Characterization of the AD7C-NTP cDNA expression in Alzheimer's disease and measurement of a 41-kD protein in cerebrospinal fluid.

We have isolated a novel Alu sequence-containing cDNA, designated AD7c-NTP, that is expressed in neurons, and overexpressed in brains with Alzheimer's disease (AD). The 1,442-nucleotide AD7c-NTP cDNA encodes an approximately 41-kD protein. Expression of AD7c-NTP was confirmed by nucleic acid sequencing of reverse transcriptase PCR products isolated from brain. AD7c-NTP cDNA probes hybridized with 1. 4 kB mRNA transcripts by Northern blot analysis, and monoclonal antibodies generated with the recombinant protein were immunoreactive with approximately 41-45-kD and approximately 18-21-kD molecules by Western blot analysis. In situ hybridization and immunostaining studies localized AD7c-NTP gene expression in neurons. Using a quantitative enzyme-linked sandwich immunoassay (Ghanbari, K., I. Beheshti, and H. Ghanbari, manuscript submitted for publication) constructed with antibodies to the recombinant protein, AD7c-NTP levels were measured under code in 323 clinical and postmortem cerebrospinal fluid (CSF) samples from AD, age-matched control, Parkinson's disease, and neurological disease control patients. The molecular mass of the AD7c-NTP detected in CSF was approximately 41 kD. In postmortem CSF, the mean concentration of AD7c-NTP in cases of definite AD (9.2+/-8.2 ng/ml) was higher than in the aged control group (1.6+/-0.9; P < 0.0001). In CSF samples from individuals with early possible or probable AD, the mean concentration of AD7c-NTP (4.6+/-3.4) was also elevated relative to the levels in CSF from age-matched (1.2+/-0.7) and neurological disease (1.0+/-0.9) controls, and ambulatory patients with Parkinson's disease (1.8+/-1.1) (all P < 0.001). CSF levels of AD7c-NTP were correlated with Blessed dementia scale scores (r = 0. 66; P = 0.0001) rather than age (r = -0.06; P > 0.1). In vitro studies demonstrated that overexpression of AD7c-NTP in transfected neuronal cells promotes neuritic sprouting and cell death, the two principal neuroanatomical lesions correlated with dementia in AD. The results suggest that abnormal AD7c-NTP expression is associated with AD neurodegeneration, and during the early stages of disease, CSF levels correlate with the severity of dementia.

Probability distribution function-based classification of structural MRI for the detection of Alzheimer's disease.

High-dimensional classification methods have been a major target of machine learning for the automatic classification of patients who suffer from Alzheimer's disease (AD). One major issue of automatic classification is the feature-selection method from high-dimensional data. In this paper, a novel approach for statistical feature reduction and selection in high-dimensional magnetic resonance imaging (MRI) data based on the probability distribution function (PDF) is introduced. To develop an automatic computer-aided diagnosis (CAD) technique, this research explores the statistical patterns extracted from structural MRI (sMRI) data on four systematic levels. First, global and local differences of gray matter in patients with AD compared to healthy controls (HCs) using the voxel-based morphometric (VBM) technique with 3-Tesla 3D T1-weighted MRI are investigated. Second, feature extraction based on the voxel clusters detected by VBM on sMRI and voxel values as volume of interest (VOI) is used. Third, a novel statistical feature-selection process is employed, utilizing the PDF of the VOI to represent statistical patterns of the respective high-dimensional sMRI sample. Finally, the proposed feature-selection method for early detection of AD with support vector machine (SVM) classifiers compared to other standard feature selection methods, such as partial least squares (PLS) techniques, is assessed. The performance of the proposed technique is evaluated using 130 AD and 130 HC MRI data from the ADNI dataset with 10-fold cross validation(1). The results show that the PDF-based feature selection approach is a reliable technique that is highly competitive with respect to the state-of-the-art techniques in classifying AD from high-dimensional sMRI samples.

Single-strand conformational polymorphisms (SSCP): studies of the genetic polymorphisms of exon 4 of apolipoprotein C III.

OBJECTIVE: We used single-strand conformational polymorphism (SSCP). To screen for mutations/polymorphisms in exon 4 of the apolipoprotein C III in 45 patients with hypertriglyceridemia and 46 control individuals, single-strand conformational polymorphism was investigated using restriction endonuclease and amplification refractory mutations systems (ARMS). RESULTS: SSCP identified six patterns corresponding to six genotypes. We confirmed that the different genotypes result from the two polymorphic sites at positions 3175 and 3206 of the apo C III gene. Only three of four possible haplotypes were found in the study population. This resulted in the identification of 6 of the 10 possible genotypes. CONCLUSIONS: SSCP is a useful method to screen for both known and unknown mutations/polymorphisms and should have increasing applications in clinical laboratories involved with the study of genetic markers of a wide variety of diseases.

EDTA-plasma vs serum differences in cholesterol, high-density-lipoprotein cholesterol, and triglyceride as measured by several methods.

To investigate EDTA-plasma/serum (P/S) differences, we collected paired samples from 25 volunteers and measured total cholesterol (TC), triglyceride (TG) and high-density-lipoprotein cholesterol (HDLC), using the Cobas FARA, Ektachem 700, DuPont Dimension, and Baxter Paramax Analyzers. The mean (SD) P/S ratios for TC, HDLC, and TG concentrations were, respectively: 0.980 (0.0171), 1.063 (0.0704), and 0.961 (0.363) for Paramax; 0.976 (0.0189), 1.034 (0.1091), and 0.950 (0.557) for Dimension; 1.003 (0.0221), 1.059 (0.0304), and 0.988 (0.0179) for Ektachem; and 0.993 (0.0162), 1.063 (0.0830), and 1.013 (0.0410) for Cobas. We conclude that P/S ratios vary by analytical methods, and that HDLC ratios tend to be larger in magnitude and in the opposite direction from TC and TG. Both effects lead to significant biases in computed disease risk.

Biochemical assay for AD7C-NTP in urine as an Alzheimer's disease marker.

A reliable and specific immunoassay has been developed to detect and measure AD7C-NTP, a biochemical marker for Alzheimer's disease, in urine. The urine samples are first processed by centrifugation and ultrafiltration to fractionate and concentrate AD7C-NTP. The urinaryAD7C-NTP has the same molecular weight asAD7C-NTP in brain and cerebrospinal fluid by size exclusion chromatography. It has also retained the binding properties to the monoclonal and polyclonal antibodies developed against recombinantly produced AD7C-NTP. This assay is an enzyme linked sandwich immunoassay (ELSIA) using 96 well microtiter plates. The plate surface is coated with a monoclonal antibody (N314) which has a high affinity and specificity for AD7C-NTP, capturing it effectively from the samples. The detection was achieved using a polyclonal antibody (ADRI). The utility of the assay has been demonstrated using urine specimens from Alzheimer's disease (AD) patients and non-Alzheimer's controls. UrinaryAD7C-NTP in the AD group (2.5 ng/mL, n=66) was significantly higher than the non-AD group (0.8 ng/mL, n=134). Using 1.5 ng/mL as cut off, in this patient population, specificity and sensitivity of urinary AD7C-NTP were comparable to CSFAD7C-NTP.

Acridinium esters as high-specific-activity labels in immunoassay.

A chemiluminescent acridinium ester has been synthesized that reacts spontaneously with proteins to yield stable, immunoreactive derivatives of high specific activity. The compound has been used to prepare chemiluminescent monoclonal antibodies to human alpha 1-fetoprotein having average incorporation ratios as great as 2.8 mol of label per mole of antibody, which corresponds to a detection limit of approximately 8 X 10(-19) mol. These antibodies have been used in the preliminary development of a two-site immunochemiluminometric assay for human alpha 1-fetoprotein, which requires only a 30-min incubation and a quantification time of 5 s per sample.

Determination of a T/G polymorphism at nucleotide 3206 of the apolipoprotein C III gene by amplification refractory mutation system.

We used the amplification refractory mutation system (ARMS)--a polymerase-chain-reaction-based method--to determine the 3206 T-to-G polymorphism on exon 4 of the apolipoprotein (apo) C III gene. Apo C III is an inhibitor of the enzyme lipoprotein lipase (EC 3.1.1.34). Previous studies have demonstrated that a polymorphism at nucleotide 3175 on exon 4 of this gene is associated with hypertriglyceridemia. We studied 45 hypertriglyceridemic and 46 age-matched controls for the 3206 T-to-G polymorphism. The results showed a significant difference in the distribution of the genotypes with respect to this allele between the hypertriglyceridemic and control individuals. We also determined the presence of the SacI site at nucleotide 3175 in these same individuals and found no significant difference in SacI genotypes between the two groups. This study reaffirms the usefulness of ARMS as a simple, reliable method for detecting mutations and polymorphisms in clinical and epidemiological studies.