Immunochemical analyses of nucleic acids.
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Animals↗
Biomedical subjects
B D Stollar
Publications and source records attributed to B D Stollar.
Involvement of dipeptidyl peptidase IV in an in vivo immune response.
Dipeptidyl peptidase IV (DP IV) is a serine protease that selectively cleaves X-Pro dipeptides from polypeptides and proteins. Among blood cells, this enzyme occurs preferentially on the surface of CD4+ T cells and the amount of enzyme activity increases with T cell activation. In previous work, two potent and specific peptidyl-boronic acid inhibitors of DP IV, Ala-boroPro and Pro-boroPro, were synthesized and Pro-boroPro was shown to suppress antigen-specific proliferative responses of T cells in vitro. In this study, we tested the in vivo effects of these inhibitors. Subcutaneous injection of Ala-boroPro or Pro-boroPro into BALB/c mice inhibited DP IV activity in serum and spleen cell suspensions. Repeated injections of more than 10 micrograms of Ala-boroPro or Pro-boroPro at 12 h intervals maintained in vivo DP IV activity at less than 30% of the normal level. Repeated injections of the inhibitors during the primary, secondary or tertiary immune response to bovine serum albumin (BSA) reduced anti-BSA antibody production. Without inhibitor, immunization with BSA was followed by a temporary decrease in serum DP IV activity and then by enhanced serum enzyme activity after several days. These results provide the first direct evidence that DP IV plays an important role in immune response in vivo.
Animals↗
VH-gene representation in autoantibodies reflects the normal human B-cell repertoire.
The recurrence in the V-gene repertoire of individual germline VH genes can now be extended from the restricted B-cell populations of the fetus, autoantibodies and B-cell malignancies to the expressed V-gene repertoire of normal adults. Why the human B cell preferentially utilizes these individual VH genes remains speculative. However, it is apparent that the population of VH genes used to encode autoantibodies reflects the normal expressed repertoire (Fig. 7). Even so, the overrepresentation of other V genes such as Dxp'1 in anti-DNA antibodies and the presence of somatic mutation in the pathogenic autoantibodies of autoimmune disease continues to suggest an antigenic influence on V-gene selection. We postulate that only a fraction of available germline V genes are utilized in the expressed repertoire, and that polyspecificity of naturally occurring antibodies and somatic mutation of CDR3 compensate for the loss of diversity entailed by the limited use of the potential repertoire. The mechanisms by which germline genes become pathogenic remains unclear but they presumably relate to mutation, loss of regulatory control or perhaps environmental factors (Isenberg et al. 1992). What then are the mechanisms which lead to escape of these VH genes from normal control? What antigenic drive if any produces anti-DNA specificity in SLE? Why indeed is the expressed repertoire using only a fraction of the available germline? To answer these questions, further study of the V-gene repertoire of selected populations of antigen-binding cells and of pathogenic IgG autoantibodies is necessary and is ongoing. The contribution of individual V genes to antigen binding and idiotype is also being dissected and promises to yield important information about the relative contribution of VH genes to autoimmunity.
Adult↗
Immunoglobulin heavy chain gene expression in peripheral blood B lymphocytes.
cDNA libraries for IgM heavy chain variable regions were prepared from unmanipulated peripheral blood lymphocytes of two healthy people. Partial sequencing of 103 clones revealed VH gene family use and complete CDR3 and JH sequences. The libraries differed in the two subjects. In one person's cDNA the VH5 family was overexpressed and the VH3 family underexpressed relative to genomic complexity. In the second person's cDNA, VH3 was most frequently expressed. In both libraries, JH4 was most frequent. VH segments of several clones were closely related to those in fetal repertoires. However, there was also evidence of mutation in many cDNAs. Three clones differed from the single nonpolymorphic VH6 germline gene by 7-13 bases. Clones with several differences from VH5 germline gene VH251 were identified. CDR3 segments were highly diverse. JH portions of several CDR3's differed from germline JH sequences. 44% of the clones had DH genes related to the DLR and DXP families, most with differences from germline sequences. In 11 DLR2-related sequences, several base substitutions could not be accounted for by polymorphism. Thus, circulating IgM-producing B cell populations include selected clones, some of which are encoded by variable region gene segments that have mutated from the germline form.
Amino Acid Sequence↗
The molecular biology of anti-DNA antibodies and their idiotopes.
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Amino Acid Sequence↗
Construction of representative immunoglobulin variable region cDNA libraries from human peripheral blood lymphocytes without in vitro stimulation.
We have developed a sensitive and specific method for preparation of representative IgM and IgG cDNA libraries of peripheral blood lymphocytes without in vitro stimulation of B cells and without primer-based bias. The procedure involves preparation of double-stranded cDNA from initial C mu and C gamma constant region primers. Linkers are attached to the cDNA and nonselective polymerase chain reaction (PCR) amplification is performed with the linkers as primers. This product is ligated to M13 RF DNA, and selective PCR amplification is achieved with a nested constant region primer and a vector primer. The product is cloned into M13 phage. More than 85% of the white plaques are positive with a JH probe. Diversity of the library is confirmed by hybridization with VH gene probes and by DNA sequencing. A sample of 7-30 ml of blood from a normal human adult can yield both mu and gamma cDNA libraries. Analysis of the libraries yields a picture of Ig variable gene usage at the time of sampling.
Base Sequence↗
Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies.
The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.
Amino Acid Sequence↗
Autoantibodies and autoantigens: a conserved system that may shape a primary immunoglobulin gene pool.
Formation of certain autoantibodies is associated with a variety of autoimmune diseases, but the production of small amounts of autoantibodies also occurs in the normal immune system. Germline-encoded IgM antibodies that are autoreactive and bind to diverse antigen structures with low affinity are prominent in the primary antibody repertoire. Many IgG disease-related autoantibodies differ in structure and binding properties from these normally occurring IgM autoantibodies. The two sets may arise independently, but some properties, such as shared idiotypes, link some members of the two populations. Many autoantigen targets of both sets of autoantibodies are structurally conserved among species, as are certain features of the autoantibodies themselves. These elements, interacting before exposure of the system to foreign antigens, may constitute a conserved system that contributes to shaping and maintaining a primary immunoglobulin gene pool.
Autoantibodies↗
Ultraviolet cross-linking of helical oligonucleotides to two monoclonal MRL-1pr/1pr anti-DNA autoantibodies. Variations in H and L chain binding to DNA.
Experiments were performed to determine whether both H and L chains of different anti-native DNA autoantibodies are uniformly involved in binding to DNA. Two purified monoclonal mouse (MRL-1pr/1pr) IgG autoantibodies, H241 and 2C10, were tested. They both bound synthetic helical oligonucleotides of 10 to 20 base pairs in a gel electrophoresis retardation assay but differed in their preferences for given base sequences. Exposure of antibody-radiolabeled oligonucleotide mixtures to UV light (254 nm) for 10 min led to specific covalent cross-linking of oligonucleotide to both the H and the L chains of H241 but only to the H chain of 2C10. Single labeling events were detected without higher aggregation. The oligonucleotides were not cross-linked to unrelated IgG, even after 2 h of irradiation. Cross-linked (radioactively labeled) H and L chains of H241 and 2C10 were isolated from denaturing electrophoresis gels and digested with lysyl endopeptidase and/or staphylococcal V8 protease. H241 and 2C10 H chains each yielded a major labeled peptide fragment, but the peptides from the two antibodies were different. These experiments measured only some of the antibody-DNA interactions, probably with bases in the major groove of the DNA. They indicated that two MRL-1pr/1pr IgG anti-native DNA antibodies differ in their H and L chain contacts with DNA and provide an approach to identifying affinity-labeled binding sites in the antibodies.
Animals↗
Characterization of anti-Z-DNA antibody binding sites on Z-DNA by nuclear magnetic resonance spectroscopy.
Two monoclonal anti-Z-DNA antibodies, Z22 and Z44, were shown to bind to the oligonucleotides, d(CG)2 and d(CG)3, and to interact with different parts of the helix. 1H nuclear magnetic resonance spectroscopy showed that Fab fragments stabilize an ordered structure in the tetranucleotide d(CG)2. Nuclear Overhauser effects measured in the presence of Z22 Fab indicate a syn conformation of guanine residues of d(CG)2. Intermolecular transfer of saturation between the Fabs and bound d(CG)3 was detected by a saturation of the protein spectrum and observation of changes in the DNA spectrum. Antibodies with deuterated aromatic amino acids were prepared to eliminate the protein aromatic resonances and thereby allow a more detailed analysis of the transfer to the DNA base protons. The greatest transfer with Z44 was to the dC-5 protons although all of the base protons interact with this antibody. Little, if any, transfer to the DNA base protons was observed with Z22. These results are consistent with a Z44 binding site on the convex surface of the Z-helix (analogous to the major groove of B-DNA) and a Z22 binding site on the sugar-phosphate backbone.
Animals↗
The presence of an antigen reactive with a human autoantibody in Trichosia pubescens (Diptera: Sciaridae) and its association with certain transcriptionally active regions of the genome.
The antigens in HeLa and Trichosia pubescens cells, recognized by sera from patients with rheumatic diseases containing anti-Ku antibodies, were compared by means of immunoprecipitation of labeled cell extracts. The autoantibodies present in the tested sera precipitate at least two polypeptides of approximately Mr = 70,000 and Mr = 80,000 in HeLa cell extracts and a polypeptide of approximately Mr = 72,000 in Trichosia salivary gland cell extracts. The distribution of the insect antigen in chromatin was studied in salivary gland polytene chromosomes by indirect immunofluorescent staining with sera from two different patients. Both sera react with certain transcriptionally active chromosomal sites. The presence of the antigen in polytene chromosomes is strictly dependent on transcription, as no reaction is observed in the same sites before or after gene activation. Other sites, such as the nucleolar organizing region, are very active in transcription but never reacted with the anti-Ku positive sera. These results show that the insect antigen is associated with transcription-related processes of a subset of the chromosomal loci of T. pubescens. The anti-Ku positive sera react with a highly conserved antigen, which may serve a very important and similar role in the cellular metabolism of both insect and mammalian cells.
Animals↗
The biochemistry and genetics of DNA and anti-DNA antibodies.
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Amino Acid Sequence↗
VH genes of human autoantibodies.
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Amino Acid Sequence↗
The recurrent expression of variable region segments in human IgM anti-DNA autoantibodies.
RNA sequences for the V regions of human hybridoma-produced autoantibodies were determined by primer extension with reverse transcriptase. The sequencing of IgM autoantibodies from a leprosy patient revealed examples of recurrent use of V region gene segments in different autoantibodies from this patient and a previously studied patient with SLE. Moreover, several gene segments used in these autoantibodies show little alteration from germ-line sequences. mAb TH3, from a patient with leprosy, binds denatured DNA and poly(dT). The center of its H chain CDR35 has a sequence identical to that found previously in two anti-DNA antibodies from a lupus patient; these identities and their overlapping with two other published sequences define a human D-gene segment of approximately 25 nucleotides. Autoantibody TH9, from a leprosy patient, does not bind DNA. Its VH sequence has 87% identity with a VHI anti-DNA antibody, but differs from it markedly in the CDR1 region. TH9 also has a different H chain CDR3. The closely related JH4 or JH5 gene segments are expressed in five lupus or leprosy autoantibodies. In four of the antibodies, examples of V kappa 1, V kappa 3, or V kappa 4 and J kappa 2, or J kappa 5 segments were found. Two distinct leprosy-derived anti-DNA antibodies, 8E10 and TH3, share a completely identical V kappa sequence. This sequence differs in only two positions from that of a germ-line RF L chain gene. Several gene segments that are close to the germ line in sequence encode Ig V regions with autoantibody reactivity. These results provide a base line for determining whether these genes are precursors of more highly diversified antibodies that may be pathogenic in patients with SLE.
Amino Acid Sequence↗
Restriction fragment length polymorphism analysis of the V kappa locus in human lupus.
To evaluate the degree of genetic polymorphism of the V kappa repertoire in systemic lupus erythematosus (SLE), we performed Southern blot hybridizations with human gene probes corresponding to the four human V kappa gene families. In a comparative analysis, non-lymphoid cell DNA samples from three patients with idiopathic SLE, eight subjects with susceptibility to drug-induced lupus and seven control individuals were digested with the restriction endonucleases Bam HI, Bg 1 II, Eco RI and Hind III, and hybridized sequentially to the four V kappa family-specific probes. The restriction patterns on Southern blots revealed a low degree of polymorphism of the human V kappa gene repertoires of SLE patients and control individuals. This analysis, together with previous parallel studies of the V kappa locus in lupus-prone mice, implies that autoantibody hyperproduction in lupus is not associated with major modifications in the structure or genomic organization of immunoglobulin light chain genes.
Antipsychotic Agents↗
Cross-reactions of nucleic acids with monoclonal antibodies to phosphatidylinositol phosphate and cholesterol.
Four monoclonal IgM antibodies to phosphatidylinositol phosphate (PIP), four antibodies to cholesterol and one antibody to liposomes containing phosphatidylcholine, cholesterol and dicetyl phosphate were tested for reactivity with denatured DNA. Three of four antibodies to PIP cross-reacted strongly with denatured DNA. The other antibodies did react with denatured DNA but only very weakly. The binding to DNA was competed by synthetic polynucleotides. In competitive assays, one of the anti-PIP antibodies was particularly reactive with poly(dT) and another with poly(I) and poly(dG). Binding of an anti-cholesterol antibody to ssDNA was also inhibited by poly(I) and poly(dG). Two of the anti-PIP antibodies were also reactive with mononucleotides, and all four bound inositol hexaphosphate. High concns of nucleosides did not compete for binding, indicating that phosphate is involved in the binding site. Phospholipids, particularly those containing inositol phosphate, also competed for binding to DNA, but to varying extents, indicating a variable overlap in the antibody binding site for DNA and phospholipid determinants. These antibodies, induced by immunization with liposomes, showed cross-reactivity characteristics often found with certain types of autoantibodies, but they did not bear the H130 idiotype, which was identified on IgM anti-DNA autoantibodies from MRL-lpr/lpr mice.
Animals↗
Immunochemistry of DNA.
Since the first reports of anti-DNA antibodies in sera of patients with systemic lupus erythematosus (SLE) in 1957, studies of nucleic acid immunochemistry have grown in two directions. One has been the analysis of the specificity, the nature and the origins of these autoantibodies. The second has been exploration of anti-nucleic acid antibodies that can be induced experimentally, their specificities, and their application as biochemical reagents. Although the properties of autoantibodies and experimentally induced antibodies differ in certain respects, these two lines of research are complementary and provide important information for each other. For example, the production of autoantibodies by adjuvant-stimulated B cells yields a background that has to be considered in evaluating the specificity of weak responses to experimental nucleic acid immunogens: in turn, the possibilities and limitations of experimental immunization should be considered in evaluating possible stimuli for autoantibody production. Several aspects of nucleic acid immunochemistry have been described and evaluated in previous reviews. Following some general statements of historical perspective, this review will emphasize questions addressed and findings of about the last five years.
Antibodies, Antinuclear↗