Toward Precision and Reproducibility of Diffusion Tensor Imaging: A Multicenter Diffusion Phantom and Traveling Volunteer Study.

BACKGROUND AND PURPOSE: Precision medicine is an approach to disease diagnosis, treatment, and prevention that relies on quantitative biomarkers that minimize the variability of individual patient measurements. The aim of this study was to assess the intersite variability after harmonization of a high-angular-resolution 3T diffusion tensor imaging protocol across 13 scanners at the 11 academic medical centers participating in the Transforming Research and Clinical Knowledge in Traumatic Brain Injury multisite study. MATERIALS AND METHODS: Diffusion MR imaging was acquired from a novel isotropic diffusion phantom developed at the National Institute of Standards and Technology and from the brain of a traveling volunteer on thirteen 3T MR imaging scanners representing 3 major vendors (GE Healthcare, Philips Healthcare, and Siemens). Means of the DTI parameters and their coefficients of variation across scanners were calculated for each DTI metric and white matter tract. RESULTS: For the National Institute of Standards and Technology diffusion phantom, the coefficients of variation of the apparent diffusion coefficient across the 13 scanners was <3.8% for a range of diffusivities from 0.4 to 1.1 × 10-6 mm2/s. For the volunteer, the coefficients of variations across scanners of the 4 primary DTI metrics, each averaged over the entire white matter skeleton, were all <5%. In individual white matter tracts, large central pathways showed good reproducibility with the coefficients of variation consistently below 5%. However, smaller tracts showed more variability, with the coefficients of variation of some DTI metrics reaching 10%. CONCLUSIONS: The results suggest the feasibility of standardizing DTI across 3T scanners from different MR imaging vendors in a large-scale neuroimaging research study.

A founder mutation in presenilin 1 causing early-onset Alzheimer disease in unrelated Caribbean Hispanic families.

CONTEXT: Genetic determinants of Alzheimer disease (AD) have not been comprehensively examined in Caribbean Hispanics, a population in the United States in whom the frequency of AD is higher compared with non-Hispanic whites. OBJECTIVE: To identify variant alleles in genes related to familial early-onset AD among Caribbean Hispanics. DESIGN AND SETTING: Family-based case series conducted in 1998-2001 at an AD research center in New York, NY, and clinics in the Dominican Republic. PATIENTS: Among 206 Caribbean Hispanic families with 2 or more living members with AD who were identified, 19 (9.2%) had at least 1 individual with onset of AD before the age of 55 years. MAIN OUTCOME MEASURE: The entire coding region of the presenilin 1 gene and exons 16 and 17 of the amyloid precursor protein gene were sequenced in probands from the 19 families and their living relatives. RESULTS: A G-to-C nucleotide change resulting in a glycine-alanine amino acid substitution at codon 206 (Gly206Ala) in exon 7 of presenilin 1 was observed in 23 individuals from 8 (42%) of the 19 families. A Caribbean Hispanic individual with the Gly206Ala mutation and early-onset familial disease was also found by sequencing the corresponding genes of 319 unrelated individuals in New York City. The Gly206Ala mutation was not found in public genetic databases but was reported in 5 individuals from 4 Hispanic families with AD referred for genetic testing. None of the members of these families were related to one another, yet all carriers of the Gly206Ala mutation tested shared a variant allele at 2 nearby microsatellite polymorphisms, indicating a common ancestor. No mutations were found in the amyloid precursor protein gene. CONCLUSIONS: The Gly206Ala mutation was found in 8 of 19 unrelated Caribbean Hispanic families with early-onset familial AD. This genetic change may be a prevalent cause of early-onset familial AD in the Caribbean Hispanic population.

Screening for PS1 mutations in a referral-based series of AD cases: 21 novel mutations.

BACKGROUND: Mutations in the presenilin-1 gene (PS1) account for a majority of patients with early-onset familial AD. However, the clinical indications and algorithms for genetic testing in dementia are still evolving. METHODS: The entire open reading frame of the PS1 gene was sequenced in a series of 414 consecutive patients referred for diagnostic testing, including 372 patients with AD and 42 asymptomatic persons with a strong family history of AD. RESULTS: Forty-eight independent patients screened had a PS1 mutation including 21 novel mutations. In addition, 3% of subjects (11/413) had a known polymorphism, the Glu318Gly substitution. The majority of the mutations were missense substitutions but there were three insertions and Delta exon 10 mutation. With six exceptions (codons 35, 178, 352, 354, 358, and 365) most of the mutations occurred at residues conserved in the homologous PS2 gene or in PS1 of other species. CONCLUSIONS: Eleven percent of a referral-based series of patients with AD can be explained by coding sequence mutations in the PS1 gene. The high frequency of PS1 mutations in this study indicates that screening for PS1 mutations in AD is likely to be successful, especially when directed at patients with a positive family history with onset before 60 years (90% of those with PS1 mutations were affected by age 60 years). This will also have significance for the secondary identification of at-risk relatives who might be candidates for future prophylactic therapies for AD.

Diagnostic approaches to Alzheimer's disease.

The importance of obtaining an accurate and early diagnosis for Alzheimer's disease is now becoming recognized. Nonpharmacological as well as pharmacological therapies can be best initiated once a diagnosis is obtained. Biochemical markers to identify Alzheimer's disease have been sought for many years, with many candidates proposed. Recently criteria were established to evaluate putative diagnostic tests. Several biomarkers now show utility in identifying those with Alzheimer's disease. The ApoE e4 allele, while a risk factor rather than a deterministic gene, in the context of an individual with suspicion of AD has a positive predictive value of 94-98% and may come to have utility in predicting response to certain classes of pharmacological agents. Independent groups have shown that the markers in cerebrospinal fluid tau and Ab42 are, respectively, elevated and reduced in patients with AD versus other patient groups and that the lumbar puncture itself is usually well tolerated. For early-onset AD, sequencing presenilin 1 has come into use and the positive frequency is similar to that found in other genetic-based laboratory tests.

High cerebrospinal fluid tau and low amyloid beta42 levels in the clinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype.

OBJECTIVE: To evaluate cerebrospinal fluid (CSF) levels of amyloid beta protein ending at amino acid 42 (Abeta42) and tau as markers for Alzheimer disease (AD) and to determine whether clinical variables influence these levels. DESIGN: Cohort study. SETTING: Six academic research centers with expertise in dementia. SUBJECTS: Eighty-two patients with probable AD, including 24 with very mild dementia (Mini-Mental State Examination score >23/30) (AD group); 60 cognitively normal elderly control subjects (NC group); and 74 subjects with neurological disorders, including dementia (ND group). MAIN OUTCOME MEASURES: Levels of Abeta42 and tau were compared among AD, NC, and ND groups. Relationships of age, sex, Mini-Mental State Examination score, and apolipoprotein E (Apo E) genotype with these levels were examined using multiple linear regression. Classification tree models were developed to optimize distinguishing AD from NC groups. RESULTS: Levels of Abeta42 were significantly lower, and levels of tau were significantly higher, in the AD group than in the NC or ND group. In the AD group, Abeta42 level was inversely associated with Apo E epsilon4 allele dose and weakly related to Mini-Mental State Examination score; tau level was associated with male sex and 1 Apo E epsilon4 allele. Classification tree analysis, comparing the AD and NC subjects, was 90% sensitive and 80% specific. With specificity set at greater than 90%, the tree was 77% sensitive for AD. This tree classified 26 of 74 members of the ND group as having AD. They had diagnoses difficult to distinguish from AD clinically and a high Apo E epsilon4 allele frequency. Markers in CSF were used to correctly classify 12 of 13 patients who later underwent autopsy, including 1 with AD not diagnosed clinically. CONCLUSIONS: Levels of CSF Abeta42 decrease and levels of CSF tau increase in AD. Apolipoprotein E epsilon4 had a dose-dependent relationship with CSF levels of Abeta42, but not tau. Other covariates influenced CSF markers minimally. Combined analysis of CSF Abeta42 and tau levels discriminated patients with AD, including patients with mild dementia, from the NC group, supporting use of these proteins to identify AD and to distinguish early AD from aging. In subjects in the ND group with an AD CSF profile, autopsy follow-up will be required to decide whether CSF results are false positive, or whether AD is a primary or concomitant cause of dementia.

A sensitive, nondestructive assay for transfected genes.

We have adapted the fibrin overlay assay for plasminogen activators (Jones et al., 1975) into a gene transfer expression assay which has the advantage of being very sensitive and nondestructive. In this assay plasminogen activators convert plasminogen to plasmin, which then degrades fibrin, resulting in clearings in a fibrin overlay. Furthermore, the assay can be used as a signal indicating the efficiency of gene transfer or the loss of introduced genetic elements in unstable cell lines.

The synthesis and in vivo assembly of functional antibodies in yeast.

The yeast Saccharomyces cerevisiae can synthesize, process and secrete higher eukaryotic proteins. We have investigated the expression of immunoglobulin chains in yeast and demonstrate here the synthesis, processing and secretion of light and heavy chains, the glycosylation of heavy chain, the intracellular localization of these foreign proteins by immunofluorescence, and the detection of functional antibodies in cells co-expressing both chains. This may provide the basis of a microbial fermentation process for the production of monoclonal antibodies. The co-expression of light and heavy chains in Escherichia coli has been reported but functional antibodies were not assembled in vivo. Furthermore, only low-level assembly of these chains was found in vitro.

Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells.

The variable regions of immunoglobulin heavy chains are encoded in the germ line by three discrete DNA segments: VH (variable) elements, D (diversity) elements and JH (joining) elements. During the differentiation of B lymphocytes, individual segments from each group are brought together by recombination to form the complete VHDJH variable region. To understand these processes better, we have now isolated and sequenced molecular clones representing intermediates (DJH fusions) and final products (VH-to-DJH joins) of heavy-chain gene rearrangement in two cell lines that represent analogues of cells at early stages of B-lymphocyte differentiation. Heavy-chain gene assembly in one cell line but not in the other is accompanied by the appearance of short nucleotide insertions at the recombinational junctions. The generation of such insertions is positively correlated with the expression of terminal deoxynucleotidyl transferase in these lines.

Assembly of functional antibodies from immunoglobulin heavy and light chains synthesised in E. coli.

Genes for a murine mu heavy chain and a lambda light chain immunoglobulin have been inserted into bacterial expression plasmids containing the Escherichia coli trp promoter and ribosome binding site. Induction of transcription from the trp promoter results in accumulation of both light and heavy chain polypeptides in appropriate host strains. Both proteins were found as insoluble products. Following extraction and purification of the immunoglobulin containing fractions, antigen binding activity was recovered. The activity demonstrates essentially the same properties as the antibody from the hybridoma from which the genes were cloned.

The influence of messenger RNA secondary structure on expression of an immunoglobulin heavy chain in Escherichia coli.

A gene for murine mu heavy chain immunoglobulin has been inserted into a bacterial expression plasmid containing the Escherichia coli trp promoter and ribosome binding site. A low level expression of mu protein was detected. Secondary structure analysis showed the presence of a hairpin loop burying the mu initiation codon. Alteration of secondary structure at this site by oligonucleotide replacement mutagenesis revealed a correlation between mu expression levels and accessibility of the ribosome binding site. Abolition of secondary structure increased mu protein expression over ninety-fold, to a level approximately equal to that of a trpE -mu fusion protein using the native trpE ribosome binding site.

Localization of the Abelson murine leukemia virus protein in a detergent-insoluble subcellular matrix: architecture of the protein.

We examined the interaction of Abelson murine leukemia virus protein P120 with other cellular components after extraction with the nonionic detergent Triton X-100. Most of the Abelson murine leukemia virus P120-associated kinase activity was found in the detergent-insoluble matrix in both lymphoid and fibroblast cell lines. The P120 labeled during a short exposure of cells to [35S]-methionine was mainly in the detergent-insoluble matrix (lymphoid cells) or equally distributed in the detergent-insoluble matrix and the soluble fraction (fibroblasts). Steady-state-labeled P120 was distributed equally in the two fractions (lymphoid cells) or mostly in the soluble portion (fibroblasts). Thus, there was an apparent movement of P120 from the detergent-insoluble matrix to the detergent-soluble fraction and a concomitant loss of enzymatic activity. When the detergent-insoluble matrix was incubated with [32P]ATP in situ, phosphorylation of tyrosine residues of P120 was observed. We found an 80,000-molecular-weight fragment of P120 (designated F80) after extraction of fibroblast cells with detergent. F80 was not found in extracted lymphoid cells, but mixing labeled lymphoid cells and unlabeled fibroblasts before extraction produced the fragment. F80 contained the gag determinants of P120 but did not react with Abelson-specific serum. These data allowed us to assign various features of the protein to regions of the P120 molecule and to localize the Abelson-specific antigenic determinants to the C-terminal region of the molecule.

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.

Partial maturation and light chain restriction of Abelson virus-transformed B cell precursors.

The induction of partial maturation in an in vitro derived Abelson virus-transformed murine lymphoid cell subline (ABC-1/AT1) is described. Pre-B (cytoplasmic, mu chain-positive) lymphocytes were induced from presumptive B cell precursors by prostaglandin E1, butyric acid, lipopolysaccharide and interferon. Maturation was independent of alterations in cellular growth rate and could be achieved in the absence of cell division. The AT1 subline was found to be restricted to the expression of a single light chain type (lambda) indicating a possible B cell lineage-committed precursor as the target for viral transformation.

Differentiation-linked expression of cell surface markers on HL-60 leukemic cells.

The cell surface antigenic phenotype of HL-60, a human acute promyelocytic leukemia cell line, has been analyzed before and after maturation induction with dimethylsulfoxide (DMSO) using a panel of markers including a "library" of monoclonal antibodies and "conventional" antisera in conjunction with the fluorescene-activated cell sorter. HL-60 cells express granulocyte and "leukocyte" differentiation antigens but not antigens of the lymphoid, platelet, and erythroid lineages. DMSO-induced morphological maturation was found to be associated with a decrease in the proportion of cells in mitotic cycle, induction of C3d receptors, increased expression of granulocytic and leukocyte antigens, and diminished expression of HLA-A,B,C and beta 2-microglobulin determinants. HL-60 cells have no detectable expression of HLA-DR-associated determinants as assayed by rabbit anti-p28,33 monoclonal anti-HLA-DR (monomorphic determinant), and HLA-DRw typing alloantisera. The relationship of these changes in cell surface properties to normal granulocytic differentiation is discussed.

Expression of binding sites for peanut agglutinin during murine B lymphocyte differentiation.

Haemopoietic cells from several murine organs were examined for the presence of peanut agglutinin (PNA) receptors. Foetal liver and adult bone marrow contained a number of cells, which were PNA+ but did not stain with conventional T and B markers. Some of the PNA+ cells, however, were pre-B cells, as shown by the presence of cytoplasmic IgM in the absence of cell surface Ig. Cells in the T lineage retained their PNA receptors during maturation, although these became masked by sialic acid on mature peripheral blood T cells. Cells of the B lineage, in contrast, gradually lost their PNA receptors as they matured from pre-B to mature surface immunoglobulin positive B lymphocytes.