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ABSTRACT: Alzheimer's disease (AD) is a progressive dementing disorder characterized by age-related amyloid-beta (Abeta) deposition, neurofibrillary tangles, and synapse and neuronal loss. It is widely recognized that Abeta is a principal pathogenic mediator of AD. Our goal was to develop an immunotherapeutic approach, which would specifically lead to the clearance and/or neutralization of Abeta in the triple transgenic mouse model (3xTg-AD). These mice develop the amyloid and tangle pathologies and synaptic dysfunction reminiscent of human AD. Using a human single-chain variable fragment (scFv) antibody phage display library, a novel scFv antibody specific to Abeta was isolated, its activity characterized in vitro, and its open reading frame subsequently cloned into a recombinant adeno-associated virus (rAAV) vector. Three-month-old 3xTg-AD mice were intrahippocampally infused with serotype-1 rAAV vectors encoding Abeta-scFv or a control vector using convection-enhanced delivery (CED). Mice receiving rAAV1-Abeta-scFv harbored lower levels of insoluble Abeta and hyperphosphorylated tau, and exhibited improved cognitive function as measured by the Morris Water Maze (MWM) spatial memory task. These results underscore the potential of gene-based passive vaccination for AD, and provide further rationale for the development of Abeta-targeting strategies for this debilitating disease.
Molecular Therapy 08/2010; 18(8):1471-81. · 6.87 Impact Factor
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ABSTRACT: The Candida albicans adhesin, Als3p, was identified as a potential cognate antigen for previously described human antibody fragments [single-chain variable fragment (scFv)] based on similarity of the binding pattern of the scFv to the distribution of this protein on the hyphal surface. Although all scFv bound avidly to wild type, scFv3 showed no detectable binding via immunofluorescence assay to strain 1843, containing a homozygous deletion of ALS3. Binding to the ALS3 reintegrant strain, 2322, was preserved, and scFv3 also bound to Saccharomyces cerevisiae expressing ALS3. Other scFv retained binding to 1843, but with a markedly altered pattern. To determine if scFv3 could interfere with Als3p function, adhesion assays were conducted using human epithelial or endothelial cells as target. Treatment of wild-type C. albicans with scFv3 reduced adhesion of the fungus to both cell types to levels comparable to the als3Delta/als3Delta mutant. These experiments confirm that phage display is a viable method to isolate human scFv specific to an antigen implicated in C. albicans virulence, and that the scFv interfere with adhesion to human cells. The altered pattern of immunostaining with other scFv that retain binding to the als3Delta/als3Delta mutant suggest that Als3p may also have a role in structural organization of the C. albicans cell surface.
FEMS Immunology & Medical Microbiology 08/2008; 54(2):195-202. · 2.44 Impact Factor
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Paul Denny,
Fred K Hagen,
Markus Hardt,
Lujian Liao,
Weihong Yan,
Martha Arellanno,
Sara Bassilian,
Gurrinder S Bedi,
Pinmannee Boontheung,
Daniel Cociorva, [......],
Julian P Whitelegge,
H Ewa Witkowska,
Lawrence Wolinsky,
Yongming Xie,
Tao Xu,
Weixia Yu,
Jimmy Ytterberg,
David T Wong,
John R Yates,
Susan J Fisher
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ABSTRACT: Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.
Journal of Proteome Research 06/2008; 7(5):1994-2006. · 5.11 Impact Factor
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ABSTRACT: A unifying characteristic of prion diseases is the conversion of a normal cellular protein (PrP(c)) to an abnormal pathogenic conformation, designated PrP(sc). Antibodies directed against PrP(c), when added to scrapie-infected cell cultures or passively administered in vivo, can result in elimination of PrP(sc) or prevent its replication, respectively. In our efforts to develop an approach with potential prophylactic utility we employed a recombinant adeno-associated vector type 2 (rAAV2) viral vector platform to express PrP(c)-specific single-chain fragment variable (scFv) antibodies within the central nervous system (CNS) of susceptible mice that were subsequently inoculated peripherally with infectious prions. Vector expressed scFvs delayed onset of prion pathogenesis as evidenced by improvements in clinical signs and rotarod performance, in extended incubation periods, and in decreased PrP(sc) burden in the CNS. This novel antibody delivery platform enables the in vivo translation of prion prophylactics to other species afflicted by transmissible spongiform encephalopathies (TSEs) and which also has relevance to the development of therapeutics for other protein-misfolding diseases such as Alzheimer's or Parkinson's disease.
Molecular Therapy 04/2008; 16(3):481-6. · 6.87 Impact Factor
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ABSTRACT: Parkinson's disease (PD) is a common neurodegenerative disease of unknown etiology. Evidence suggests a role for protein misfolding in disease pathogenesis. One pathologic feature observed in dopaminergic neurons is the intracytoplasmic eosinophilic inclusions known as Lewy bodies. One component of Lewy bodies, the presynaptic protein, alpha-synuclein forms oligomers and higher order aggregates and is proposed to be involved in dopaminergic neuronal death. In an effort to discriminate between alpha-synuclein conformational forms as well as design potential disruptors of pathogenic misfolding we panned a human phage antibody library for anti-synuclein single chain antibodies (scFvs). We identified six scFvs which recognize different conformers of alpha-synuclein in both an ELISA and Western blot analysis. These scFvs may further our understanding of alpha-synuclein's role in PD.
Biochemical and Biophysical Research Communications 12/2006; 349(4):1198-205. · 2.48 Impact Factor
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ABSTRACT: RNA interference (RNAi) was used to characterize the requirement of protein glycosylation for cell membrane stability during cytokinesis in the early embryo. This screen targeted 13 enzymes or components of polypeptide sugar transferases that initiate either N-glycosylation or three different pathways of O-glycosylation. RNAi of genes in the mucin-type and epidermal growth factor-fringe glycosylation pathways did not affect cytokinesis. However, embryos deficient in N-glycosylation exhibited a variable inability to complete cytokinesis. The most potent block in early embryonic cell division was obtained by RNAi of the polypeptide xylose transferase (ppXyl-T), which is required to initiate the proteoglycan modification pathway. Two generations of ppXyl-T RNAi-feeding treatment reduced the body size, mobility, brood size, and life span of adult animals. Embryos escaping ppXyl-T and Gal-T2 RNAi lethality develop to adulthood but have cytokinesis-deficient offspring, suggesting that glycosyltransferases in the proteoglycan pathway are maternal proteins in the early embryo. Gal-T2::GFP fusions and anti-Gal-T2 antibodies revealed a perinuclear staining pattern, consistent with the localization of the Golgi apparatus. RNAi in green fluorescent protein (GFP)-tagged strains to follow tubulin, PIE-1, and chromatin showed that deficient proteoglycan biosynthesis uncouples the stability of newly formed cell membranes from cytokinesis, whereas cleavage furrow initiation, mitotic spindle function, karyokinesis, and partitioning of intrinsic components are intact.
Molecular Biology of the Cell 10/2005; 16(9):4202-13. · 4.94 Impact Factor
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ABSTRACT: Molecular Therapy (2005) 11, S373|[ndash]|S373; doi: 10.1016/j.ymthe.2005.07.509
966. AAV Delivered Single-Chain Antibodies as Passive Immuno-Therapy for Prion Disease
Charles A. Wuertzer1, Mark A. Sullivan2 and Howard J. Federoff11Center for Aging and Developmental Biology, University of Rochester, Rochester, NY2Center for Human Genetics and Molecular Pediatric Disease, University of Rochester, Rochester, NY
Molecular Therapy 04/2005; · 6.87 Impact Factor
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ABSTRACT: High throughput approaches to structural genomics requires expression, purification, and crystallization of proteins derived from predicted open reading frames cloned into a host organism, typically E. coli. Early results from this approach suggest that the success rate of obtaining well diffracting crystals from eukaryotic proteins is disappointingly low. A proven method of improving the odds of crystallization is formation of a complex with a conformation-stabilizing partner of known structure that is easily crystallized. Such complexes are also able to engage in different crystal contacts than the original protein by itself. Fab fragments derived from monoclonal antibodies have been successfully used for this purpose for a variety of proteins, however conventional methods for the isolation of monoclonal antibodies from hybridomas are time consuming and expensive. We are exploring the use of phage display to generate recombinant antibodies to target proteins that can be used to obtain co-complexes to facilitate crystallization and structural determination. We are using a large, human single-chain Fv (scFv) library to select for antibodies that bind to a panel of Leishmania major target proteins. Thirteen out of 16 target proteins yielded good binders after three rounds of enrichment. A total of 55 distinct scFvs were identified, with five targets each yielding at least five different scFvs. Individual clones were analyzed for binding specificity and soluble scFv can be readily produced and purified via the appended His(6) epitope tag. Using immunoaffinity chromatography, eight scFv target protein pairs were identified that exhibit stable complex formation and are suitable for co-crystallization trials.
Journal of Structural and Functional Genomics 02/2005; 6(2-3):171-5.
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ABSTRACT: Molecular Therapy (2004) 9, S208–S208; doi: 10.1016/j.ymthe.2004.06.471
552. Single-Chain Antibodies for Viral-Based Passive Immuno-Therapy for Prion Disease: Linear Epitope Mapping and Clearance from Thalamic Injection
Charles A. Wuertzer1, Mark A. Sullivan2 and Howard J. Federoff11Center For Aging and Developmental Biology, University of Rochester, Rochester, NY2Center For Human Genetics and Molecular Pediatric Disease, University of Rochester, Rochester, NY
Molecular Therapy 04/2004; · 6.87 Impact Factor
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ABSTRACT: To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.
Journal of Clinical Microbiology 04/2003; 41(3):1152-60. · 4.15 Impact Factor
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ABSTRACT: We developed a forage allocation model using a deterministic, linear optimization module in a commercially available spreadsheet package to help resource managers in Theodore Roosevelt National Park (TRNP), North Dakota determine optimum numbers of four ungulate species, bison (Bison bison), elk (Cervus elaphus), mule deer (Odocoileus hemionus), and feral horses, in the Park. TRNP staff actively managed bison, elk, and feral horse numbers within bounds suggested by our model from 1983 to 1996. During this period, we measured vegetation at 8 grassland and 12 wooded sites at 1-3 year intervals to determine if model solutions were appropriate for maintaining stable conditions in important plant communities in the Park. The data we recorded at these sites indicated minimal change in plant communities from 1983 to 1996. Changes in most vegetation categories that we expected when animal numbers exceeded model optimums for short periods (decreases in coverage/stem numbers of palatable plant species, increases in bare ground or unpalatable plant species) did not occur consistently under high or low precipitation conditions. The lack of sensitivity of our model to decreases in overall production of palatable plant species that occurred due to drought, fire, expansion of black-tailed prairie dog (Cynomys ludovicianus) colonies, and the spread of leafy spurge (Euphorbia esula) in areas of the Park where we did not have monitoring sites suggested that the model under-estimated the total number of ungulates that the Park could support. Management for population levels of ungulates defined by the model probably led to over protection of common plant communities and insufficient protection of rare plant communities. Detecting changes in rare plant communities could have been accomplished by re-designing our vegetation monitoring program, but changing emphasis to protection of rare plants would have likely promoted under use of grazing-tolerant habitat types, dissatisfaction in tourists visiting the Park to see large mammals, and large increases in cost and intrusiveness of management activities such as fencing and control of ungulate populations. The model was a flawed representation of grazing dynamics in TRNP, but we believe it succeeded in making management personnel aware of the biological constraints they face when making management decisions.
Journal of Environmental Management 03/2002; 64(2):153-69. · 3.24 Impact Factor
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ABSTRACT: A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were conserved among different strains of C. albicans and several other Candida species. Two scFv clones detected antigens specifically expressed by C. albicans blastoconidia; the third detected antigens in both blastoconidia and filamentous forms of C. albicans. The antigens containing the epitopes recognized by all three scFv could be extracted from blastoconidia by dithiothreitol, suggesting attachment to the cell wall via sulfhydryl bonds. Epitope detection by the scFv was sensitive to treatment of C. albicans blastoconidia with sodium periodate, but not proteinase K, indicating the cognate epitopes were composed of carbohydrate. Antigenic determinants for each of the three scFv were detected by immunohistochemical staining of skin sections from a model of cutaneous candidiasis, demonstrating expression in vivo. Through selection for the ability to bind intact organisms, the phage display system provides a means to rapidly identify monoclonal binding ligands to Candida surface antigens. Being entirely human, mature antibodies generated from the scFv have potential utility in the treatment of candidiasis.
Journal of Immunological Methods 12/2001; · 2.20 Impact Factor
