FrankRA2003Biological

Neurobiology of Aging 24 (2003) 521–536 Rapid communication Biological markers for therapeutic trials in Alzheimer’s disease Proceedings of the biological markers working group; NIA initiative on neuroimaging in Alzheimer’s disease Richard A. Frank a,∗ , Douglas Galasko b,1 , Harald Hampel c,2 , John Hardy d,3 , Mony J. de Leon e,4 , Pankaj D. Mehta f,5 , Joseph Rogers g,6 , Eric Siemers h,7 , John Q. Trojanowski i,8 a Pharmacia Corporation, Mailstop 134, Peapack, NJ 07977, USA Department of Neurosciences, UCSD, VA Medical Center, 3350 LaJolla Village Drive, SanDiego, CA 92161, USA c Department of Psychiatry, Alzheimer Memorial Center and Geriatric Psychiatry Branch, Ludwig-Maximilian University, Nussbaumstr. 7, 80336 Munich, Germany Laboratory of Neurogenetics, National Institute on Aging, National Institutes of Health, Building 10, Room 6C103, MSC1589, Bethesda, MD 20892, USA e Department of Psychiatry, Center for Brain Health, NYU School of Medicine, Millhauser Wing HN400, 560 First Ave., New York, NY 10016, USA f Division of Immunology, Department of Developmental Neurobiology, Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, USA g Sun Health Research Institute, 10515 West Santa Fe Drive, Sun City, AZ 85351, USA h Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA i Department of Pathology and Laboratory Medicine, Institute on Aging, Alzheimer’s Disease Center, Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, HUP, Maloney 3rd Floor, 36th and Spruce Streets, Philadelphia, PA 19104-4283, USA b d Received 16 December 2002; accepted 17 December 2002 Keywords: Alzheimer’s disease; Mild cognitive impairment (MCI); Biomarkers; Surrogate endpoints; Disease modification; Drug development; Dementia; Screening; Diagnostics; Prognostics; Enrichment strategy; Bioassay; Progression; CSF; Blood 1. Introduction and aims of the present review Alzheimer’s disease (AD) is the most common neurodegenerative disease and afflicts about 10% of the population over 60. While diagnostic accuracy for the disease has improved, the differential diagnosis for the disorder is still Corresponding author. Tel.: +1-908-901-7986; fax: +1-908-672-0165. E-mail addresses: Richard.Frank@Verizon.net (R.A. Frank), dgalasko@ucsd.edu (D. Galasko), hampel@psy.med.uni-muenchen.de (H. Hampel), hardyj@mail.nih.gov (J. Hardy), mony.deleon@med.nyu.edu (M.J. de Leon), pdmehta@worldnet.att.net (P.D. Mehta), Joseph.Rogers@sunhealth.org (J. Rogers), esiemers@lilly.com (E. Siemers), trojanow@mail.med.upenn.edu (J.Q. Trojanowski). 1 Tel.: +1-858-552-8585. 2 Tel.: +49-89-5160-5814. 3 Tel.: +1-301-451-3829. 4 Tel.: +1-212-263-5805. 5 Tel.: +1-718-494-5159. 6 Tel.: +1-623-876-5466. 7 Tel.: +1-317-433-7144. 8 Tel.: +1-215-662-6399. ∗ problematic. In the very early stages of disease, frequently classified as mild cognitive impairment (MCI), delineating disease process from “normal ageing” may be difficult; in later stages of the disease distinguishing AD from a number of neurodegenerative diseases associated with dementia may also be difficult. Furthermore, the disease progression is slow and there is variability of performance on clinical measures, making it difficult to monitor change effectively. Current criteria for the clinical diagnosis of AD are largely based on the exclusion of other dementing disorders [93]. Although a relatively high accuracy rate of 80–90% applying clinical criteria are reported [44,68,87,150], these studies do not represent typical clinical practice, but instead are based on diagnoses in later stages of the disease as confirmed by longitudinal follow up; classification as definite AD requires confirmation by autopsy. Diagnostic accuracy will be lower at the earlier clinical, and especially pre-symptomatic, stages of the disease. Since disease modifying therapy is likely to be most effective early in the course of disease, early diagnosis is highly desirable before neurodegeneration becomes severe 0197-4580/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0197-4580(03)00002-2 522 R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 and widespread. Thus, there is a great need for biomarkers that could substantially aid early diagnosis of AD. Criteria for an ideal biomarker of AD have been proposed by another consensus group on molecular and biochemical markers of AD; “The ideal biomarker for AD should detect a fundamental feature of neuropathology and be validated in neuropathologically-confirmed cases; it should have a diagnostic sensitivity >80% for detecting AD and a specificity of >80% for distinguishing other dementias; it should be reliable, reproducible, non-invasive, simple to perform, and inexpensive. Recommended steps to establish a biomarker include confirmation by at least two independent studies conducted by qualified investigators with the results published in peer-reviewed journals” [49]. Beyond these criteria for early and accurate diagnosis, it would be especially useful if the biomarker could also capture the beneficial effect of disease modifying therapy. These two problems hamper the clinical development of treatments for AD. We need to be able to make accurate diagnoses early in the disease process and we need to be able to monitor, effectively and inexpensively, whether treatments are working. For these reasons the National Institute on Aging (NIA) commissioned a working group on biomarkers as part of its Initiative on Neuroimaging in AD. This review is the report of that group’s deliberations. As such, this article is an opinion piece; it lacks new data and is not an Table 1 List of biological measures Analyte Feasible, core Tau (total, phospho) Matrix CSF Method ELISA Sources and vendors Sources: p-tau231 : Molecular Geriatrics Corporation (Vernon Hills, IL); t-tau and p-tau181 : Innogenetics NV (Gent, Belgium); p-tau199 : Mitsubishi Kagaku Institute of Life Sciences (Tokyo, Japan) Innogenetics (for A 1–42 in CSF only, no other reliable kits) consider all A , e.g. C-terminus-truncated Vendors: Alerchek (Portland, ME), Alpha Diagnostic (San Antonio), American Diagnostica (Greenwich, CT), United Biotech (Mountain View, CA). hsCRP may be included in the standard laboratory analyses Vendors: ELISA assays can be made using antibodies from DAKO, Quidel, or Advanced Research Technology IL-6RC = IL-6/IL-6R/gp130; vendors: IL-6, sIL-6R and sgp130 “QuantikineTM Immunoassays” (R&D Systems, Minneapolis, MN) Central lab options include Indiana University and the Coryell Institute See published reports for academic laboratories, especially University of Bonn Standard clinical laboratory analyses with B12, folate, and RBC folate See published reports for academic or PhRMA laboratories and sources of antibody See published reports for academic laboratories; see also DNA/RNA above See published reports for academic laboratories Commercial kit from Boehringer-Ingelheim (nephelometry); Aby for ELISA commonly available but no commercial kit See published reports for academic laboratories See published reports for academic laboratories Vendor SYN-X pharma, Canada See published reports for academic laboratories Vendor = Linco, Inc. (“lipid-omics”) Vendor = Nymox, Canada See published reports for academic laboratories See published reports for academic or PhRMA laboratories See published reports for academic laboratories See published reports for academic laboratories vendor: antibody from ATCC, Rockville, MD Commercially available antibody (22C11) See published reports for academic laboratories See published reports for academic laboratories Antibodies are commercially available; see published reports for academic laboratories See published reports for academic laboratories See published reports for academic laboratories A total, 1–42 hsCRP Plasma, CSF Serum, plasma, CSF ELISA ELISA C1q IL-6RC DNA, RNA Oxysterols Homocysteine APP Apo E Isoprostane Alpha-antichymotrypsin Nitro-tyrosine Feasible, non-core A antibodies Glutamine synthetase GFAP Sulfatide AD7C (NTP) Kallikrein Serum, plasma, CSF Serum, CSF Blood Plasma, CSF Plasma CSF DNA, plasma Plasma, CSF, urine Blood, CSF CSF Serum, plasma, CSF Serum, CSF CSF CSF CSF, plasma CSF, plasma ELISA ELISA Genotyping GC/MS ELISA ELISA GC/MS ELISA HPLC, ED ELISA ELISA ELISA MS ELISA ELISA ELISA Immunoblot Immunoblot, densitometric Immunoblot ELISA ELISA ELISA HPLC, GC/MS Uncertain feasibility for multi-center clinical trials A 1–40 Blood, CSF CD59 Serum, plasma, CSF Melanotransferrin (p97) Serum, CSF APP platelet ratio Synaptic markers S100 Neurofilaments, phospho Alpha-synuclein 8OH-Guanidine Platelets CSF Blood, CSF CSF CSF CSF, plasma, urine R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 523 evidence-based review of all available literature. There also were three other working groups established, one each for Magnetic Resonance Imaging (MRI, volumetric), Positron Emission Tomography (PET), and Subjects and Protocol Design. The proceedings of those working groups are not reported here. The mission of the Working Group on Biological Measures was to provide the NIA with a list of biological measures suitable for a multi-center, longitudinal study of AD (http://ocm.od.nih.gov/drc/rfp.htm), with special consideration given to MCI. These measures are considered to have potential value in diagnosis, prognosis, or assessing the beneficial effects of treatment. A wide range of biological measures with possible relevance to AD were considered and then classified into categories of “Feasible, core,” “Feasible, non-core” and “Uncertain feasibility” as listed in Table 1. The relevance of each measure is summarized in Sections 2–4. Feasibility was determined by the availability of a validated assay for the biological measure in question, with properties that included high precision and reliability of measurement, where reagents and standards were well-described. Core analytes were those judged by the group to have reasonable evidence for association with key mechanisms of pathology implicated in AD, while non-core were felt to be less clearly connected with mechanisms of pathogenesis or neurodegeneration in AD. There could be several different reasons to support measuring a biochemical marker, ranging from increasing diagnostic accuracy, enhancing the prediction of progression from MCI to clinical AD, or providing insight into a pathway influenced by a drug treatment for AD. It is likely that no single marker could serve all these utilities, hence the need for a panel of measures. Part of the purpose of publishing the groups’ deliberations is to give the opportunity for others to correct any omissions in our deliberations and to criticize the rationale for any of these biological measures. 2. Feasible, core 2.1. Amyloid β peptide 40 and 42 (Aβ40 and Aβ42) The major component of neuritic plaques is the amyloid (A ) protein, a small 42 residue protein derived through proteolytic processing of a larger membrane bound glycoprotein, the amyloid protein precursor (APP) [128]. Secreted soluble A is a product of normal cell metabolism, and found in various body fluids including plasma and CSF [91]. Recent studies have shown that in AD brain, A protein ending at residue 42 (A 42) is deposited first and is the predominant form in senile plaques; whereas A protein ending at residue 40 (A 40) is deposited later in the disease and is prominent in vascular amyloid deposits [161]. Of all A normally released from cells, A 40 accounts for approximately 90% while A 42 accounts for approximately 10%. 2.1.1. Aβ in cerebrospinal fluid (CSF) Many different home-made ELISA tests have been developed for the measurement of A 42 in CSF and plasma [69,98], and there is currently one commercially available ELISA-kit for the measurement of A 42 in CSF [154]. Data showed that A 42 concentrations are decreased in CSF of AD patients, and a number of studies have confirmed the findings [4,5,64,145]. The specificity to distinguish patients with AD from controls has varied from 42 to 88%, and the sensitivity has varied from 72 to 100% in these studies. The levels were lower in AD patients with the Apo E ε4 allele than those without Apo E ε4 allele [147]. The sensitivity of A 42 measurement was 83.6% for AD patients carrying an Apo E ε4 allele, whereas in AD patients without Apo E ε4 allele it was 54.2%. Decreased CSF A 42 levels are not specific for AD since studies showed that half of the patients with vascular dementia had decreased CSF A 42 [64]. A number of studies have shown that CSF A 40 concentrations in AD and controls were similar [90,145]. In one study [69] however, a decrease in CSF A 40 values was found with significant overlap between the groups. 2.1.2. Aβ in plasma Studies have shown that plasma A 40 and A 42 levels were 2–3-fold higher in patients with familial AD and with presenilin mutations than sporadic AD and controls, and that A levels are approximately 100-fold lower in plasma than CSF [125]. Recent studies have shown that plasma A 40 and A 42 levels are similar in AD and control groups [144]. However, others showed that plasma A 40 levels are increased more in AD patients with Apo E ε4 allele than in those without this allele and in age-matched controls [92]. Because of the considerable overlap between the two groups, the measurement of plasma A 40 levels is not useful as a diagnostic tool to distinguish patients with sporadic AD from elderly non-demented controls. Plasma A 42 levels are similar between AD and controls [92,144]. Recent longitudinal study of unrelated individuals showed that those who subsequently developed AD had higher plasma A 42 levels at entry than those who remained free of dementia [84]. The results indicate that elevated plasma A 42 levels may be detected several years before the onset of symptoms supporting the role of extracellular A 42 in the pathogenesis of AD. In summary, although blood is easy to obtain, it is still unclear if there are systemic changes specific for AD, and to what extent changes in blood composition reflect pathological changes seen in the brain. Many studies have consistently showed that CSF A 42 is reduced in probable AD than controls. Plasma A 42 levels showed no difference between AD and controls, whereas data with A 40 are controversial (either increase in AD or no change). Many studies have 524 R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 showed that CSF A 42 levels were lower in patients with MCI than controls [6]. However, one study found increased levels of CSF A 42 in patients with MCI than controls. Longitudinal study showed that elevated plasma A 42 levels occur before the onset of MCI in some individuals [84]. 2.2. APP APP is the parent molecule of A . There are three main isoforms of APP, of which APP695 is predominantly expressed in neurons and APP751 and 770 are predominantly expressed in astrocytes [11]. APP mRNA is expressed in several non-neuronal tissues, and there are high levels in platelets. The large N-terminus of APP (ectodomain) is released into the extracellular compartment when APP is cleaved by the - and -secretases. The total amounts of secreted APP (sAPP), or specifically cleaved forms sAPP and sAPP can be measured, and are abundant in CSF. In principle, if APP mismetabolism underlies AD, then indices of sAPP could have diagnostic utility. In addition, measuring CSF APP could provide indices of APP processing. For example, administration of an inhibitor of the -secretase enzyme should lead to decreased levels of sAPP in CSF. Although APP has been measured by Western blots in several studies [105,129,156], this method is not truly quantitative, lacks the precision and reliability of other methods, and does not permit the facile analysis of large numbers of samples. Antibodies specific for sAPP and have been developed, which in principle would allow ELISAs to be developed for quantitation of APP. One study made use of such an ELISA to quantify sAPP, and found no difference in CSF levels between patients with AD and controls [59]. Several studies have found that CSF levels of sAPP appear to be slightly decreased in AD compared to controls [105,129,156], with too much overlap for sufficient diagnostic utility. Levels of sAPP do not appear to differ [129]. The CSF concentration of APP appears to decrease with advancing dementia severity, consistent with a smaller pool of surviving neurons producing sAPP. 2.3. Tau proteins One of the major neuropathological hallmarks of AD are neurofibrillary tangles composed of paired helical filaments (PHF). The principal protein subunit of PHF is abnormally hyperphosphorylated tau (p-tau). Normally, the 6 brain tau isoforms are located mainly in axons, associated with the cytoskeleton and intracellular transport systems. Total tau and truncated forms of monomeric and phosphorylated tau can be measured in the CSF. Using antibodies that detect all isoforms of tau proteins independent of phosphorylation, or specific phosphorylation sites, ELISA have been developed to measure total (t-) and phosphorylated CSF tau protein (p-tau) concentrations (for review see [12,55,137]). 2.3.1. Total tau (t-tau) The t-tau, a general marker of neuronal destruction, has been intensely studied in more than 2000 AD patients and 1000 age-matched elderly controls over the last 5–10 years [70]. The most consistent finding is a statistically significant increase of CSF t-tau protein in AD. The mean level of CSF t-tau protein concentration is about 300% higher in AD compared to elderly controls. Across the reviewed studies, sensitivity and specificity levels varied due to the different control groups and statistical methods used. Specificity levels were between 65 and 86% and sensitivity between 40 and 86%. In several studies, a significant elevation was found in patients with early dementia as well. Therefore, in mild dementia, the potential of CSF t-tau protein to discriminate between AD and normal aging is high, with a mean sensitivity of 75% and specificity of 85%. An age-associated increase of t-tau protein has been shown in non-demented subjects. Therefore, the effect of age should be considered when t-tau protein levels are diagnostically employed. Age-dependent reference values for t-tau protein have already been established for subjects between 21 and 50 years old at <300 pg/ml, between 51 and 70 years old at <450 pg/ml, and between 70 and 93 years old at <500 pg/ml [19]. 2.3.2. Phospho tau (p-tau) Currently, promising efforts are under way to establish p-tau in CSF as a putative disease specific biological marker for AD. Immunoassays have been developed specifically detecting tau at different epitopes, such as threonine 231 (p-tau231 ), serine 199 (p-tau199 ) and threonine 181 (p-tau181 ) [12]. Evidence from these studies indicates that quantification of tau phosphorylated at these specific sites may improve early detection, differential diagnosis and tracking of disease progression in AD. In a pilot study, CSF p-tau231 distinguished between AD-patients and subjects with other neurological disorders with a sensitivity of 85% and a specificity of 97% [73]. In an independent large-scale multi-center study, p-tau231 significantly improved differential diagnosis between AD and other non-AD groups (sensitivity 90%, specificity 80%). In AD versus fronto-temporal dementia, p-tau231 raised sensitivity levels compared to t-tau from 57.7 to 90.2% at a specificity level of 92.3% for both markers [22]. In the differentiation between AD and geriatric major depression, specificity levels were raised from 68% for t-tau to 85% using p-tau231 at a sensitivity level of 90% for both markers. Discriminative power of p-tau231 was significantly higher compared to t-tau [21]. Itoh et al. reported that CSF p-tau199 discriminates between AD and the combined group of non-AD subjects with a sensitivity and a specificity of 85% [67]. CSF p-tau181 was elevated in AD compared to other dementias and healthy controls and has been proposed as a potential marker for discriminating AD patients from patients suffering from dementia with Lewy bodies [136] or vascular dementia [127]. In a 6-year serial CSF longitudinal study, p-tau231 concentrations decreased linearly with time in AD patients. Furthermore, rate R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 525 of change of p-tau231 was correlated with the MMSE score at baseline, with a more pronounced rate of decline with advanced cognitive impairment. This was not found for t-tau [50]. Another promising value of p-tau231 may be its ability to predict cognitive decline and conversion to AD from MCI. A 1-year longitudinal MCI study showed progressive elevation of p-tau231 concentrations in MCI subjects compared to healthy controls [29]. High CSF p-tau231 levels significantly correlated with subsequent cognitive decline and conversion to AD. In addition to p-tau231 , old age and apolipoprotein E ε4 carrier status independently predicted cognitive decline in this sample of 77 MCI subjects. The whole model explained 27% of variance [20]. Confirmation of assay performance at autopsy in prospective, population-based studies is warranted to fully establish CSF p-tau proteins as putative specific AD biomarkers for routine diagnostic use. To compare diagnostic accuracy and combined evaluation of CSF concentrations of p-tau231 , p-tau181 and p-tau199 a large-scale multi-center comparative study was recently completed. Comparative data on diagnostic accuracy and assay performance will be published soon [51]. 2.4. Apolipoprotein E (Apo E) Apolipoprotein E (Apo E) is a 34 kDa polymorphic protein that is involved in the transport and redistribution of lipids among various tissues. Apo E binds avidly to A , and its immunoreactivity has been shown in amyloid plaques cores and vascular amyloid. The Apo E4 allele is a significant risk factor for the development of sporadic and familial late-onset AD [123]. In order to understand the relationship between Apo E polymorphism and Apo E concentrations in blood and CSF, investigators have quantitated the levels in AD and controls. Reports on the concentrations of serum and CSF Apo E in AD are conflicting. Some studies showed higher levels of Apo E in CSF of AD than controls, however, others showed that the levels were similar. There was no correlation between Apo E isoforms and Apo E levels in AD [109]. Serum Apo E levels were decreased in AD relative to controls [134], wheras others reported that the levels were increased or similar between AD and controls [139,142]. There are no published report on the levels of Apo E between MCI and controls and its relationship with Apo E genotype. 2.5. Isoprostanes Growing evidence implicates oxidative/nitrative damage in the pathogenesis of AD and other neurodegenerative disorders [46,110]. Thus, neurons at risk for AD degeneration have increased lipid peroxidation (LPO), nitration, free carbonyls, and nucleic acid oxidation. Since specific isoprostanes (iPs), i.e. 8,12-iso-iPF2 -VI appear to be sensitive markers of LPO in AD patients, subsequent studies were undertaken showing that 8,12-iso-iPF2 -VI levels are elevated in urine, blood and CSF of AD patients and that these values correlate with memory impairments, CSF tau levels and the number of Apo E4 alleles [110–112]. This suggests that 8,12-iso-iPF2 -VI is a useful biomarker in AD, and this is supported by data on levels of 8,12-iso-iPF2 -VI in urine, plasma and brain of a transgenic mouse model of AD amyloidosis, which correlated with increasing brain A levels and deposits [113]. Analysis of LPO due to oxidative stress is performed by measuring isoprostane levels in urine and brain as described by Pratico et al. [110–113]. Briefly, urine is collected from subjects into plastic tubes containing BHT. Samples are spiked with internal standard (d4 -8,12-iso-iPF2 -VI) and extracted on a C18 cartridge column. The eluent is purified by thin layer chromatography and assayed by negative ion chemical ionization gas chromatography/mass spectrometry. A urine aliquot (0.1 ml) is used for measurement of creatinine levels by a commercially available standardized automated colorimetric assay. Levels are expressed as ng per mg of creatinine. Finally, CSF, plasma and postmortem brain tissue samples prepared as described by Pratico and co-workers are used for isoprostane analysis by the procedures already described. While additional studies are needed to confirm and extend these findings in larger cohorts of MCI and AD patients, 8,12-iso-iPF2 -VI appears to be a promising peripheral marker for the onset and progression of AD, and it remains to be determined if this analyte will informative for monitoring the response of AD patients to new therapies in clinical trials. 2.6. α1-Antichymotrypsin (ACT) ACT, one of the serine proteinase inhibitors, plays an important role in inflammation. Concentrations of ACT are elevated in the brains of AD, and ACT is one of the components of the senile plaque [1,75]. Many studies have been published in AD CSF and serum or plasma but the findings are controversial. Some studies showed no difference in ACT levels between AD and controls, whereas others showed higher levels in AD than controls [76,83,108]. Recent report showed that the levels correlate with severity of dementia [77]. Further studies are essential to confirm these findings. There are no studies published on patients with MCI. 2.7. The soluble interleukin-6-receptor-complex (IL-6, IL-6R, gp130) Basic research and clinical studies have indicated that immunological and inflammatory mechanisms may play an important role in the pathophysiology of AD. It has been shown that amyloid within senile plaques is associated with activated microglia and astrocytes that express inflammatory proteins and neuroregulatory factors such as interleukin-6 (IL-6). IL-6 has been consistently detected in the frontal-, parietal- and occipital-cortex and hippocampus of AD patients but not of non-demented elderly subjects. 526 R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 In further investigations IL-6 has been demonstrated in diffuse early plaques without neuritic pathology of isocortical (frontal-, temporal- and parietal-cortex) and hippocampal brain samples of AD patients. IL-6 immunoreactivity was rare in classical plaques and absent in compact or burned-out plaques. Therefore, it has been suggested that IL-6 expression may appear before neuritic changes rather than follow neuritic degeneration (for reviews see [3,52]). Basic studies show that IL-6 exerts its biological actions only by complex interactions with specific soluble or membrane bound receptors, forming the biologically active IL-6 receptor complex (IL-6RC). The involved proteins, besides the 19.5 kDa cytokine IL-6, are two membrane glycoproteins, an 80 kDa protein referred to as the ligand-binding -subunit (gp80, IL-6R, or CD126) and a 130 kDa protein referred to as the non-ligand binding, affinity converting and signal transducing -receptor (gp130 or CD130). All members of the IL-6 cytokine family (IL-6, IL-11, oncostatin M (OSM), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1)) share gp130 as a component critical for signal transduction. In the nervous system IL-6 can be secreted by microglia, astroglia, neurons and endothelial cells, the IL-6R by neurons and gp130 by all cells. gp130 neuropil immunoreactivity was observed in telencephalic structures including the hippocampus, cerebral cortex and caudate-putamen. Activation of membrane bound gp130 by IL-6 and the soluble IL-6R was reported to generate a neuronal differentiation signal. Soluble forms of the two receptors (sIL-6R, sgp130) arise by limited proteolysis (shedding) or differential splicing (sIL-6R with 38 kDa and sgp130 with 68 kDa). It has been reported that this soluble complex (sIL-6RC) forms a hexameric structure in solution, consisting of the three different proteins with a 2:2:2 stoichiometry. There is a complex regulatory interaction between all sIL-6R components. sIL-6R enhances IL-6 effects by making the ligand accessible to the membrane-bound signal-transducing -subunit, however, it has also been shown to augment the action of sgp130, which neutralizes IL-6 signals. There are commercially available bioassays (ELISA) to detect the IL-6RC in biological fluids. Using such an assay statistically significant decreased CSF concentrations of sIL-6R [54] and sgp130 [56], in the presence of unchanged IL-6 concentrations [53] in AD-patients compared to healthy age-matched controls were reported. In addition, these data indicate that the application of multivariate discriminant analysis using combined CSF total tau protein (t-tau) and sIL-6RC components may add more certainty to the diagnosis of AD [56]. The reported method, however, needs to be extended to an independent group of patients, comparisons and control subjects to assess the true diagnostic applicability. Interpretations regarding the relationship between CSF and brain levels of the IL-6RC at present remain speculative and require studies based on simultaneous measurement of corresponding CSF and brain samples. 2.8. C-reactive protein (CRP) CRP is a pentraxin acute phase reactant that can activate complement in an antibody-independent fashion. It is upregulated in AD brains compared to samples from non-demented (ND) individuals [85]. High sensitivity CRP (hsCRP) in serum is a well known biomarker for presence and risk of coronary artery disease [8,117], and has recently been used to assess effectiveness of a statin in a clinical trial [116]. In a follow-up of the Honolulu-Asia Aging Study, Schmidt et al. [126] found that men in the upper three quartiles for serum hsCRP had a three-fold, significantly increased risk for all dementias combined, AD, and vascular dementia. Over 1000 cases were examined. Remarkably, the serum samples that were assayed from these cases had been taken and stored some 20–25 years earlier, long before onset of dementia symptoms in any subject. Alternatively, in a study of 11 AD and 11 ND patients, Licastro et al. [78] observed comparable levels of CRP in AD and ND patients. 2.9. C1q C1q is the first component of the classical complement pathway. Normally synthesized in the liver, there is strong evidence that C1q can also be produced in brain by endogenous cells [131]. In pathologically-vulnerable regions of the AD brain, C1q levels have been widely demonstrated to be dramatically elevated in AD compared to ND samples [3,16]. C1q avidly binds A and A -related peptides (including intact APP) that contain an accessible human A 1–16 sequence [158], and is potently activated, in an antibody-independent fashion, by A [86,118,159]. C1q also avidly binds to and is activated by tau aggregates [131]. Of the many different inflammatory mediators that enhance A aggregation, C1q is one of the most potent [160]. Collectively, the above findings suggest that the A - and aggregated tau-laden AD brain might serve as a sink for C1q. Consistent with this hypothesis, significant decreases of C1q have been reported in AD compared to ND CSF, and these CSF C1q deficits exhibited significant correlations with cognitive deficits over a wide range of tests [140]. 2.10. Homocysteine Homocysteine is a sulfur-containing amino acid, derived from the metabolism of methionine. In homocystinuria, an inherited metabolic disorder, very high levels of homocysteine in the blood are associated with widespread premature atherosclerosis. This led to the question of whether mild elevation of homocysteine may promote vascular disease in adult life. Studies involving a large number of subjects found that concentrations above the upper limit of normal (about 15 umol/l) are associated with vascular disease and with increasing mortality [15,103]—as much as 10% of population risk for coronary heart disease cold be attributed to homocysteine. Vascular injury, and factors such as hypertension have R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 527 been implicated in some studies of AD. One of the first associations between homocysteine and AD came from a study comparing autopsied patients with AD versus controls. Homocysteine levels in the highest tertile were associated with a greater than four-fold increase in the relative risk of AD [25], and low vitamin B12 and folate levels were also found in AD. The extent of risk has varied across further studies. In the Framingham study, plasma homocysteine levels greater than 14 umol/l almost doubled the risk of AD [130]. Another survey found that, independent of low folate levels, higher levels of homocysteine were associated with worse memory performance in a large group of older subjects [97]. A recent magnetic resonance imaging (MRI) study suggested that increased homocysteine was a risk factor for cerebrovascular disease independent of AD [93], raising the question of whether homocysteine is directly linked to mechanisms of AD or indirectly, via cerebrovascular disease. While homocysteine is not a diagnostic test, it may represent a modifiable risk factor for dementia, because supplementation with B group vitamins lowers homocysteine levels by up to 30% [24]. Clinical trials are beginning to explore the impact of such treatment on vascular disease, and recent studies have begun in patients with AD. In these trials, levels of vitamin B12, folate and homocysteine will need to be monitored as predictors of outcome. There are a variety of assays to measure homocysteine levels, including immunoassays and HPLC-based methods, which have similar performance and good precision [94,166]. In the US, fortification of cereal grain products with folic acid was mandated in the late 1990s, and there has already been a decrease in mean values of homocysteine in older individuals. This will make it more difficult to evaluate whether further supplementation has benefits in dementia. 2.11. Oxysterols and cholesterol metabolism Converging evidence links cholesterol metabolism and AD [58]. The Apo E4 protein, involved in cholesterol transport in the brain and body, is a risk factor for AD. In observational studies, the use of cholesterol-lowering drugs (HMG-CoA reductase inhibitors, or statins) is associated with a decreased risk of AD [162]. Cholesterol-lowering drugs decrease neuronal production of A in cultured cells and in animals [39,114,115], while high cholesterol diets enhance amyloid deposition in animals [141]. Cholesteryl ester levels in the brain are directly correlated with A production [114], and the -secretase enzyme, which cleaves APP to give rise to A , is found in cholesterol-rich lipid rafts [58]. The relationship between serum cholesterol and lipid levels and the risk of AD is not clear. In one prospective study, high serum cholesterol in midlife appeared to increase the risk of incident AD [102], while another population-based study found no clear relationship between cholesterol levels and AD [119]. Several oxysterols can be measured as indices of brain cholesterol metabolism. Sensitive and reliable assay meth- ods are available, and utilize chromatography/mass spectrometry [36]. Lathosterol is a major precursor of cholesterol, and can be measured in CSF. 24S-Hydroxycholesterol is an index of brain elimination of cholesterol, whose levels in CSF and plasma reflect brain production. The plasma levels of 24S-hydroxycholesterol overlap markedly between patients with AD and controls [17], although a slight increase in CSF levels was recently reported in patients with mild AD compared to more severe AD or to controls [106]. Although measures of cholesterol metabolism do not appear to be diagnostically useful in AD, they may serve as indices of treatment effects and mechanisms in clinical trials. The mounting evidence implicating cholesterol pathways in AD has led to preliminary studies of statins in patients with AD [40,135]. Following treatment with a statin, CSF levels of lathosterol and 24S-hydroxycholesterol decreased, but CSF levels of A were not found to be altered. Further clinical trials of cholesterol-lowering agents in AD have recently been initiated. 2.12. 3-Nitrotyrosine In the presence of reactive oxygen species and nitric oxide, 3-nitrotyrosine (3NT) may be formed in constituent proteins in the brain. The predominant pathway appears to involve nitric oxide in the presence of superoxide ion to form peroxynitrite. Peroxynitrite in turn reacts with tyrosine residues in proteins or with free tyrosine to form 3NT [61]. 3NT can be found in CSF in specific proteins, e.g. superoxide dismutase [7], or can be found as free 3NT. With normal aging, 3NT concentrations in CSF increase modestly from about 0.75 nM at 40 years to about 2 nM at 80 years [151]. In the brains of patients with AD, regionally specific increases in 3NT have been found in the neocortex and cerebellum [60]. In the CSF of patients with AD, the concentration of 3NT is reported by Tohgi et al. [151] to be 11.4 nM, or about six-fold higher than age-matched controls. In this study, concentrations of 3NT in CSF were inversely correlated with Mini-mental Status Exam (MMSE) scores, but did not correlate with duration of disease. Analysis of 3NT is relatively straightforward when done by high pressure liquid chromatography with electrochemical detection (HPLC/ED) [61]. The stability of 3NT in plasma proteins however is not well established, and thus the significance of 3NT concentrations in this compartment is less clear [61,149]. A volume of 400 l is necessary for CSF analysis, and acid precipitation of proteins is sufficient for the analysis of free 3NT [61,151]. Increased concentrations of 3NT in CSF, along with changes in isoprostanes and 8-hydroxy-2-deoxyguanosine (8OH2DG) as outlined in this review, are consistent with the suggestion that oxidative stress may play an important role in the pathogenesis of AD. What is not yet clear is whether the increase in reactive oxygen species with oxidative stress are proximal or distal in the pathophysiologic cascade leading to cell death. As with a number of proposed 528 R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 biomarkers for AD, longitudinal studies are necessary to determine if changes such as those in 3NT in CSF increase monotonically with disease progression, or might have a more complex relationship with disease severity. Even in the absence of a linear relationship with disease severity, measurement of 3NT and other markers of oxidative stress may provide an indirect marker of drug efficacy in sub-acute clinical trials using putative disease-modifying agents. 3. Feasible, non-core 3.1. Glutamine synthetase Glutamine synthetase is a widely expressed enzyme. In the brain, it is made by astrocytes, and plays a role in detoxifying ammonia. Its levels increase after injury, and the temporal lobe of patients with AD show increased immunostaining for glutamine synthetase, most likely due to astrogliosis [153]. Levels in CSF were increased in a small number of patients with AD relative to controls, but intermediate increases were also seen in vascular dementia and amyotrophic lateral sclerosis. Production by brain tissue is likely to be the main contributor to CSF glutamine synthetase, because levels in CSF are considerably higher than those in serum. A sensitive sandwich ELISA has recently been developed, allowing re-evaluation of this marker in serum [143]. In a preliminary study, increased levels (greater than 2SDs above the mean for normals) were found in 24/24 AD patients tested, while only 1/13 patients with other causes of dementia showed an increase. It is not clear whether other brain disorders or systemic conditions may also influence levels of this marker in serum, and further clinical studies are in progress. 3.2. Human antibodies against Aβ-related proteins Recent work in transgenic mouse models has suggested that antibodies directed at A , generated by passive or active immunization, may help clear A and reduce cognitive/mnemonic deficits [9,124]. Although this approach does not so far appear viable in humans, owing to uncontrolled inflammatory responses following multiple administrations of the immunogen, it has generated ancillary interest in the possibility that humans may naturally develop antibodies to A . Whether such antibodies might be helpful, harmful, or neutral with respect to the development and progression of AD remains undetermined. Likewise, it is unclear what the conditions that induce formation of such antibodies might be, or how specific they are to AD. In serum derived from a single AD patient, Gaskin et al. [45] found four Epstein–Barr virus (EBV) transformed B cell lines that secreted antibodies reactive with AD A deposits. Subsequent studies by the group suggested that the antibodies were generated via an antigen-driven process [38], and that there was a significantly higher frequency of anti-A antibody-producing EBV-transformed B cell lines in AD (2.3%) than non-demented elderly controls (ND) (0.3%) serum [163]. Two additional studies, one published and one unpublished, have directly assayed anti-A antibodies in AD and ND serum. Analysis of 50 probable AD and 50 control serum samples using a plaque-killing assay for presence of anti-A antibodies revealed that approximately 50% of AD and 50% of control cases were positive (J. Rogers and B. Mueller-Hill, unpublished data). These findings are generally consistent with the report of Hyman et al. [65] who found low but detectable A autoantibodies in just over 50% of all patients, and modest levels in under 5% of all patients. In CSF, however, significantly lower titers of anti-A antibodies have been observed in AD compared to ND patients using an ELISA [35]. 3.3. Glial fibrillary acidic protein (GFAP) and antibodies to GFAP GFAP is a major intermediate filament cytoskeleton protein expressed primarily by astrocytes [37,89]. It forms an important component of the plaques in AD. Because GFAP is considered a brain specific marker, investigators have measured the levels in CSF and serum from AD patients. Investigators have shown higher levels of GFAP in CSF of AD than controls, however, there was significant overlap between the groups [157]. Several studies have found higher frequency of antibodies to GFAP CSF and serum of AD than controls or OND [90,146,148]. Whether GFAP antibodies are useful as a marker in early or late onset AD, additional studies are needed. There is no published report on GFAP or antibodies to GFAP levels in patients with MCI. 3.4. Sulfatide While many of the biomarkers currently utilized in AD are peptides or amino acid derivatives, lipids other than cholesterol metabolites have not been extensively studied. Recently, the concentration of a lipid known as sulfatide was reported to be reduced in patients with CDR scores of 0.5 compared to age-matched volunteers [62]. The alteration in concentration was found after screening CSF using electrospray ionization mass spectroscopy for changes in various lipid concentrations. The concentration of sulfatide was normalized to the concentration of phosphotidyl inositol (PI) in CSF. Using this method, the sulfatide/PI ratio in patients with a CDR value of 0.5 was 0.51 ± 0.19 (N = 12) as compared to 0.88 ± 0.07 in volunteers (N = 16). Very little overlap in values was seen in this small study. Studies have also shown a reduction in brain sulfatide in patients with a CDR value of 0.5 as well as in patients with more advanced dementia due to AD [57]. Approximately 1 ml of CSF is required for analysis of sulfatide by nanospray ionization mass spectroscopy. R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 529 Freeze-thaw effects on sulfatide measurements have not been specifically reported. The initial data in cross-sectional studies with small sample sizes for this compound suggest that alterations in sulfatide production or metabolism occur in AD. Additional larger studies will be necessary to confirm this effect and to determine the sensitivity and specificity of sulfatide in CSF as a diagnostic marker of the disease. Additional longitudinal studies will be necessary to determine if additional changes in CSF sulfatide occur with disease progression. The fact that the sulfatide/PI ratio in CSF is decreased by 42% in patients with a CDR value of 0.5 would suggest that changes in this ratio occur early in the disease process. 3.5. AD7C/NTP AD7C refers to a series of antibodies developed against an approximately 41 kDa protein originally called neuronal thread protein (NTP), and used to establish a commercial ELISA. Antibodies to AD7C/NTP stain neuronal processes surrounding plaques in AD [27]. The relevance of this molecule to pathogenic mechanisms in AD is otherwise unclear, although roles have been proposed in neuritic sprouting and apoptosis [28]. Levels of AD7C in CSF are significantly increased in AD compared to controls, including patients with multiple sclerosis and Parkinson’s disease [27,70,96]. To date, few studies have evaluated this marker. In one study, sensitivity for AD appeared to be similar to that of total tau levels [70]. The increased CSF levels of AD7C correlated with dementia severity. A competitive ELISA was also developed to try to measure AD7C in urine. A preliminary publication reports on details of assay methodology, but the validity of the urine assay has not been established against rigorously diagnosed patients with AD and controls [100]. Further understanding of the biology of AD7C and its relationship to AD, and more clinical studies are necessary to establish the potential utility of this marker. 3.6. Kallikrein 6 Kallikrein 6 is a serine protease that was identified through work designed to isolate -secretase, using a strategy that included the use of PCR primers for serine proteases [79]. After the initial discovery of kallikrein 6, in vitro studies using human embryonic kidney cells did show that transfection of these cells with APP and kallikrein 6 resulted in the formation of truncated forms of A . Subsequent immunohistochemical studies have shown that kallikrein 6 is highly expressed in cells of the choroid plexus [30]. Consistent with this finding, kallakrein 6 concentrations are normally 5–10-fold higher in CSF than in plasma [30]. In patients with AD, CSF concentrations of kallakrein 6 are reported to be increased about three-fold compared to controls [30]. Some overlap of values is present due to skewed distributions and some relatively high values among volunteers. In the brain, levels of kallakrein 6 are reduced to about one-third of those in healthy controls [166]; in plasma a substantial reduction has also been reported [30]. Kallikrein 6 concentrations can be determined in CSF, plasma and brain extracts using a modified ELISA technique that has been reported in detail [31,165]. Approximately 100 l of CSF are required. Many biomarkers related to structural brain components decrease in AD, and kallikrein 6 levels are diminished in brain and plasma. Conversely, the increase in kallikrein 6 in CSF in AD patients suggests that egress of this enzyme from cells in the choroid plexus increases or that clearance from CSF is reduced. Whether the changes in kallakrein 6 in brain, CSF and plasma are a primary part of the pathology or are a secondary phenomenon can not as yet be determined. The relationship of these changes to the hydrocephalus ex vacuo that inevitably develops with AD is not clear. Evidence suggesting that the change in kallikrein 6 could be a primary part of the pathology includes the formation of truncated forms of A using in vitro systems expressing kallikrein 6 and APP [80]. Studies of CSF from patients with MCI or from pre-symptomatic patients carrying a mutation for autosomal dominant AD could help to determine whether changes in kallikrein 6 are an early or late abnormality in the course of AD. 4. Uncertain feasibility for multi-center clinical studies 4.1. APP isoforms in platelet membranes APP is abundant in platelets, predominantly as the 770 isoform. After the platelets are activated, soluble forms of cleaved APP are released, analogous to processing in neurons, after platelets are activated [23]. Two research groups have measured relative amounts of two platelet isoforms in platelet membrane preparations, and reported a decrease in the amount of the higher (130 kDa) band compared to the lower (110 kDa band) [10,33]. It is not clear why systemic abnormalities of short-lived cells such as platelets should be found in patients with sporadic AD. Both groups used the same commercially available antibody against APP to visualize APP on Western blots, followed by densitometry. This method will be difficult to standardize between different laboratories and is not amenable to scaling up to analyze large numbers of samples. A relatively large volume of blood is needed (about 30 ml), with careful handling, rapid processing and preparation of platelets, and prompt freezing of samples before immunoblotting. There are no data on how reliably these procedures can be carried out in a multi-center study. The findings are intriguing: the ratio of the 130:110 kDa bands is decreased in patients with AD and even in MCI, relative to controls, and the extent of decrease is correlated with indices of dementia severity [10,31,32,104]. To interpret this potential biomarker more clearly, we need a better understanding 530 R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 of the biochemical nature of the platelet isoforms of APP, and their relationship to pathophysiological steps in AD. 4.2. Neurofilament (NF) proteins NF subunits are highly abundant axonal proteins, and high (NFH), middle (NFM) and low (NFL) molecular weight NFs are found in specific classes of neurons, where they play roles in axonal structure. Their content is highest in those neurons with the largest axons. The release of NF proteins into CSF could be an index of axonal or neuronal damage. However, this release would not be expected to be specific to any particular disease process. In fact, increased levels of NF proteins have been found in CSF in diverse conditions, including ALS, multiple sclerosis, brain trauma, AD and vascular dementia [120,121,136,138]. Phosphorylation regulates various properties of NF, especially NFH and NFM, including their interactions with other molecules. Since abnormal kinase activity, and hyperphosphorylation of tau protein is a hallmark of AD, phosphorylated NFs could possibly be more directly related to AD and although they are not the building block proteins of neurofibrillary tangles, NF proteins do accumulate in the tangles of AD and other tauopathies where experimental studies in transgenic mice suggest they may play a role as pathological chaperones. A sensitive ELISA was developed, capable of quantifying low levels of NF-light in CSF [120]. Subsequent studies showed that this marker was increased in many conditions, including AD, as already noted. In patients with AD and vascular dementia, there were slightly higher levels of CSF NF in patients with more extensive white matter changes [136]. Also, levels tended to be higher in fronto-temporal dementia than in AD [138]. Although measuring NF levels does not appear to have specific diagnostic utility, this marker could potentially be used to assess whether treatment protects axons from axonal degeneration: this benefit could lead to a decrease in CSF levels of NF over time. Recently, a sensitive ELISA for phosphorylated forms of NF high and medium was established [63]. A preliminary study showed that levels of phosphorylated NF proteins were significantly increased in AD compared to normal and disease controls. This interesting finding requires confirmation and extension. 4.3. Synaptic markers Loss of synapses or impaired synaptic function are prominent findings in AD, and may correlate more strongly with the severity of dementia than other pathology indices [82]. There are many synaptic proteins, and it is possible that they could be identified in CSF in patients undergoing synaptic dysfunction in AD. In one preliminary study, CSF was concentrated, followed by isoelectric focusing, then Western blotting with a panel of antibodies against synaptic components [26]. Immunoreactive bands for rab3a, synaptotagmin, growth-associated protein (GAP-43), synaptosomal-associated protein (SNAP-25) and neurogranin were detected in CSF. Sensitive assays will need to be developed to allow AD and controls to be compared. 4.4. Alpha-synuclein Alpha-synuclein (AS) is a small highly charged protein expressed in CNS neurons and predominately localized at presynaptic terminals, and it belongs to a family of proteins, which presently comprises two other members: beta-synuclein and gamma-synuclein [101]. However, only AS is found in fibrillar pathological lesions, and cytoplasmic pathological inclusions comprised of filamentous AS are seen in AD with plaques and tangles, but these lesions are most characteristic of neurodegenerative disorders such as Parkinson’s disease (PD) that are collectively known as alpha-synucleinopathies [47,74,101,152]. While AS inclusions are located predominantly in the perikarya (LBs) or processes (Lewy neurites, LN) of brain stem nuclei and diencephalic neurons in PD, LBs and LNs have a far more widespread cerebral distribution in dementia with LBs (DLB) which can be indistinguishabfle clinically from AD during life [47,101,152]. Indeed, DLB often is associated with sufficient plaques and tangles to justify a concurrent diagnosis of AD, and the combination of DLB and AD often is referred to as the LB variant of AD (LBVAD). The accurate diagnosis of neurodegenerative diseases can be challenging due to overlapping clinical manifestations, and it can be difficult to differentiate diseases having dementia as a common clinical feature during life. For example, DLB is the second most common neurodegenerative dementing illness of the elderly after AD, but it often is misdiagnosed. Presently, there is no bioassay to assist in the diagnosis of alpha-synucleinopathies, and the development of a reliable biomarker that could facilitate the detection of these disorders could improve the specificity and sensitivity of diagnosis. 4.5. 8OH 2 Deoxyguanosine Studies of 8-hydroxy-2 -deoxyguanosine (8OHDG) have been performed related to AD, PD and Huntington’s disease (HD). The formation of 8OHDG is thought to be a result of attack by reactive oxygen species (ROS) on DNA. In particular, the actions of the hydroxyl ion [2] and peroxynitrite [66] have been suggested, implying a possible role of hydrogen peroxide and nitric oxide. Increased levels of 8OHDG have been found in mitochondrial DNA [88] and in DNA from brain and CSF of patients with AD [80]. Additionally, levels of 8-hydroxy guanine (8OHG) in DNA are increased in the brain and CSF of AD patients [43,81]. Conversely, decreased concentrations of free 8OHDG [79] and 8OHG [81] have been reported in the CSF of patients with AD obtained at autopsy. Increased 8OHDG has also been reported in the brains of patients with PD [122], HD R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 531 [18] and ALS [42] and in urine and CSF of patients with ALS [14]. Measurements of 8OHDG and 8OHG provide an opportunity to assess damage by ROS directly to DNA, a determination not generally provided by other biomarkers. The ability to assess 8OHDG in small volumes of urine also is attractive. Although the cross-sectional data to date in four different neurodegenerative diseases would suggest that this marker may be fundamentally related to neurodegenerative processes, the usual caveats concerning the lack of longitudinal data should be noted. Finally, the methodology necessary to measure 8OHDG and 8OHG appears quite complex at this time [13], reducing the ability to readily obtain these measurements. Auto-oxidation of deoxy-guanosine can occur during sample preparation if appropriate precautions are not taken [95]. Additional methodological work as well as longitudinal clinical data would greatly improve the utility of these analytes. 4.6. Melanotransferrin Melanotransferrin (also known as p97) is a protein related to transferrin that may have roles in iron transport in the brain. After an antibody to p97 was shown to stain microglia surrounding AD plaques, a study was undertaken to measure p97 in CSF and serum. The initial study found almost perfect discrimination between AD and controls in CSF and serum [71], but used a complex assay that underwent several changes of format in later studies. A second study by the same group found a much wider normal rage, and elevation in AD relative to controls [41]. Another study used a dot-blot assay and found increased immunoreactivity with a melanotransferrin antibody, with high sensitivity and specificity for AD [72]. The different assay methods and specificity of the assays need to be reconciled, and more knowledge is needed about the role of p97 in the brain in AD, to allow p97 to be better understood as a potential biomarker related to AD. 4.7. CD59 CD59 (membrane inhibitor of reactive lysis) is a complement regulatory protein that inhibits fixation of the lytic membrane attack complex on homologous cell membranes. Significant decreases of CD59 have been reported for homogenates of AD compared to ND cortex [164]. This is potentially of great importance since deficiencies in CD59 would be permissive for enhanced complement attack, and full complement activation is known to occur in AD cortex [3,118,132,159]. One of the difficulties with CD59 and, indeed, almost all inflammation-related molecules as biomarkers for AD is their potential susceptibility to confounding with other inflammatory conditions and intercurrent infections. Under such circumstances, multiple samples over several months may be needed to establish a reliable baseline. 4.8. S100 β S100 is a calcium binding protein expressed primarily by astrocytes in the brain [34]. It has been implicated in the process of cell growth and differentiation. Activated astrocyes have been reported to be associated with amyloid plaques and shown to contain elevated levels of S100 [99,155]. CSF levels of S100 were higher in patients with AD and FTD than controls [48,133]. Recent report indicated that CSF S100 were higher in mild to moderate AD than advanced stage AD [107]. There are no published studies on MCI. 5. Discussion The value of the samples of physiologic fluids to be collected will be greatly enhanced by the breadth and scope of imaging and classical clinical assessments to be available in the same well-characterized subjects. For this reason it is expected that, in general, samples for biological measures would be collected at each time point for which there are also images and cognitive data collected. Furthermore, patient documentation should include potential confounding factors such as chronobiology and nutritional supplementation, as well as concomitant diseases and medications. A limited range of samples were proposed for collection, preparation, and storage under a range of conditions in anticipation of new assays being developed. Informed consent should anticipate both current and possible future uses for these samples and the data derived from them, including novel research. The importance of quality assurance was emphasized. One approach would be to establish central laboratories for each analysis, taking into account the costs and risks of shipping samples. A tracking system would be needed for each sample and the assay results, irrespective of the number of laboratories involved. A tracking system is particularly important for the banked samples. The Working Group also proposed that the NIA consider the value of autopsy material, which would require re-consenting and tracking of patients over a duration beyond the scope of the planned trial. Excepting autopsy material, sites participating in the trial should be required to achieve certain minimum levels of achievement in the collection of useful samples of each type; criteria should be higher for cross-sectional than longitudinal collections, and sites failing these criteria should be closed. 6. Conclusion Such a resource as this database and sample repository would be of unique value in the development of new medications and should be freely available to any applicant, public or private. Special additional considerations would need to be given to immortalized cell lines as a source of 532 R.A. Frank et al. / Neurobiology of Aging 24 (2003) 521–536 [8] Auer J, Rammer M, Berent R, Weber T, Lassnig E, Eber B. Relation of C-reactive protein levels to presence, extent, and severity of angiographic coronary artery disease. Indian Heart J 2002;54:284–8. [9] Bard F, Cannon C, Barbour R, Burke RL, Games D, Grajeda H, et al. 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The amyloid precursor protein of Alzheimer’s disease is released by human platelets. J Biol Chem 1990;265:15977–83. [24] Clarke R, Collins R. Can dietary supplements with folic acid or Vitamin B6 reduce cardiovascular risk? Design of clinical trials to test the homocysteine hypothesis of vascular disease. J Cardiovasc Risk 1998;5:249–55. [25] Clarke R, Smith AD, Jobst KA, Refsum H, Sutton L, Ueland PM. Folate, Vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch Neurol 1998;55:1449–55. [26] Davidsson P, Puchades M, Blennow K. Identification of synaptic vesicle, pre- and postsynaptic proteins in human cerebrospinal fluid using liquid-phase isoelectric focusing. Electrophoresis 1999;20:431–7. DNA. It is recognized, however, that the physical samples in storage, as distinguished from the imaging data, would represent a finite resource and therefore a review body would need to be established for the consideration of proposed uses. Such a review body would not restrict access for novel research nor would it preclude the development of innovative products based on that novel research. 7. Conflict of interest statement The following conflicts of interest were declared by the authors with respect to publication of this paper: Richard A. Frank is an employee of Pharmacia Corporation. Douglas Galasko is a paid consultant of Elan Pharmaceuticals Inc., Pharmacia Inc., and Takeda Pharmaceuticals Inc. Mony de Leon is an employee of NYU School of Medicine and holds a patent on CSF biomarker dilution factor corrections by MRI. Joseph Rogers holds equity in Elan Pharmaceuticals and a patent on the use of anti-inflammatory drugs as a treatment for Alzheimer’s disease. Eric Siemers is an employee of, and holds equity in, Eli Lilly and Company. Harold Hampel, John Hardy, Pankaj D. Mehta and John Q. Trojanowski had no conflicts of interest to declare. 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