B D Stollar

Ajou University, Seoul, Seoul, South Korea

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Publications (148)1181.27 Total impact

  • Y J Jang, B D Stollar
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    ABSTRACT: Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus.
    Cellular and Molecular Life Sciences CMLS 03/2003; 60(2):309-20. · 5.62 Impact Factor
  • B D Stollar, F Stephenson
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    ABSTRACT: Drs Berden and Koutouzov presented evidence that nucleosomes are antigens in lupus pathogenesis and that apoptotic cells are the source of nucleosomes. Berden's group measured persistence of circulating nucleosomes and nucleosome-antibody complexes in autoimmune mice and demonstrated nucleosomal deposition in skin of SLE patients as well as in renal lesions. Koutouzov reported that anti-nucleosomes are among the earliest autoantibodies in MRL+/+ mice, appearing several weeks before anti-DNA antibodies. Treatment of the mice with a pro-apoptotic drug, taxotere, accelerated autoantibody production and development of lesions. Herrmann proposed that persistent immunization results from reduced dead cell clearance and reduced production of immunosuppressive cytokines by defective scavenger macrophages. He also described accumulation of apoptotic cells in germinal follicles in SLE patients and attachment of nuclear antigens that are produced in apoptosis to the surface of follicular dendritic cells. Apoptosis-derived nucleosomes may be important in both the immunizing and effector arms of pathogenesis.
    Lupus 02/2002; 11(12):787-9. · 2.78 Impact Factor
  • Source
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    ABSTRACT: It has been proposed that autoreactivity of modest affinity contributes to positive selection of a preimmunization B cell repertoire, whereas high-affinity autoreactivity leads to negative selection. This hypothesis predicts that a B cell producing a physiologically selected unmutated ssDNA-binding Ab should be a precursor of cells that respond to diverse exogenous Ags. To test this prediction, we prepared transgenic mice bearing the rearranged V(H) domain of an IgM Ab from a nonautoimmune mouse immunized with a DNA-protein complex, poly(dC)-methylated BSA. The Ab, dC1, binds both poly(dC) and ssDNA. It is encoded by V(H) and V(L) gene segments with no mutations, suggesting that the producing cell may have been selected before and activated during immunization. The dC1V(H) transgene was targeted to the IgH locus. In heterozygous mice, on a nonautoimmune C57BL/6 background, the transgene allotype was expressed on B cell surfaces and in serum Ig, but about one-third of B cells expressed the endogenous allele instead. Total serum Ig concentrations were normal and included both transgene- and endogenous gene-coded IgM and IgG. The transgene V(H) D(H)J(H) was expressed in splenic IgM cDNA with few or no mutations, and in IgG cDNA with multiple mutations. The transgene allotype was also expressed in Abs formed on immunization with thyroglobulin, pneumococcal polysaccharide, and ssDNA-methylated BSA. Consistent with the hypothesis, cells with a rearranged autoreactive V(H) domain selected for reactivity with a form of ssDNA did serve as precursors for cells producing IgM and IgG Abs to diverse Ags.
    The Journal of Immunology 11/2001; 167(7):3746-55. · 5.52 Impact Factor
  • K C O'Connor, K Nguyen, B D Stollar
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    ABSTRACT: Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.
    Journal of Molecular Recognition 01/2001; 14(1):18-28. · 3.01 Impact Factor
  • X Wang, B D Stollar
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    ABSTRACT: This protocol describes application of single cell reverse transcription polymerase chain reaction (RT-PCR) to the study of human immunoglobulin V region usage. The procedure begins with separation of peripheral blood mononuclear cells (PBMC) from human blood. The PBMC are stained with the B cell selective marker, anti-CD19. Stained B cells are sorted by flow cytometry and deposited, consecutively, one cell into each of an array of tubes. cDNA for one or more antibody variable regions (VH and/or VL) is synthesized with a primer (or primers) complementary to sequence(s) within the constant region (Cmu, Cgamma, Ckappa and/or Clambda). The cDNA is used as template for PCR amplification with gene or gene family specific primers. A second PCR is then performed with two nested primers to increase both the specificity and quantity of V region PCR products. The purified PCR products are sequenced directly and aligned to V region germline database and the Genbank database. Single cell RT-PCR is a fast and convenient way to analyze V region gene expression. It avoids the bias that may be introduced into V region cDNA library construction by the presence of highly variable levels of mRNA in different cells. The PCR products are obtained in quantities that can be cloned into bacterial expression vectors for production of recombinant V region protein domains.
    Journal of Immunological Methods 11/2000; 244(1-2):217-25. · 2.23 Impact Factor
  • E Breitbart, B D Stollar
    The Israel Medical Association journal: IMAJ 10/2000; 2(9):703-7. · 0.98 Impact Factor
  • K C O'Connor, S Ghatak, B D Stollar
    Analytical Biochemistry 03/2000; 278(2):239-41. · 2.58 Impact Factor
  • B D Stollar
    Clinical Reviews in Allergy & Immunology 03/2000; 18(1):41-50. · 5.59 Impact Factor
  • Xiaowei Wang, B.David Stollar
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    ABSTRACT: Immune responses change in aging humans, but it is not known whether there is an age-associated change in the expressed B cell repertoire. We compared Ig VH cDNA libraries from circulating B cells of five elderly and three young human adults. As in young persons, nearly two-thirds of the cDNA clones from older subjects had zero to three V(H) mutations, although there was more individual variation among the elderly. V(H)4 family expression increased in older subjects, both in unmutated and in mutated cDNA clones, whereas V(H)3 family expression predominated in young adults. To test for bias toward activated cells in the cDNA libraries, we studied two older persons by both cDNA library analysis and single-cell RT-PCR. In one subject, more than 85% of VH segments were unmutated by either analysis. In the second, mutated Ig segments were much more frequent in cDNA clones than in consecutive single cells; however, V(H) family usage and high representation of particular genes were similar in both analyses. While aging humans continue to produce naive B cells with unmutated Ig genes, a shift to greater use of the V(H)4 family members and expression of particular genes may reflect changes in selection of developing B cells before affinity maturation toward reactivity with foreign antigen.
    Clinical Immunology 12/1999; 93(2):132-42. · 3.77 Impact Factor
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    ABSTRACT: We have used single and multiple site-directed mutagenesis, and molecular modeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous replacement of four Arg residues in the CDR3H abolished binding activity. With one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exceptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and Asn35 to A1a in CDRIH decreased DNA binding, whereas replacement of Arg31 with A1a had negligible effect. Ala substitution of any one of a cluster of Asp residues in CDR1L increased DNA binding three to six-fold, confirming previous findings that the L-chain of MAb 2C10 is not favorable for DNA binding. The L-chain does participate in shaping the selectivity of antigen binding, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specificity of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10.
    Molecular Immunology 01/1999; 35(18):1207-17. · 2.65 Impact Factor
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    ABSTRACT: Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed VH3-23 gene segment with varied DH and JH segments. The other clones contained unmutated VH3-7, VH3-9, VH3-53, and VH4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a VH3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.
    The Journal of Immunology 09/1998; 161(3):1274-83. · 5.52 Impact Factor
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    ABSTRACT: The properties of duplex CTG.CAG and CGG.CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 x 10(-19) erg.cm for CTG and 1.27 x 10(-19) erg.cm for CGG, approximately 40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were approximately 60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 x 10(-19) erg.cm and 10.4 base pairs (bp)/turn for CTG and 2.4 x 10(-19) erg.cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224-245 bp in length (64-71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.
    Journal of Biological Chemistry 08/1997; 272(27):16783-92. · 4.65 Impact Factor
  • B D Stollar, J M Lecerf, Y Hirabayashi
    Annals of the New York Academy of Sciences 05/1997; 815:30-9. · 4.38 Impact Factor
  • B D Stollar
    Lupus 02/1997; 6(3):327-8. · 2.78 Impact Factor
  • B D Stollar
    Lupus 02/1997; 6(3):346-8. · 2.78 Impact Factor
  • B D Stollar
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    ABSTRACT: Bacterial production of recombinant Fab or Fv domains of antibodies is an important tool for analyzing structural correlates of antigen binding or idiotype expression. Bacterial products may be Fab or Fv molecules that assemble from separate chains in the periplasm or a single-chain Fv protein. This article describes properties and applications of a plasmid vector used for production of single-chain Fv (scFv). The expression cassette, initially designed for production of the Fv domain of anti-Z-DNA mAb Z22, has a bacterial secretion signal that permits secretion of soluble scFv into growth medium. A single B domain of staphylococcal protein A facilitates affinity purification and generic assay of products independent of antigen-binding activity. Gene segment swapping and directed mutagenesis identified structural features important for Z-DNA binding and revealed the structural similarity of autoantibody and immunization-induced antibody. The H and L regions of mAb Z22 are readily replaced by those of any cloned Ig, a library of V regions, or other proteins. Modified forms of the vector code for production of separate H or L chain V regions, which can associate with each other to form functional Fv complexes. Although there is large variation in the yield with different V regions, most clones provide enough soluble product for antigen-binding assays. Current developments are aimed at increasing the yield consistency to allow production of enough material for three-dimensional structural analysis.
    Methods 02/1997; 11(1):12-9. · 3.64 Impact Factor
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    ABSTRACT: To investigate the autoantibody repertoire associated with SLE, we have created phage display IgG Fab libraries from two clinically active SLE patients and from the healthy identical twin of one of these patients. The libraries from the lupus discordant twins were found to both include unusually large representations of the V(H)5 gene family. By panning with DNA, the SLE libraries each yielded IgG anti-double-stranded (ds) DNA autoantibodies, which are characteristic of lupus disease. These included a V(H)5 autoantibody from the affected twin, that has a targeted cluster of mutations that potentially improves binding affinity. The recovered IgG anti-dsDNA autoantibodies expressed the same idiotypes associated with the in vivo IgG anti-dsDNA response of the respective SLE donor. Heavy-light chain shuffling experiments demonstrated a case in which the in vitro creation of anti-dsDNA binding activity required restrictive pairing of a heavy chain with Vlambda light chains similar to those in circulating anti-dsDNA autoantibodies. By contrast, IgG anti-ds autoantibodies could not be recovered from the library from the healthy twin, or from shuffled libraries with heavy chains from the healthy twin. These repertoire analyses illustrate how inheritance and somatic processes interplay to produce lupus-associated IgG autoantibodies.
    Journal of Clinical Investigation 01/1997; 98(12):2827-37. · 12.81 Impact Factor
  • Y J Jang, J M Lecerf, B. David Stollar
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    ABSTRACT: Antibodies H241 and 2C10 are lupus mouse IgG autoantibodies that bind native DNA. In previous experiments, oligonucleotide antigens affinity-labeled both H and L chains of H241 but only the H chain of antibody 2C10. Primary structures of the V regions of the 2C10 H and L chains and the H241 L chain, determined from cDNA, help to explain the previous affinity-labeling experiments. The 2C10 L chain CDRs had several Asp residues and a net negative charge of five, whereas the 2C10 H chain CDRs had four Arg residues and a net positive charge of five. The L chain CDRs of H241 had a net positive charge of one. [The H241 H chain cDNA sequence was published previously by Gangemi et al. (1993) J. Immun. 151, 4660-4671]. Plasmid vectors were used for bacterial expression of H and L chains of 2C10 alone and in combinations in single chain Fv (scFv) molecules. The H chain alone bound native DNA as well as or better than the H-plus-L chain scFv. The H chain alone also bound Z-DNA. Combination of the 2C10 H chain with the L chain of an anti-Z-DNA antibody maintained the selectivity for Z-DNA, whereas its combination with the 2C10 L chain (in the 2C10 Fab) yielded selective B-DNA binding. The results with 2C10 match other examples in which the H chain is sufficient for DNA binding but selectivity is modulated by the L chain. The H chain binding to autoantigen may reflect selective events in early stages of B cell development.
    Molecular Immunology 03/1996; 33(2):197-210. · 2.65 Impact Factor
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    ABSTRACT: The relationships between the antigen-binding specificities of four human monoclonal anti-DNA antibodies and the structural aspects of the combining sites of two of these were examined. Competition ELISAs were used to examine the reactivities of two IgM MAbs (WRI-176 and RT-79) and two IgG mAbs (D5 and B3) to a wide range of polynucleotides. The mAbs WRI-176 and RT-79 were found to bind predominantly ssDNA, with a preference for poly (dT), whilst D5 and B3 bound components of both ss- and dsDNA, and Z-DNA. The mAb B3 also exhibited a preference for A(T) rich nucleotides. Computer models were generated for the Fv regions of WRI-176 and B3. Models for RT-79 and D5 were not generated as the structure of the long CDR-H3 loops in these mAbs could not be predicted. The B3 combining site contains a groove flanked by three arginines at positions CDR-L1-27A, CDR-L2-54 and CDR-H2-53. Using interactive molecular graphics, B-DNA was docked into the B3 antigen combining site along the plane of the VH/VL interface, whilst Z-DNA was best-fitted at approximately 90 degrees to this direction. The models provide a hypothesis to explain the ability of a single autoantibody to bind two different antigens. In addition, aspects of the base specificity of B3 may be explained. The model of the WRI-176 Fv region revealed a relatively flat surface, on which a large number of hydrophobic and aromatic residues were present. Trp-H52, in particular, is prominent on the surface. This may participate in ssDNA binding through base stacking interactions. The models allow identification of potential targets for site-directed mutagenesis.
    Molecular Immunology 01/1996; 33(4-5):471-83. · 2.65 Impact Factor
  • B D Stollar
    Annals of the New York Academy of Sciences 10/1995; 764:265-74. · 4.38 Impact Factor

Publication Stats

5k Citations
1,181.27 Total Impact Points

Institutions

  • 1999–2003
    • Ajou University
      • Institute for Medical Sciences
      Seoul, Seoul, South Korea
  • 1975–2002
    • Tufts University
      • • Department of Biochemistry
      • • Department of Medicine
      • • Division of Hematology/Oncology
      Medford, MA, United States
  • 1994
    • Tokyo Medical and Dental University
      • Department of Health Science Policies
      Edo, Tōkyō, Japan
  • 1979–1993
    • Boston Medical Center
      Boston, Massachusetts, United States
    • Harvard Medical School
      • Division of Immunology
      Boston, MA, United States
  • 1992
    • University of Massachusetts Medical School
      • Department of Medicine
      Worcester, MA, United States
  • 1984–1990
    • New England Baptist Hospital
      Boston, Massachusetts, United States
  • 1987
    • Ben-Gurion University of the Negev
      Be'er Sheva`, Southern District, Israel
  • 1983
    • Harvard University
      Cambridge, Massachusetts, United States
  • 1982
    • University of Massachusetts Boston
      Boston, Massachusetts, United States