Fibrinogen isoforms as potential blood-based biomarkers of Alzheimer's disease using a proteomics approach.

Objective: Alzheimer's disease (AD), the commonest form of dementia which is characterized by progressive decline in cognitive function, can only be definitively diagnosed after death. Although biomarkers may aid diagnosis, currently available AD biomarkers, which are predominantly based on cerebrospinal fluid and neuroimaging facilities, are either invasive or costly. Blood-based biomarkers for AD diagnosis are highly sought after due to its practicality at the clinic. This study was undertaken to determine the differential protein expression in plasma amongst Malaysian AD, mild cognitive impairment (MCI) and non-AD individuals. Methods: A proteomic approach which utilized two-dimensional differential in gel electrophoresis (2 D DIGE) was performed for blood samples from 15 AD, 14 MCI and 15 non-AD individuals. Results: Mass spectrometry (MS)-based protein identification via MALDI ToF/ToF showed that fibrinogen-ß-chain (spot 64) and fibrinogen-γ-chain (spot 91) with differential expression ratio >1.5 were significantly upregulated (p < 0.05) in AD patients when compared to non-AD individuals. Further data analysis using Pearson correlation found that the upregulated fibrinogen-γ-chain was weakly but significantly (p < 0.05) and inversely correlated with cognitive decline. Conclusion: Fibrinogen isoforms may play important roles in the vascular pathology of AD as well as neuroinflammation. As such, fibrinogen appears to be a promising blood-based biomarker for AD. Further validation of the present findings in larger population is now warranted.

Evidence that blood-CSF barrier transport, but not inflammatory biomarkers, change in migraine, while CSF sVCAM1 associates with migraine frequency and CSF fibrinogen.

OBJECTIVE: Our objective is to explore whether blood-cerebrospinal fluid (CSF) barrier biomarkers differ in episodic migraine (EM) or chronic migraine (CM) from controls. BACKGROUND: Reports of blood-brain barrier and blood-cerebrospinal fluid barrier (BCSFB) disruption in migraine vary. Our hypothesis is that investigation of biomarkers associated with blood, CSF, brain, cell adhesion, and inflammation will help elucidate migraine pathophysiology. METHODS: We recruited 14 control volunteers without headache disorders and 42 individuals with EM or CM as classified using the International Classification of Headache Disorders, 3rd edition, criteria in a cross-sectional study located at our Pasadena and Stanford headache research centers in California. Blood and lumbar CSF samples were collected once from those diagnosed with CM or those with EM during two states: during a typical migraine, before rescue therapy, with at least 6/10 level of pain (ictal); and when migraine free for at least 48 h (interictal). The average number of headaches per month over the previous year was estimated by those with EM; this enabled comparison of biomarker changes between controls and three headache frequency groups: <2 per month, 2-14 per month, and CM. Blood and CSF biomarkers were determined using antibody-based methods. RESULTS: Antimigraine medication was only taken by the EM and CM groups. Compared to controls, the migraine group had significantly higher mean CSF-blood quotients of albumin (Qalb : mean ± standard deviation (SD): 5.6 ± 2.3 vs. 4.1 ± 1.9) and fibrinogen (Qfib mean ± SD: 1615 ± 99.0 vs. 86.1 ± 55.0). Mean CSF but not plasma soluble vascular cell adhesion molecule-1 (sVCAM-1) levels were significantly higher in those with more frequent migraine: (4.5 ng/mL ± 1.1 in those with <2 headache days a month; 5.5 ± 1.9 with 2-14 days a month; and 7.1 ± 2.9 in CM), while the Qfib ratio was inversely related to headache frequency. We did not find any difference in individuals with EM or CM from controls for CSF cell count, total protein, matrix metalloproteinase-9, soluble platelet-derived growth factor receptor ß, tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-6, IL-8, IL-10, or C-reactive protein. CONCLUSIONS: The higher Qalb and Qfib ratios may indicate that the transport of these blood-derived proteins is disturbed at the BCSFB in persons with migraine. These changes most likely occur at the choroid plexus epithelium, as there are no signs of typical endothelial barrier disruption. The most striking finding in this hypothesis-generating study of migraine pathophysiology is that sVCAM-1 levels in CSF may be a biomarker of higher frequency of migraine and CM. An effect from migraine medications cannot be excluded, but there is no known mechanism to suggest they have a role in altering the CSF biomarkers.

Increased cerebrospinal fluid fibrinogen in major depressive disorder.

Major depressive disorder (MDD) presumably includes heterogeneous subgroups with differing pathologies. To obtain a marker reflecting such a subgroup, we analyzed the cerebrospinal fluid (CSF) levels of fibrinogen, which has been reported to be elevated in the plasma of patients with MDD. Three fibrinogen-related proteins were measured using aptamer-based analyses and CSF samples of 30 patients with MDD and 30 controls. The numbers of patients with an excessively high level (>99 percentile of the controls) was significantly increased (17 to 23%). Measurement reproducibility of these results was confirmed by an ELISA for fibrinogen (Pearson's r = 0.77). In an independent sample set from 36 patients and 30 controls, using the ELISA, results were similar (22%). When these two sample sets were combined, the number of patients with a high fibrinogen level was significantly increased (15/66; odds ratio 8.53; 95% confidence interval 1.9-39.1, p = 0.0011). By using diffusion tensor imaging, we found white matter tracts abnormalities in patients with a high fibrinogen level but not those patients with a normal fibrinogen level, compared with controls. Plasma fibrinogen levels were similar among the diagnostic groups. Our results point to a subgroup of MDD represented by increased CSF fibrinogen and white matter tract abnormalities.

Proteomic profiling in multiple sclerosis clinical courses reveals potential biomarkers of neurodegeneration.

The aim of our project was to perform an exploratory analysis of the cerebrospinal fluid (CSF) proteomic profiles of Multiple Sclerosis (MS) patients, collected in different phases of their clinical course, in order to investigate the existence of peculiar profiles characterizing the different MS phenotypes. The study was carried out on 24 Clinically Isolated Syndrome (CIS), 16 Relapsing Remitting (RR) MS, 11 Progressive (Pr) MS patients. The CSF samples were analysed using the Matrix Assisted Laser Desorption Ionisation Time Of Flight (MALDI-TOF) mass spectrometer in linear mode geometry and in delayed extraction mode (m/z range: 1000-25000 Da). Peak lists were imported for normalization and statistical analysis. CSF data were correlated with demographic, clinical and MRI parameters. The evaluation of MALDI-TOF spectra revealed 348 peak signals with relative intensity ≥ 1% in the study range. The peak intensity of the signals corresponding to Secretogranin II and Protein 7B2 were significantly upregulated in RRMS patients compared to PrMS (p<0.05), whereas the signals of Fibrinogen and Fibrinopeptide A were significantly downregulated in CIS compared to PrMS patients (p<0.04). Additionally, the intensity of the Tymosin ß4 peak was the only signal to be significantly discriminated between the CIS and RRMS patients (p = 0.013). Although with caution due to the relatively small size of the study populations, and considering that not all the findings remained significant after adjustment for multiple comparisons, in our opinion this mass spectrometry evaluation confirms that this technique may provide useful and important information to improve our understanding of the complex pathogenesis of MS.

Cerebrospinal fluid biomarkers of neuropathologically diagnosed Parkinson's disease subjects.

OBJECTIVES: Parkinson's disease (PD) afflicts approximately 1-2% of the population over 50 years of age. No cures or effective modifying treatments exist and clinical diagnosis is currently confounded by a lack of definitive biomarkers. We sought to discover potential biomarkers in the cerebrospinal fluid (CSF) of neuropathologically confirmed PD cases. METHODS: We compared postmortem ventricular CSF (V-CSF) from PD and normal control (NC) subjects using two-dimensional difference gel electrophoresis (2D-DIGE). Spots exhibiting a 1·5-fold or greater difference in volume between PD patients and controls were excised from the two-dimensional gels, subjected to tryptic digestion and identification of peptides assigned using mass spectrometric/data bank correlation methods. RESULTS: Employing this strategy six molecules: fibrinogen, transthyretin, apolipoprotein E, clusterin, apolipoprotein A-1, and glutathione-S-transferase-Pi, were found to be different between PD and NC populations. DISCUSSION: These molecules have been implicated in PD pathogenesis. Combining biomarker data from multiple laboratories may create a consensus panel of proteins that may serve as a diagnostic tool for this neurodegenerative disorder.

Altered cerebrospinal fluid index of prealbumin, fibrinogen, and haptoglobin in patients with Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.

OBJECTIVES: Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) are autoimmune diseases of the peripheral nervous system. A clinical hallmark of GBS and CIDP is the albumino-cytologic dissociation in the cerebrospinal fluid (CSF). Changes in the CSF levels of proteins other than albumin in patients with GBS and CIDP are not as well studied. If altered, aberrant levels of CSF proteins may render it possible to establish useful biomarkers for GBS and CIDP. MATERIALS AND METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of prealbumin, fibrinogen, haptoglobin, apolipoprotein E, apolipoprotein A4 in both CSF and plasma samples from 19 patients with GBS and eight with CIDP, 24 controls with multiple sclerosis (MS) as well as 20 patients with other non-inflammatory neurological disorders (OND). RESULTS: The levels of prealbumin in both the plasma and the CSF were elevated in patients with GBS and MS compared with the controls. The higher levels of fibrinogen were seen in the CSF of patients with GBS and CIDP, but not in the plasma. The levels of CSF prealbumin and fibrinogen, measured by the CSF index of these proteins, were lower in patients with GBS and that of fibrinogen in patients with CIDP compared with controls with OND. Haptoglobin levels in the CSF rather than in the plasma were higher in patients with GBS and CIDP than in controls. The CSF haptoglobin index was higher in patients with CIDP and MS, but not in those with GBS. No correlation was found between levels of CSF proteins and clinical parameters in patients with GBS and CIDP. CONCLUSIONS: Our data provide preliminary evidence that GBS is associated with low CSF index levels of prealbumin and fibrinogen, but normal levels of haptoglobin, whereas CIDP is associated with normal CSF index levels of prealbumin, low fibrinogen, and high levels of haptoglobin. Further studies are needed to identify the underlying mechanisms behind these CSF protein alterations and to clarify whether prealbumin, fibrinogen, and haptoglobin can serve as useful biomarkers for GBS and CIDP.

Identification of SPARC-like 1 protein as part of a biomarker panel for Alzheimer's disease in cerebrospinal fluid.

We have used proteomic fingerprinting to investigate diagnosis of Alzheimer's disease (AD). Samples of lumbar cerebrospinal fluid (CSF) from clinically-diagnosed AD cases (n = 33), age-matched controls (n = 20), and mild cognitive impairment (MCI) patients (n = 10) were used to obtain proteomic profiles, followed by bioinformatic analysis that generated a set of potential biomarkers in CSF samples that could discriminate AD cases from controls. The identity of the biomarker ions was determined using mass spectroscopy. The panel of seven peptide biomarker ions was able to discriminate AD patients from controls with a median accuracy of 95% (sensitivity 85%, specificity 97%). When this model was applied to an independent blind dataset from MCI patients, the intensity of signals was intermediate between the control and AD patients implying that these markers could potentially predict patients with early neurodegenerative disease. The panel were identified, in order of predictive ability, as SPARC-like 1 protein, fibrinogen alpha chain precursor, amyloid-ß, apolipoprotein E precursor, serum albumin precursor, keratin type I cytoskeletal 9, and tetranectin. The 7 ion ANN model was further validated using an independent cohort of samples, where the model was able to classify AD cases from controls with median accuracy of 84.5% (sensitivity 93.3%, specificity 75.7%). Validation by immunoassay was performed on the top three identified markers using the discovery samples and an independent sample cohort which was from postmortem confirmed AD patients (n = 17).

Hydrocephalus and spinal cord tumors: a review.

INTRODUCTION: Hydrocephalus secondary to intraspinal tumors is a well-known but rare condition since about 1% of patients with spinal cord tumors have various degrees of hydrocephalus at initial presentation. DISCUSSION: The mechanism of development of intracranial hypertension and hydrocephalus in patients with spinal cord tumor is not exactly known. The problematic aspects of this condition, with regard to clinical presentation and pathophysiology, are discussed and the relevant literature is reviewed. This uncommon association should always be kept in mind in the differential diagnosis of hydrocephalus of unknown etiology for three main reasons: the possibility of neurological deterioration if the patient is shunted prior tumor removal, the possibility to treat the hydrocephalus without shunting by simply removing the tumor, and the possible role of hydrocephalus as an early sign of intracranial metastasis in patients previously operated upon for removal of intramedullary gliomas. Due to the very slow evolution of the disease, a careful and close clinical and neuroradiological follow-up are essential for many years afterward. The presence of intracranial hypertension in a patient previously operated for a spinal tumor should be considered and investigated as an early sign of neoplastic intracranial seeding.

Shunting in hydrocephalus due to tuberculous meningitis. Cases presenting with high cerebrospinal fluid proteins in pediatric age.

AIM: Management of hydrocephalus due to tuberculous meningitis is still a challenging issue. High concentrations of cerebrospinal fluid proteins and elevated cell counts will affect ventricular shunt function. The aim of this prospective study was to evaluate the results of shunt function in cases having high concentrations of cerebrospinal fluid proteins. METHODS: Between January 1995 and January 2001, 84 children were treated with the diagnosis of tuberculous meningitis in Istanbul Pediatric Clinics. Hydrocephalus occurred in 32 (38%) of them. The cerebrospinal fluid samples obtained from these patients via lumbar puncture, ventricular puncture or trapping of the shunt pumps in operated patients and analyses included protein electrophoresis and differential cell counts. RESULTS: Mean cerebrospinal fluid protein level was 236 mg/dL (range 26-643 mg/dL) and mean cell count was 36/mm (range 12-132/mm?) with a dominancy of the lymphocytes (70-90%). Protein electrophoresis consisted of low molecular weight proteins with a mean of 93.1% (range 85.7-96.7 %). The average follow-up period was 45 months (range 26-67 months). All the clinical and radiological findings of hydrocephalus showed a regression after the shunt operations, despite the worries of shunt dysfunction preoperatively. The cerebrospinal fluid proteins level and cell counts had dramatically declined with neurological improvements after shunt insertion. CONCLUSIONS: This prospective study showed that in certain limits, covering mostly low molecular weighted proteins like albumin, prealbumin, even high cell counts in cerebrospinal fluid, does not affect shunt function.

Fibrinogen gamma-A chain precursor in CSF: a candidate biomarker for Alzheimer's disease.

BACKGROUND: Cerebrospinal fluid (CSF) may be valuable for exploring protein markers for the diagnosis of Alzheimer's disease (AD). The prospect of early detection and treatment, to slow progression, holds hope for aging populations with increased average lifespan. The aim of the present study was to investigate candidate CSF biological markers in patients with mild cognitive impairment (MCI) and AD and compare them with age-matched normal control subjects. METHODS: We applied proteomics approaches to analyze CSF samples derived from 27 patients with AD, 3 subjects with MCI and 30 controls. The AD group was subdivided into three groups by clinical severity according to clinical dementia rating (CDR), a well known clinical scale for dementia. RESULTS: We demonstrated an elevated level of fibrinogen gamma-A chain precursor protein in CSF from patients with mild cognitive impairment and AD compared to the age-matched normal subjects. Moreover, its expression was more prominent in the AD group than in the MCI and correlated with disease severity and progression. In contrast, fibrinogen gamma-A chain precursor protein was detected very low in the age-matched normal group. CONCLUSION: These findings suggest that the CSF level of fibrinogen gamma-A chain precursor may be a candidate biomarker for AD.

Proteomic identification of potential protein markers in cerebrospinal fluid of GBS patients.

Increased protein level in the cerebrospinal fluid (CSF) is a characteristic of patients with Guillain-Barré syndrome (GBS), an acute inflammatory autoimmune disorder in the peripheral nervous system (PNS). However, the molecular mechanisms underlying the disease remain poorly understood and so far no reliable disease-related markers are available. By comparing the CSF proteome of GBS patients with control subjects suffering from other neurological disorders, it may be possible to identify proteins that involve in the disease process and thus to study the pathogenesis of GBS. We used two-dimensional difference gel electrophoresis (2D DIGE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), to determine the abnormal CSF proteins in GBS patients. Our data showed that the levels of six proteins and their isoforms in CSF were significantly altered in GBS patients compared with controls. Haptoglobin, apolipoprotein A-IV and PRO2044 (unnamed protein) were considerably increased in the CSF of GBS patients, whereas transthyretin, apolipoprotein E and fibrinogen were considerably decreased. We concluded that these six proteins may be involved in the pathogenesis of GBS and call for further studying the role of these proteins in the pathogenesis of the disease.

Proteome study of human cerebrospinal fluid following traumatic brain injury indicates fibrin(ogen) degradation products as trauma-associated markers.

Traumatic brain injury (TBI), like other central nervous system pathologies, causes changes in the composition of cerebrospinal fluid (CSF). Consequently analysis of the CSF components is important to better understand the pathological processes involved in such diseases. The aim of this work was to identify specific markers of severe TBI. Proteomic analysis including two-dimensional gel electrophoresis combined with mass spectrometry analysis was used to compare the CSF protein profile of severe TBI patients and controls. Proteins (alpha 1 antitrypsin, haptoglobin 1 alpha1, alpha2, and beta) belonging to the acute phase response showed an increased expression in severe TBI patients. Two other proteins, identified as proteolytic degradation products of the carboxyl-terminal portion of the fibrinogen beta, were present only in TBI patients. The presence of these markers could correlate with a post-traumatic local increase in fibrinolysis as well as to an inflammatory event following CNS tissue injury.

Low dose intraventricular fibrinolytic treatment to prevent posthaemorrhagic hydrocephalus.

Posthaemorrhagic ventricular dilatation (PHVD) is thought to be due to clots from intraventricular haemorrhage obstructing cerebrospinal fluid pathways involved in reabsorption. Over 60% of infants with progressive PHVD have gone on to require surgical shunt placement. Previous treatments all have major problems. The object of this pilot study was to achieve enough fibrinolysis to restore pathways of cerebrospinal fluid reabsorption and so avoid shunt surgery. Nine preterm infants with progressive PHVD were treated with intraventricular infusion of streptokinase for 12-72 hours. All the infants survived and surgical shunting was required in only one case. A 200% increase in fibrinolytic activity was demonstrated in both ventricular and spinal fluid during streptokinase treatment. There were no cases of infection. Minor rebleeding occurred in one case and was not a serious problem. This represents the first direct therapeutic approach to the pathology of PHVD.

Fibrinolytic activity after subarachnoid haemorrhage and the effect of tranexamic acid.

Seventy-four patients with recent subarachnoid haemorrhage were randomly allocated to placebo or tranexamic acid treatment. Fibrinolytic activity in the blood and cerebrospinal fluid was assessed before treatment, one week later and two weeks later. The natural history of fibrinolysis following subarachnoid haemorrhage was obtained from analysis of the placebo group. Following subarachnoid haemorrhage, fibrin degradation products and plasminogen activity in the cerebrospinal fluid were elevated. Subsequently, fibrin degradation products in the cerebrospinal fluid fell progressively over the following 2 weeks. Changes in cerebrospinal fluid plasminogen activity correlated with those of blood plasminogen activity. Complications such as rebleeding, hydrocephalus or cerebral thrombosis could not be predicted from analysis of fibrinolytic activity. Tranexamic acid treatment resulted in a reduction in cerebrospinal fluid and blood plasminogen activity. The relevance of fibrinolysis in cerebrospinal fluid and blood to the management of subarachnoid haemorrhage is discussed.

Activation of the coagulation system in the subarachnoid space after subarachnoid haemorrhage: serial measurement of fibrinopeptide A and bradykinin of cerebrospinal fluid and plasma in patients with subarachnoid haemorrhage.

Fibrinopeptide A (FPA) levels as an indicator of thrombin activity in the cerebrospinal fluid (CSF) and plasma of 25 patients with subarachnoid haemorrhage (SAH) were measured serially by radioimmunoassay (RIA). FPA levels in CSF were extremely high on days 0-1 (1253 +/- 269 ng/ml, mean +/- standard error) but decreased rapidly (11.3 +/- 3.9 ng/ml on days 2-4, 10.7 +/- 5.9 ng/ml on days 5-7, and 6.3 +/- 1.5 ng/ml on days 8-14). In the controls the FPA concentration in CSF was 1.2 +/- 0.9 ng/ml (mean +/- standard deviation). Plasma FPA levels in patients with SAH showed no statistically significant changes with time. The bradykinin (BK) concentration in CSF and plasma in 27 patients with SAH was measured serially by RIA. The concentrations in CSF were 122.7 +/- 22.7 pg/ml (mean +/- standard error) on day 0, 38.6 +/- 6.1 pg/ml on day 1, 22.7 +/- 6.3 pg/ml on day 2, and 17.1 +/- 3.0 pg/ml or less thereafter. Plasma BK levels in patients with SAH were higher than those in the control group, but there was no statistically significant change over time. From the measurement of FPA it was apparent that the coagulation system in the subarachnoid space is strongly activated in the early stage of SAH. The formation of BK in CSF after SAH is thought to be due to the contact activation of Hageman factor (intrinsic factor) in the subarachnoid space. Trabeculae as collagen bundles in the subarachnoid space were considered to have a possible role in activating the Hageman factor of the coagulation system in SAH.

Local hemostasis in subarachnoid hemorrhage.

Fibrinopeptide A (FPA), platelet-secreted protein, platelet factor 4 and beta-thromboglobulin were determined in the cerebrospinal fluid of patients who had suffered from subarachnoid hemorrhage and were treated with 6 g tranexamic acid or 4 million KIU aprotinin to prevent rebleeding. Platelet-secreted proteins and FPA were cleared from the cerebrospinal fluid within 3 days after bleeding. Their vasoactive and thrombotic capability is limited to the initiation period of vasospasm that usually comes to clinical observation 3-8 days after bleeding. Increased thrombotic activity of the cerebrospinal fluid, as reflected by high levels of FPA and platelet-secreted protein, seemed to promote the occurrence of neurological deficits.