels are not raised immediately following acute ischaemic stroke and peak 3 days after symptom onset. However, serum S100B levels correlate well with infarct volume and are higher in stroke patients at risk of malignant infarction or haemorrhagic transformation after thrombolysis. In addition, serum S100B levels correlate well with functional outcome. Conclusion: The evidence suggests that S100B is not a valuable biomarker for diagnosing acute ischaemic stroke. Instead, it may have a more promising role in non-specialist hospitals, as an additional tool for identifying patients at increased risk of specific early neurological complications after stroke and as a surrogate marker of cerebral damage and functional outcome, particularly in a research setting.

Objectives In this study, the concentrations of uric acid, purine profile and creatinine in samples of cerebrospinal fluid and serum of multiple sclerosis (MS) patients were measured by HPLC and compared with corresponding values recorded in patients without MS (cerebrospinal fluid) and healthy subjects (serum). Design and methods All samples were deproteinized with ultrafiltration (which ensures minimal sample manipulation and efficient protein removal) and then assayed for the synchronous HPLC separation of uric acid, hypoxanthine, xanthine, inosine, adenosine, guanosine and creatinine. Results The values of all compounds assayed were significantly higher in both biological fluids of MS patients with respect to values measured in controls. In particular, serum hypoxanthine, xanthine, uric acid and sum of oxypurines were, respectively, 3.17, 3.11, 1.23 and 1.27-fold higher in these patients than corresponding values recorded in controls ( p Conclusions Differently from what previously reported, we here demonstrate that all purine compounds, including uric acid, are elevated in biological fluids of MS patients. Reinforced by the trend observed for creatinine, this corroborates the notion of sustained purine catabolism, possibly due to imbalance in ATP homeostasis, under these pathological conditions. These results cast doubt on the hypothesis that uric acid is depleted in MS because of increased oxidative stress, rather suggesting that this disease causes a generalized increase in purine catabolism. As observed in other pathological states, uric acid, purine compounds and creatinine, can be considered markers of metabolic energy imbalance rather than of reactive oxygen species, even in MS.

AimsAxonal pathology extends to the axonal cytoarchitecture leaving its signature on axoskeletal proteins. This study investigated whether neurofilament (NfH) phosphorylation would relate to the dynamics of axonal pathology in multiple sclerosis (MS).MethodsNfH phosphoforms (SMI32, SMI34, SMI35) were quantified by ELISA from microdissected samples of control and MS brain and spinal cord. Individual axons were analysed by electron microscopy, densitometrically and morphologically in adjacent tissue sections. Experiments were carried out pre- and post enzymatic dephosphorylation.ResultsIn control tissue a rostro-caudal gradient of NfH indicated an increase in axonal density from the brain gray matter towards the spinal cord. The highest levels of phosphorylated and hyperphosphorylated NfH were found in acute lesions of brain and spinal cord, in contrast to chronic lesions where levels were lower than in white matter, consistent with axonal loss. Dephosphorylated NfH was higher, but less densly packed in MS white matter axons compared to control tissue.ConclusionsThe findings suggest that a less organised/compact axoskeleton or impaired axonal transport may represent an early sign of axonal pathology within the normal appearing white matter in MS. Subsequently a proportional increase of dephosphorylated NfH, aberrant phosphorylation and/or aggregation may occur whilst the protein is transported through the white matter towards the MS plaque, where hyperphosphorylated NfH dominates.

There is increasing interest in imaging cadavers for noninvasive autopsies for research purposes. However, the temperature is well below that of in vivo imaging, and a variety of interesting 'cold brain' effects are observed. At lower temperatures conventional FLAIR sequences no longer produce dark cerebrospinal fluid (CSF); T(1) is reduced from about 4.0 sec in vivo to 1.7 sec at 1 degrees C. The diffusion coefficient (DC) of CSF is much reduced (from 3.1 10(-9) m(2)s(-1) in vivo to 1.1 at 1 degrees C). DC values therefore provide a noninvasive thermometer to measure brain core temperature to within 1.0 degrees C. In three cadavers DC values were 1.1-1.5 10(-9) m(2)s(-1), indicating brain core temperatures of 1-10 degrees C, consistent with external thermocouple measurements. An improved inversion time (TI(0)) can then be found for FLAIR. At 10 degrees C this Cold FLAIR sequence (TI(0) = 1.5 sec) gave black CSF. Expressions for CSF DC and T(1) as a function of temperature were produced. A measurement of CSF DC could be converted directly to temperature and the required TI(0) found. In vitro values of CSF DC were about 1% lower than that of water. Thus, FLAIR imaging can be optimized for cadaveric brains at low and unknown temperatures, thereby improving value for autopsy purposes and facilitating comparisons with in vivo imaging.

Secondary ischaemic deficit adversely affects outcome in patients with subarachnoid hemorrhage (SAH). Astrocytes are vulnerable to ischemia, releasing glial fibrillary acidic protein (GFAP) when challenged. In this study, we followed nine patients with SAH who underwent extra-ventricular drainage for the management of secondary hydrocephalus. Cerebrospinal fluid (CSF) was collected daily for up to 14 days. CSF GFAP was quantified using a standard ELISA. In the patients, we found that the CSF GFAP values were pathologically elevated in 83/89 (93%) of the CSF samples. The levels were highest on day 1 (median = 47.64 ng/mL) and decreased to 11.19 ng/mL on day 3, leveling out at approximately 1 ng/mL after 10 days. In non-survivors, a secondary rise of GFAP levels became significant during the high-risk period for vasospasm, with median levels of 21.76 ng/mL compared to 2.62 ng/mL in the survivors (p = 0.037) on day 6. This study suggests that CSF GFAP levels are of prognostic value in SAH. Additionally, the difference in the slope of GFAP levels between survivors (rapid wash-out) and non-survivors (secondary peaks) may allow difierentiation between primary brain injury from secondary brain damage due to delayed cerebral ischaemia.

A method for the diagnosis of the congenital disorders of glycosylation type I (CDG-I) by SELDI-TOF-MS of serum transferrin immunocaptured on protein chip arrays is described. The underglycosylation of glycoproteins in CDG-I produces glycoforms of transferrin with masses lower than that of the normal fully glycosylated transferrin. Immobilisation of antitransferrin antibodies on reactive-surface protein chip arrays (RS100) selectively enriched transferrin by at least 100-fold and allowed the detection of patterns of transferrin glycoforms by SELDI-TOF-MS using approximately 0.3 microL of serum/plasma. Abnormal patterns of immunocaptured transferrin were detected in patients with known defects in glycosylation (CDG-Ia, CDG-Ib, CDG-Ic, CDG-If and CDG-Ih) and in patients in whom the basic defect has not yet been identified (CDG-Ix). The correction of the N-glycosylation defect in a patient with CDG-Ib after mannose therapy was readily detected. A patient who had an abnormal transferrin profile by IEF but a normal profile by SELDI-TOF-MS analysis was shown to have an amino acid polymorphism by sequencing transferrin by quadrupole-TOF MS. Complete agreement was obtained between analysis of immunocaptured transferrin by SELDI-TOF-MS and the IEF profile of transferrin, the clinical severity of the disease and the levels of aspartylglucosaminidase activity (a surrogate marker for the diagnosis of CDG-I). SELDI-TOF-MS of transferrin immunocaptured on protein chip arrays is a highly sensitive diagnostic method for CDG-I, which could be fully automated using microtitre plates and robotics.

We describe a semi-quantitative method for measuring the relative affinity of antigen-specific oligoclonal IgG bands separated by isoelectric focusing followed by blotting onto antigen-coated membrane and incubation with sodium thiocyanate. When the developed blot is digitised in greyscale, densitograms can be made and peak areas calculated using ImageJ freeware. By expressing peak area as a percentage of the total area under the curve we have shown that there is a statistically significant rise in percentage of peak area for a given band which persists with increasing molarities of sodium thiocyanate.

The authors describe the measurement of S100B protein in brain extracellular fluid (ECF) of patients with acute brain injury (traumatic brain injury and subarachnoid haemorrhage) using the technique of microdialysis. To our knowledge, this is the first report of S100B measurement in the human brain. Acute Brain Injury (ABI) is a leading cause of death and disability and the need for a practical and sensitive biochemical marker for monitoring these patients is urgent. The calcium binding astrocyte protein, S100B, may be a candidate for this role. Previous serum studies have shown S100B to be a sensitive predictor of mortality and rise in intracranial pressure in ABI, but it has never before been measured directly within the brain. The ECF reflects the local biochemistry of the brain parenchyma, and the use of intracerebral microdialysis opens up the possibility of studying many novel surrogate markers of injury in the laboratory, in addition to the conventional markers it measures at the bedside (lactate, pyruvate, glucose, and glycerol). In this preliminary report of two cases, the authors demonstrate the quantification of S100B in ECF microdialysate, and investigate whether changes in hourly S100B profile can be related to secondary brain injury. It is shown that extracellular concentrations of S100B change markedly in response to secondary brain injury. Further investigation is required to determine whether extracellular S100B measurement in ABI could assist in patient management.

Neurofilament phosphoforms (Nf) are principal components of the axoskeleton released during axonal injury. Cerebrospinal fluid (CSF) levels of Nf phosphoforms might be useful surrogate markers for disability in multiple sclerosis (MS), aid in distinguishing clinical subtypes, and provide valuable prognostic information.Thirty four patients with MS were included in a three year follow up study along with 318 controls with other non-inflammatory neurological diseases. CSF levels of two Nf heavy chain (NfH) phosphoforms (NfH(SMI35), NfH(SMI34)) were quantified at baseline and three year follow up using new ELISA techniques. Levels of NfH phosphoforms, the degree of phosphorylation (NfH(SMI34):NfH(SMI35) ratio), and changes in NfH levels between baseline and follow up (Delta NfH) were related to the clinical phenotype (RR or SP/PP), to three clinical scales (Kurtzke's EDSS, ambulation index (AI), and nine hole peg test (9HPT)), and to progression of disability.A significantly higher proportion (59%) of patients with SP/PPMS experienced an increase in NfH(SMI35) levels between baseline and follow up compared with those with RRMS (14%, p0.05). CSF NfH(SMI34) levels at baseline were higher in patients with SP/PP (11 pg/ml) compared with RR (7 pg/ml, p0.05) and NfH(SMI35) levels were higher at follow up in SP/PP (129 pg/ml) compared with levels below assay sensitivity in RR (p0.05). NfH(SMI35) correlated with the EDSS (r(s) = 0.54, p0.01), the AI (r(s) = 0.42, p0.05), and the 9HPT (r(s) = 0.59, p0.01) at follow up.The increase in NfH during the progressive phase of the disease together with the correlation of NfH(SMI35) with all clinical scales at follow up suggests that cumulative axonal loss is responsible for sustained disability and that high NfH(SMI35) levels are a poor prognostic sign.

Quantification of neurodegeneration in animal models is typically assessed by time-consuming and observer-dependent immunocytochemistry. This study aimed to investigate if newly developed ELISA techniques could provide an observer-independent, cost-effective and time-saving tool for this purpose. Neurofilament heavy chain (NfH(SM135)), astrocytic glial fibrillary acidic protein (GFAP), S100B and ferritin, markers of axonal loss, gliosis, astrocyte activation and microglial activation, respectively, were quantified in the spinal cord homogenates of mice with chronic relapsing experimental allergic encephalomyelitis (CREAE, n=8) and controls (n=7). Levels of GFAP were found to be threefold elevated in CREAE (13 ng/mg protein) when compared to control animals (4.5 ng/mg protein, p

This study examined the correlation between nitric oxide (NO) metabolites in the three major body fluid compartments and assessed performance of newly described vanadium-based assay for simultaneous detection of nitrite and nitrate (NO(x)) in human samples. Vanadium reduces nitrate to nitrite, which can be measured after a colorimetric reaction with Griess reagents. Cisternal cerebro spinal fluid (CSF), serum and urine samples from 10 patients with acute brain injury (ABI) were compared to control subjects. Significantly higher CSF NO(x) levels were found in brain injury patients compared to control patients (19.7+/-13.7 vs. 6.5+/-2.3 microM; p=0.01), which persisted for 10-day period of observation. The serum and urine levels of NO(x) on admission were not statistically different (42.8+/-28.2 microM; 584.1+/-337.8 micromol/g Cr, respectively) from controls (36.8+/-14.8 microM; 819.7+/-356.0 micromol/g Cr), but tended to decrease during the disease course reaching the lowest level on day 6 (serum: 19.3+/-8.4 microM, urine: 300.4+/-111.9 micromol/g Cr). CSF levels of NO(x) correlated moderately with those in serum (p=0.001, R=0.5). Serum NO(x) concentrations correlated weakly with urine levels (p=0.04, R=0.3). There was no significant correlation between CSF NO(x) and urine NO(x) levels. In conclusion, patients suffering brain injury had increased NO(x) concentrations in CSF, which remained independent from other body fluid compartments. Serum and urinary NO(x) levels cannot be used as a reliable index to assess intrathecal NO production.

S100B is a predominantly astrocytic protein with dose-dependent cytotoxic and neurotrophic properties encoded on chromosome 21q22.3. Concentrations of S100B were measured in the cerebrospinal fluid (CSF) of 31 patients with Alzheimer's disease (AD), 36 patients with frontotemporal lobe dementia (FTLD) and 49 patients with other non-inflammatory neurological diseases. Additional CSIF S100B concentrations were correlated with normalised brain volume measurements in AD and FTLD. CSF S100B was significantly higher in AD (Mean standard deviation = 0.4 +/- 0.2 ng/ml) and FTLD (0.42 +/- 0.19 ng/ml) patients when compared with control subjects (0.25 +/- 0.08, P < 0.001). In patients with AD, S100B correlated negatively with normalised brain volume (R-S = -0.53, P < 0.001). No such correlation was found for FTLD patients. This study supports the concept that S100B is of pathological relevance for degeneration of the central nervous system in AD. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Disease progression in multiple sclerosis occurs within the interface of glial activation and gliosis. This study aimed to investigate the relationship between biomarkers of different glial cell responses: (i) to disease dynamics and the clinical subtypes of multiple sclerosis; (ii) to disability; and (iii) to cross-validate these findings in a post-mortem study. To address the first goal, 51 patients with multiple sclerosis [20 relapsing remitting (RR), 21 secondary progressive (SP) and 10 primary progressive (PP)] and 51 neurological control patients were included. Disability was assessed using the ambulation index (AI), the Expanded Disability Status Scale score (EDSS) and the 9-hole PEG test (9HPT). Patients underwent lumbar puncture within 7 days of clinical assessment. Post-mortem brain tissue (12 multiple sclerosis and eight control patients) was classified histologically and adjacent sites were homogenized for protein analysis. S100B, ferritin and glial-fibrillary acidic protein (GFAP) were quantified in CSF and brain-tissue homogenate by ELISA (enzyme-linked immunosorbent assay) techniques developed in-house. There was a significant trend for increasing S100B levels from PP to SP to RR multiple sclerosis (P0.05). S100B was significantly higher in RR multiple sclerosis than in control patients (P0.01), whilst ferritin levels were significantly higher in SP multiple sclerosis than in control patients (P0.01). The S100B : ferritin ratio discriminated patients with RR multiple sclerosis from SP, PP or control patients (P0.05, P0.01 and P0.01, respectively). Multiple sclerosis patients with poor ambulation (AIor =7) or severe disability (EDSS6.5) had significantly higher CSF GFAP levels than less disabled multiple sclerosis or control patients (P0.01 and P0.001, respectively). There was a correlation between GFAP levels and ambulation in SP multiple sclerosis (r = 0.57, P0.01), and between S100B level and the 9HPT in PP multiple sclerosis patients (r = -0.85, P0.01). The post-mortem study showed significantly higher S100B levels in the acute than in the subacute plaques (P0.01), whilst ferritin levels were elevated in all multiple sclerosis lesion stages. Both GFAP and S100B levels were significantly higher in the cortex of multiple sclerosis than in control brain homogenate (P0.001 and P0.05, respectively). We found that S100B is a good marker for the relapsing phase of the disease (confirmed by post-mortem observation) as opposed to ferritin, which is elevated throughout the entire course. GFAP correlated with disability scales and may therefore be a marker for irreversible damage. The results of this study have broad implications for finding new and sensitive outcome measures for treatment trials that aim to delay the development of disability. They may also be considered in future classifications of multiple sclerosis patients.

We have developed a sensitive and specific isoelectrofocusing (IEF) method for the detection of oligoclonal (OGC) IgM in the cerebrospinal fluid (CSF) and sera. In this procedure, ampholytes, in the range pH 5-8, were used to improve the resolution, and serum was diluted in saline to avoid IgM precipitation. Samples were treated with 50 mM dithiothreitol (DTT) at pH 9.5 to reduce IgM and improve the migration of the protein. Using an anti-IgM labelled with alkaline phosphatase, the limit of detection was found to be 0.1 ng in 5 microl of sample.

18 UK patients (14 families) have been diagnosed with the carbohydrate-deficient glycoprotein syndrome (CDGS), type 1, on the basis of their clinical symptoms and/or abnormal electrophoretic patterns of serum transferrin. Eleven out of the 16 infants died before the age of 2 years. Patients from 12 families had a typical type 1 transferrin profile but one had a variant profile and another, who had many of the clinical features of CDGS type 1, had a normal profile. Eleven of the patients (10 families) with the typical type 1 profile had a deficiency of phosphomannomutase (PMM), (CDGS type 1a) but there was no correlation between residual enzyme activity and severity of disease. All these patients were compound heterozygotes for mutations in the phosphomannomutase (PMM2) gene, with 7 out of the 10 families having the common R141H mutation. Eight different mutations were found, including three novel ones. There was no correlation between genotype and phenotype, although siblings had similar phenotypes. Three patients, including the one with the normal transferrin profile, did not have a deficiency of phosphomannomutase or phosphomannose isomerase (CDGS 1b).

The intrathecal Ab response to Ag introduced into the normal brain has not been fully explored. Involvement of Ag-specific, peripheral B cells in an intrathecal response was studied using a normal rat model of Ag infusion through an indwelling cannula into defined brain sites, while maintaining a functionally intact blood-brain barrier. Specific Ab was detected in serum and cerebrospinal fluid. The intrathecal response is first detectable at day 14. Isoelectric focusing of cerebrospinal fluid reveals banding patterns consistent with local Ab production. To increase Ag-specific, circulating peripheral lymphocytes available for trafficking to Ag-stimulated brain and for enhancing intrathecal Ab synthesis, rats were preimmunized peripherally. Subsequently, Ag or saline (control) was infused through the cannula. Under this protocol, intrathecal synthesis is detectable earlier (day 5 postinfusion). Immunohistochemical studies at the infusion site assessed Ag-specific B cells, T cells, and activated APCs. Rats receiving peripheral preimmunization followed by Ag into caudate nucleus have far greater numbers of these cells, including plasma cells, within the infusion site compared with saline controls. Results confirm previous indirect evidence of intrathecal Ab synthesis in normal rat brain and provide the first direct evidence for B cell trafficking across normal brain barriers plus retention at the Ag deposition site. Our studies indicate that the normal brain microenvironment supports development of Ag-directed humoral immunity. We propose that immune privilege in normal brain is characterized by down-regulation of cell-mediated but not Ab immune responses within the central nervous system.

Two pregnancies at risk for the carbohydrate-deficient glycoprotein syndrome Type 1A (CDG1A, phosphomannomutase deficient) were monitored by enzyme and genetic linkage analyses. The index case in both families had a proven deficiency of phosphomannomutase (PMM). An unaffected fetus was predicted in family 1 following amniocentesis. Normal PMM activity was found in cultured amniotic fluid cells and there was no elevation of lysosomal enzymes in the amniotic fluid. Genetic linkage analysis using microsatellite markers closely linked to the CDG1A gene confirmed this prediction. A healthy child was born. In the second family direct assay of chorionic villi showed a profound deficiency of PMM and genetic linkage analysis showed the fetus to have the same haplotype as the proband. The pregnancy was terminated and a deficiency of PMM was confirmed in cultured fibroblasts from the fetus. Reliable prenatal diagnosis of CDG Type 1A (PMM-deficient) can be achieved by a combination of biochemical and molecular genetic tests.

Long-term morbidity from Guillain-Barre syndrome (GBS) is caused by axonal damage. This prospective study demonstrated that neurofilaments (NfHs), a biomarker for axonal damage, were of prognostic value in GBS. CSF NfH levels correlated with the F score and Medical Research Council summed score and were higher in patients with neurophysiologic evidence of axonal degeneration compared to those without. Pathologically high CSF NfH levels (>0.73 ng/mL) predicted worse motor and functional outcome.

The carbohydrate-deficient glycoprotein syndrome (CDGS) is a group of disorders characterized biochemically by abnormal glycosylation of serum and cellular glycoproteins. It has been classified into four forms on the basis of the isoelectric focusing pattern of serum transferrin and differences in clinical presentation. A deficiency of phosphomannomutase (PMM) has been reported in most patients with type 1. Seven of our eight CDGS patients, classified clinically as type 1, were shown to have a deficiency of phosphomannomutase in their fibroblast or lymphoblastoid cells (0.04-0.2 nmol/min per mg, compared with a control range of 1.0-2.1 nmol/min per mg). The eighth patient, who had many clinical features of the severe neonatal form of CDGS type 1, but lacked definite signs of CNS and ocular involvement, had a normal phosphomannomutase activity in his fibroblasts. There were approximately equal amounts of disialo- and tetrasialotransferrin and only a trace amount of asialotransferrin in the serum and ascitic fluid of this patient. The disialo- and tetrasialotransferrin isoforms were purified by ion-exchange chromatography and analysed by SDS-PAGE. The disialotransferrin had a lower molecular mass than the tetrasialotransferrin, consistent with the absence of an N-linked glycan. The N-linked glycans released enzymically from both isoforms consisted exclusively of disialylated biantennary chains, suggesting that disialotransferrin results from underglycosylation, as in the PMM-deficient CDGS type 1 patients. It is concluded that the clinical and biochemical phenotype in CDGS type 1 can result from more than one basic defect.

This study investigates whether the presence of serum and plasma anti-myelin oligodendrocyte glycoprotein (MOG) and anti-myelin basic protein (MBP) in patients presenting with a clinically isolated syndrome compatible with demyelination (CIS) predicts early conversion to multiple sclerosis (MS). Forty-seven patients with CIS (46 with optic neuritis) had anti-MOG and anti-MBP antibodies analysed at baseline, and clinical and magnetic resonance imaging assessments. There was no evidence that the MS status based on either the McDonald or Poser criteria relates to the antibody status.

Background— Visual assessment of cerebrospinal fluid (CSF) for xanthochromia (yellow color) is practiced by the majority of laboratories worldwide as a means of diagnosing intracranical bleeds. Methods— Colorimetric and spectrophotometric analysis of CSF samples for recognizing the presence of bilirubin either in low concentrations or in the presence of hemolysed blood. Results— The experiments provide the physiological and colorimetric basis for abandoning visual assessment of CSF for xanthochromia. Conclusion— We strongly recommend relying on spectrophotometry as the analytical method of choice.

One of the biochemical characteristics of carbohydrate deficient glycoprotein syndromes is the presence of abnormal glycoforms in serum transferrin. Both glycoform heterogeneity and variable site occupancy may, in principle, lead to the generation of a range of glycoforms which contain different numbers of sialic acid residues, and therefore variable amounts of negative charge. Capillary zone electrophoresis was used to resolve the glycoforms of normal human serum transferrin and also of a set of glycoforms which were prepared by digesting the sugars on the intact glycoprotein with sialidase. The sugars on the intact glycoprotein were also modified by a series of exoglycosidase enzymes to produce a series of neutral glycoforms which were-also analysed by capillary zone electrophoresis. The oligosaccharide population of human serum transferrin was analysed by a series of mixed exoglycosidase digests on the released glycan pool and quantified using a novel HPLC strategy. Transferrin was isolated from carbohydrate deficient glycoprotein syndromes type I serum and both the intact glycoforms and released sugars were resolved and quantified. The data presented here confirm the presence of a hexa-, penta- and tetra-sialoforms of human serum transferrin in both normal and carbohydrate deficient glycoprotein syndrome type I serum samples. Consistent with previous reports carbohydrate deficient glycoprotein syndrome type I transferrin also contained a di-sialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and a non-glycosylated form where both remain unoccupied. This study demonstrates that capillary zone electrophoresis can be used to resolve quantitatively both sialylated and neutral complex type glycoforms, suggesting a rapid diagnostic test for the carbohydrate deficient glycoprotein syndromes group of diseases.

Objective To determine whether vascular endothelial growth factor (VEGF) levels are associated with the presence of cerebral microbleeds (CMBs) in patients after acute ischemic stroke. Design A cross-sectional study that used blood samples obtained within 24 hours of symptom onset from patients who experienced acute stroke to measure VEGF levels by enzyme immunoassay. A validated CMB rating scale was used to analyze acutely acquired magnetic resonance images, with the rater blind to clinical details and VEGF levels. Setting Accident and Emergency Department at University College Hospital, London, England. Patients Twenty patients who experienced acute ischemic stroke. Main Outcome Measures Presence of CMBs and serum level of VEGF. Results Five of the 20 patients with acute ischemic stroke (25%) had CMBs. The median VEGF level in the CMB group was significantly higher than that in the group without CMBs (P = .003). Conclusion An increase in vascular permeability secondary to a raised VEGF level may have a role in the genesis of CMBs in patients with acute ischemic stroke.

Object The prognostic value of CSF biomarkers in patients with idiopathic normal pressure hydrocephalus (iNPH) has not been adequately studied to date. The aim of this study was to identify CSF markers of favorable surgical outcome in patients with iNPH undergoing the insertion of a ventriculoperitoneal shunt. Methods Ventricular CSF was collected intraoperatively from 22 patients with iNPH and enzyme-linked immunosorbent assay was used to analyze the levels of amyloid-β 1–42 (Aβ1–42) and total tau protein. The Black grading scale was used to assess outcomes at 6 months. Receiver operating characteristic (ROC) curves were obtained and discriminant function analysis was undertaken to provide sensitivity and specificity figures for each marker as well as their combination. Results The mean age of the patients was 71.45 years (± 9.5 years [SD]). Follow-up was achieved in 21 patients. Seventeen patients had a favorable outcome and 4 patients had unfavorable outcome at 6 months. An Aβ1–42 level of 180 pg/ml had a sensitivity of 35% and a specificity of 20% for predicting a favorable outcome at 6 months. A total tau level of 767 pg/ml will have a sensitivity of 17% and a specificity of 20% for predicting a favorable outcome at 6 months. A combination of Aβ1–42 and total tau levels predicted favorable outcomes with a sensitivity of 80% and specificity of 82.4%. Conclusions In this pilot study a combination of Aβ1–42 levels and total tau protein levels predicted favorable surgical outcomes at 6 months with adequate accuracy to be of clinical use. Further study in a larger group with longer follow-up is warranted.

New therapeutic approaches in Alzheimer's disease are urgently needed. The normal plasma protein, serum amyloid P component (SAP), is always present in cerebrospinal fluid (CSF) and in the pathognomonic lesions of Alzheimer's disease, cerebrovascular and intracerebral Aβ amyloid plaques and neurofibrillary tangles, as a result of its binding to amyloid fibrils and to paired helical filaments, respectively. SAP itself may also be directly neurocytotoxic. Here, in this unique study in Alzheimer's disease of the bis( d -proline) compound, (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), we observed depletion of circulating SAP and also remarkable, almost complete, disappearance of SAP from the CSF. We demonstrate that SAP depletion in vivo is caused by CPHPC cross-linking pairs of SAP molecules in solution to form complexes that are immediately cleared from the plasma. We have also solved the structure of SAP complexed with phosphothreonine, its likely ligand on hyperphosphorylated τ protein. These results support further clinical study of SAP depletion in Alzheimer's disease and potentially other neurodegenerative diseases.

Two brothers presented with olivopontocerebellar atrophy of neonatal onset. The clinical features (failure to thrive, hypotonia, liver disease, effusions, and visual inattention) were similar to those of the four cases already reported, as were the necropsy findings of olivopontocerebellar atrophy, hepatic steatosis and fibrosis, and microcystic renal changes. The clinical similarities between this and the disialotransferrin developmental deficiency syndrome were noted. The characteristic abnormality of serum transferrin found in the latter syndrome was also found in the two cases reported here. We suggest that both syndromes are caused by the same, or related, defects in glycoprotein metabolism.

We report a method which is capable of demonstrating the isoelectric focusing (IEF) pattern of immunoglobulin D in unconcentrated cerebrospinal fluid (CSF) samples containing as little as 0.1–0.5 ng of total IgD. The method used was an immuno-sandwich technique, with alkaline phosphatase enzyme amplification. Oligoclonal and polyclonal IgD patterns were seen in CSF samples. No cross-reactivity with other immunoglobulins (IgG, IgA and IgM) was detected.

Background: Loss of cortical neurons is a key pathological feature in neurodegenerative dementias. Cerebrospinal fluid (CSF) neurofilaments (Nf) are a biomarker for neuronal death and axonal loss. Objective: To perform a meta-analysis to investigate the value of CSF Nf levels for the laboratory-supported differential diagnosis of neurodegenerative dementias. Methods: A systematic review and meta-analysis of studies on CSF Nf heavy (NfH) and light (NfL) levels in patients with dementia. The dementia subgroups analysed were Alzheimer (AD), frontotemporal lobe dementia (FTLD), vascular dementia (SVD), minimal cognitive deficit (MCI). Results: We identified 12 studies on CSF NfH and NfL levels which met the inclusion criteria and 11 were of a quality good enough to be used in this meta-analysis. CSF data was available on 818 patients (306 AD, 106 SVD, 98 FTLD, 25 MCI, 283 controls). Overall CSF NfH and NfL levels were higher in patients with AD, FTLD and SVD when compared to controls. The size of the effect ranged from 0.71 to 1.38. The strongest effect was observed for the comparison of FTLD patients with controls, both for NfL (1.38) and NfH (0.74). CSF NfL were also able to separate patients with FTLD from those with AD. Conclusion: At present we cannot recommend CSF NfH and NfL levels for use as a screening test in the diagnosis of dementia because of the rather small effect size. However, both neurofilament proteins may be of value for targeted investigation of some patients with FTLD, SVD and AD.

Background – It has been proposed that multiple sclerosis (MS) might be a sexually transmitted disorder. There is evidence that seropositivity to herpes simplex virus type 2 (HSV-2) correlates well with the number of sexual partners. Accordingly, a raised overall HSV-2 seroprevalence in MS would lend support to this theory. Materials and methods – Serum from 497 UK subjects with clinically definite MS was tested for antibodies to HSV-2 and compared with matched historical controls from within and outside London, blood donors and genito-urinary medicine (GUM) clinics. Results – The unadjusted MS seropositivity rate was 14%. HSV-2 seroprevalence in MS patients aged 35–64 years was significantly higher overall compared with a non-London general population in an unadjusted comparison. HSV-2 seroprevalence in London MS patients compared with London blood donors was significantly greater irrespective of age, but the MS seropositive rate was lower than GUM clinic attenders. In a logistic regression analysis, increased age, female sex and MS diagnosis all independently increased the odds of seropositivity after adjustment for each other. Conclusion – It is concluded that there is increased likelihood of HSV-2 exposure in patients with MS and this may indicate a higher than average number of partners.

This study investigated whether the new Global Multiple Sclerosis Severity Scale (MSSS) correlated with cerebrospinal fluid biomarkers for axonal and glial pathology. The MSSS correlated with the phosphorylated neurofilament heavy chain (NfH-SMI35, R=0.44, P=0.016). The degree of neurofilament phosphorylation (ratio NfH-SMI34 to NfH-SMI35) was 8-fold higher in severely (median MSSS 6.5) versus mildly (MSSS 3.2) disabled patients (7.3 versus 0.9, P=0.03). The MSSS may provide a statistically powerful tool for comparing overall disease severity and be useful for validating the biomarker concept in MS.

Identification of the aetiology of central nervous system infections requires the detection of either the organism or a microbe-specific immune response within the brain or cerebrospinal fluid. We describe a screening assay to detect herpes simplex virus, varicella zoster virus, cytomegalovirus, measles and Toxoplasma gondii specific antibodies in cerebrospinal fluid. Antigen-specific immunoblotting of oligoclonal IgG and IgM was used to confirm the presence of antibody. Of 51 consecutive cerebrospinal fluid samples received by the laboratory from patients with suspected central nervous system infection 18 (35%) were screen positive for one or more antigen. In only 7 of these were antigen-specific oligoclonal IgG or IgM bands confirmed. The assay provides a simple, cheap assay to screen for microbial-specific antibody in the cerebrospinal fluid samples of patients with suspected neurological infections.

Electrically active axons degenerate in the presence of nitric oxide (NO) in vitro. High CSF NO concentrations have been observed in patients with hemorrhagic brain injury such as subarachnoid hemorrhage (SAH) and intracerebral hemorrhage (ICH). This study investigated the evidence for axonal injury in SAH and ICH and related this to CSF NO levels. In this study, neurofilament phosphoforms (NfH(SMI34), NfH(SMI35), NfH(SMI38), NfH(SMI310)), surrogate markers for axonal injury, and NO metabolites (nitrate, nitrite = NOx) were measured by ELISA in cerebrospinal fluid (CSF) from patients with SAH and ICH and from a group of controls. Injury severity was classified using the Glasgow Coma Scale, and survival was used as the outcome measure. Compared to the control group, a higher proportion of patients with SAH and ICH had elevated NfH(SMI34) levels from day 0 to day 6 (p < 0.001), elevated NfH(SMI35) levels from day 1 to 6 (p < 0.001), and elevated NfH(SMI310) levels at day 0, 1, 4, and 6 (p < 0.001). The NOx levels were higher in the SAH and ICH patients than in the controls (p < 0.05) and distinguished the non-survivors from the survivors (p < 0.05). No direct correlation was found for NOx with any of the NfH phosphoforms. This study provides evidence for primary and secondary axonal injury in patients with SAH and ICH, with non-survivors also having higher NOx levels. CSF NfH phosphoforms might emerge as a putative surrogate marker for monitoring the development for secondary axonal degeneration in neurocritical care and guiding targeted neuroprotective strategies.

To underline the importance of correctly examining cerebrospinal fluid, we describe two patients with subarachnoid haemorrhage for whom cranial computerised tomography was normal and on whom lumbar puncture was subsequently done. Both had clear and colourless cerebrospinal fluid, but subsequent spectrophotometry showed bilirubin. Cerebral angiography then showed a ruptured aneurysm in each patient. Case 1 A 28 year old woman presented with nausea and diplopia. She had global headache which had awoken her 14 days before. This was associated with neck pain and stiffness, vomiting for two days, and photophobia for five to six days. On examination, severe bilateral papilloedema, and signs of a VIth nerve palsy were present. Cranial computerised …

To the Editor: Xanthochromia, or a yellow appearance of the cerebrospinal fluid, is often used to confirm suspected subarachnoid hemorrhage.1,2 It is associated with the presence of bilirubin, a blood product that arises only in vivo by the enzymatic transformation of hemoglobin. However, visual assessment of the cerebrospinal fluid for xanthochromia is highly subjective and unreliable, especially in cases in which other blood products, such as oxyhemoglobin, are present.1,2 The differential diagnosis may be further complicated by a high protein level and a high cell count, as in meningitis and necrosis, or by the presence of other pigments . . .

This study evaluates levels of cerebrospinal fluid (C SF) brain-specific proteins (BSP) in subjects with optic neuritis (O N) who are at high risk of progression to multiple sclerosis (MS). Forty-one subjects had acute O N and 17 subjects with other neurological diseases (OND) served as controls. Twenty-o ne subjects with O N had white matter lesions on magnetic resonance imaging (MRI) and intrathecal synthesis of oligoclonal IgG bands (OB) consistent with being at high risk of progression to MS; eight of whom later were diagnosed with clinically definite MS (C DMS). Levels of S100B, ferritin and two neurofilament heavy chain phosphoforms (NfHSMI34 and NfHSMI35) were analysed using ELISA technique. A putative index of ‘axonal health’ was expressed as a ratio of NfHSMI34 to NfHSMI35. NfHSMI34 and the NfHSMI34:SMI35 were significantly elevated in subjects with O N compared to controls. No significant differences in levels of C SF BSP were seen between O N subjects with C DMS plus those at high risk of progression to MS and O N subjects with normal MRI and negative C SF analysis. In conclusion, there is evidence of axonal damage in subjects who present with O N, which is independent of the diagnosis of C DMS.

Glial fibrillary acidic protein (GFAP) is the major intermediate filament protein of the astrocyte, and body fluid levels of GFAP are an important tool for estimating astrogliosis and astrocytic activation in vivo. This paper presents a new sandwich ELISA allowing quantification of GFAP(SMI26) from the cerebrospinal fluid (CSF). The sensitivity of the GFAP(SMI26) ELISA is 5 pg/ml with a recovery of 94% and a mean within- and between-batch precision of 6% and 10%, respectively. The upper reference value for CSF GFAP(SMI26) levels (9 pg/ml) was defined as the 95% cumulative frequency from 315 CSF samples. Based on this cut-off, a significantly higher proportion of patients with subarachnoid hemorrhage (100%), traumatic brain injury (100%), dementia (76%) and normal pressure hydrocephalus (85%) had pathologically elevated CSF GFAP(SMI26) levels compared to patients with peripheral nervous system disorders (0%). In a critical review of the literature, we compare the analytical and clinical sensitivity of previous GFAP ELISA methods with particular reference to patients with dementia.

S100B is an important brain specific protein for monitoring damage and activation of astrocytes. Using a straight forward, non-resource demanding in-house ELISA technique we measured S100B in cerebrospinal fluid (CSF) and serum in patients with traumatic brain injury (TBI) (serum), subarachnoid hemorrhage (SAH) (CSF, serum), intracranial hemorrhage (ICH) (CSF, serum), normal controls (NC) (serum) and a reference population (CSF, N=409). The release and wash-out pattern found in CSF and serum are discussed in relation to the three main determinants for increased brain specific protein levels in body fluids: (i) total mass effect; (ii) pathology; and (iii) time effect.

Item does not contain fulltext A group of neurologists and clinical neurochemists representing twelve countries worked towards a consensus on laboratory techniques to improve the quality of analysis and interpretation of cerebrospinal fluid (CSF) proteins. Consensus was approached via a virtual Lotus Notes-based TeamRoom. This new approach respecting multicultural differences, common views, and minority opinions, is available in http://www.teamspace.net/ CSF, presenting the implicit, complementary version of this explicit, printed consensus. Three key recommendations were made: CSF and (appropriately diluted) serum samples should be analyzed together in one analytical run, i.e., with reference to the same calibration curve. Results are evaluated as CSF/serum quotients, taking into account the non-linear, hyperbolic relation between immunoglobulin (Ig)- and albumin-quotients rather than using the linear IgG index or IgG synthesis rate. Controls should include materials with values within the reference ranges (IgM: 0.5-1.5 mg/l; IgA: 1-3 mg/l; IgG: 10-30 mg/l and albumin: 100-300 mg/l). The physiological, methodological and clinical significance of CSF/serum quotients is reviewed. We confirmed the previous consensus on oligoclonal IgG, in particular the usefulness of the five typical interpretation patterns. The group compared current external and internal quality assurance schemes and encouraged all members to maintain national or local traditions. Values for acceptable imprecision in the CSF quality assurance are proposed.

Samples of aqueous fluid were obtained from 35 "controls" who were people undergoing routine cataract surgery. Similar samples were taken from seven patients with clinically definite multiple sclerosis (MS) and a previous history of optic neuritis, either at cataract surgery or as an elective procedure. Oligoclonal bands were found in only one subject who suffered from the MS-uveitis syndrome.