Can a herpes simplex virus type 1 neuroinvasive score be correlated to other risk factors in Alzheimer's disease?

Herpes simplex virus type 1 (HSV-1) is latent in the nervous system of most humans. Ball [Can J Neurol Sci 9 (1982) 303] first suggested the hypothesis that HSV-1 could be involved in the pathogenesis of Alzheimer's Disease (AD) by noting that regions of the brain particularly and earliest affected in AD were the same as those most damaged during HSV encephalitis. Data from Itzhaki's research suggests that HSV-1 in the brain and the carriage of an apolipoprotein E allele 4 (ApoE e4) together confer risk for AD [J Pathol 97 (2002) 395], [Mol Chem Neuropathol 28 (1996) 135], [Alzheimer's Rep 1 (1998) 173], [Biochem Soc Trans 26 (1998) 273]. Of the two other studies based on Itzhaki's findings, one showed similar results [Lancet 349 (1997) 1102], and the other showed a similar trend [Lancet 351 (1998) 1330], [Lancet 352 (1998) 1312]. To further examine the role of HSV-1 in the etiology of AD, we have formulated a Neuroinvasive Score that quantifies the presence and viral load of HSV-1 in eight brain regions. These regions are: entorhinal cortex, hippocampus, pons, cerebellum, and neocortex (temporal, parietal, occipital, and frontal). We hypothesize that the Neuroinvasive Score that encompasses the presence, amount, and extent of HSV-1 spreading (neuroinvasiveness), will correlate with the genetic risk factor, ApoE e4, in the assessment of autopsy samples from AD patients. If the neuroinvasive score can be directly correlated to the different stages of AD (mild, moderate, severe), this will strengthen the hypothesis that HSV-1 is involved in AD and that ApoE e4 also confers risk for the development and progression of AD.

Expression of Factor IX cDNA Introduced into Human Marrow Stromal Cells by Electroporation.

Management of hemophilia B with gene therapy is an attractive and potentially feasible goal since stringent regulation of the recombinant protein is not required and low circulating levels may be sufficient to prevent symptoms. We are investigating the potential of gene transfer by electroporation for a role in human gene therapy. In this study, we used electroporation to physically co-transfer human factor IX cDNA under the influence of the potent human CMV-IE promoter and a second plasmid containing a neomycin resistance gene into human bone marrow stromal cells. Following electroporation, stromal cells were selected for neomycin resistance as co-transfection of both plasmids into the cells was expected from the results of previous studies. Analysis of genomic DNA from transfected stromal cells showed stable integration of factor IX cDNA at several sites in the genome. Following electroporation, the stromal cells were shown to secrete factor IX for three weeks in culture at a maximum concentration of 17ng/10(6) cells/day. As is the case with normal, functionally active, endogenous factor IX, the glutamic acid residues in the Gla domain of the factor IX protein were found to be post-translationally modified. Our results demonstrate the feasibility of gene transfer by electroporation and the successful post-translational modification and secretion of the human factor IX protein by stromal cells. This study provides evidence of the feasibility of electroporation and the use of stromal cells for the potential correction of hemophilia B in human gene therapy.

A Bayes decision test for detecting uncovered-background and moving pixels in image sequences.

We present a ternary hypothesis test for the detection of stationary, moving, and uncovered-background pixels between two image frames in a noisy image sequence using the Bayes decision criterion. Unlike many uncovered-background detection schemes, our scheme does not require motion estimation for the differentiation between moving pixels and uncovered-background pixels. We formulate the Bayes decision rule using a single intensity-difference measurement at each pixel and using multiple intensity-difference measurements in the neighborhood of each pixel. We quantitatively evaluate our detection algorithm on an image sequence which we have generated and qualitatively on the Trevor White image sequence.

Bead transfection of adherent cells. Gene transfer into adherent mammalian cells using glass beads.

Bead transfection is a simple, rapid, efficient, and cost-effective method of gene transfer into adherent mammalian cells. It involves a brief incubation of the cells with glass beads in a solution containing the DNA to be transferred. We have optimized this technique using COS-7 (an SV40 transformed monkey kidney cell line) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Stable transfection efficiency assessed using the selectable marker gene neomycin phosphotransferase (NEOR) was 27% in COS-7 cells. As this technique delivers high transfection efficiency with little manipulation of the exogenous DNA and does not require the use of any viral sequences, it may be a useful alternative method of gene delivery in the development of gene therapy protocols.

Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.

We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.

Activated protein kinase C directly phosphorylates the CD34 antigen in acute lymphoblastic leukemia cells.

The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.

Localization of the muscle, liver, and brain glycogen phosphorylase genes on linkage maps of mouse chromosomes 19, 12, and 2, respectively.

Mammalian glycogen phosphorylases comprise a family of three isozymes, muscle, liver, and brain, which are expressed selectively and to varying extents in a wide variety of cell types. To better understand the regulation of phosphorylase gene expression, we isolated partial cDNAs for all three isozymes from the rat and used these to map the corresponding genes in the mouse. Chromosome mapping was accomplished by comparing the segregation of phosphorylase restriction fragment length polymorphisms (RFLPs) with 16 reference loci in a multipoint interspecies backcross between Mus musculus domesticus and Mus spretus. The genes encoding muscle, liver, and brain phosphorylases (Pygm, Pygl, and Pygb) are assigned to mouse chromosomes 19, 12, and 2, respectively. Their location on separate chromosomes indicates that distinct cis-acting elements govern the differential expression of phosphorylase isozymes in various tissues. Our findings significantly extend the genetic maps of mouse chromosomes 2, 12, and 19 and can be used to define the location of phosphorylase genes in man more precisely. Finally, this analysis suggests that the previously mapped "muscle-deficient" mutation in mouse, mdf, is closely linked to the muscle phosphorylase gene. However, muscle phosphorylase gene structure and expression appear to be unaltered in mdf/mdf mice, indicating that this mutation is not an animal model for the human genetic disorder McArdle's disease.

Studies on the expression and evolution of the glycogen phosphorylase gene family in the rat.

Muscle, liver, and brain glycogen phosphorylases in mammals comprise a family of closely related isozymes that are differentially expressed in a wide variety of cell types. Towards obtaining a better understanding of the mechanisms governing the tissue-specific control of expression of this isozyme family, we used an antibody generated against bovine liver phosphorylase to obtain quantitative estimates of the concentrations of the three isozymes in rat tissues by Western blot analysis. This analysis indicated that expression of these isozymes at the protein level, although widespread, was tissue-specific and each isozyme exhibited variations in expression throughout the tissues where it was produced. We also began a preliminary analysis of the evolution of the genes encoding these three isozymes. Towards this end, we isolated and sequenced a partial cDNA to the rat brain isozyme that encompassed the coding region from amino acids 569 to 729. Using known phosphorylase gene sequences, we reconstructed a phylogeny spanning three kingdoms. This phylogeny indicated that brain and muscle isozymes are more closely related to each other than to the liver isozyme and that gene duplications that give rise to the family predate the mammalian radiation. Differences in the relative rates of change of the three isozymes were observed and this may reflect different constraints on their evolution perhaps related to their functional roles and (or) tissue-specific expression.