SLE血清中抗-DNA抗体分离和性质的研究

本文用国产高分子树脂(T)接枝小牛胸腺DNA,通过亲合层析从系统性红斑狼疮SLE患者血清中纯化出抗-ds DNA抗体和抗-ss DNA抗体。酶联免疫吸附分析(ELISA)的研究表明:SLE抗-DNA抗体和DNA结合的差异性很大,是高度非均一性的。抗-ss DNA抗体不仅组成成分比抗-ds DNA抗体复杂,ss DNA/抗-ssDNA亲合能力也明显高于ds DNA/抗-ds DNA。纯化的抗-DNA抗体以IgG类抗体占主导,同时也有其它类型抗体存在(例如IgM等)。抗-ds DNA抗体有较抗-ss DNA抗体高的IgG含量(两者的IgG/IgM分别是7.0和4.0),说明IgG抗-DNA抗体更倾向于同dsDNA结合。     

Detection of human autoantibodies specific for 5'-m7GMP and m7G(5')ppp(5')N

An enzyme-linked immunosorbent assay was utilized for the detection of spontaneously occurring antibodies with apparent specificities for m7G, 5'-m7GMP, and m7G(5')ppp(5')C. From the sera of 50 patients containing anti-nuclear antibodies, 48 (96%) possessed antibodies which bound to one or more immobilized nucleoside-BSA antigens (A-, G-, C-, U-, and T-BSA). Additionally, 8 (16%) of these sera contained immunoglobulins that reacted with m7G-BSA antigen. In these latter sera, soluble competitors such as m7G, 5'm7GMP, and m7G(5')ppp(5')C (but not 5'-AMP, -GMP, -CMP, -UMP, and -TMP or m1G and m22G) effectively inhibited antibody-binding to immobilized m7G-BSA. These results indicate the existence of spontaneously occurring anti-m7G antibodies in autoimmune diseases which are distinct from anti-G antibody populations.     

Distribution and specificity of nucleotide-reactive autoantibodies in human SLE

An enzyme-linked immunosorbent assay (ELISA) was utilized to characterize nucleotide-reactive antibodies present in the sera of 67 human subjects: 27 active SLE, 20 inactive SLE, and 20 asymptomatic controls. This assay consisted of measuring the quantity of antibodies retained by a panel of immobilized 5'-nucleotide-BSA conjugates (AMP-, GMP-, CMP-, UMP-, and TMP-BSA) together with ssDNA and dsDNA antigens. Although the relative distribution of antibodies binding to nucleotide-BSA antigens (i.e., anti-GMP greater than anti-AMP greater than or equal to anti-TMP greater than anti-UMP greater than or equal to anti-CMP antibodies) was independent of clinical status, the sera of active SLE patients possessed three- and five-fold higher concentrations of these antibodies relative to those present in inactive SLE and control subjects, respectively. Affinity purification of the most dominant of these antibody populations with DNA- and GMP-agarose adsorbents suggested that the majority of anti-GMP antibodies were monospecific with respect to the guanine base moiety. For example, antibodies retained by GMP-agarose reacted with GMP-BSA and ssDNA but not with other nucleotide-BSA or dsDNA antigens. However, ELISA competition-inhibition studies with affinity-purified anti-GMP antibodies indicated that although the guanine base represents an important determinant, guanine-enriched oligo- and polynucleotides were preferred substrates (i.e., guanine-dependent, oligonucleotide specificity). This was exemplified by the finding that a 500- and 50-fold molar excess of dGMP and d(G)4 were required to achieve the same degree of inhibition as that observed with d(G)8. Finally, and as evaluated by indirect immunofluorescence with fixed HEp-2 cells, affinity-purified anti-GMP antibodies reacted with antigens restricted to nucleolar organelles.     

Antibody-nucleic acid complexes. Oligo(dG)n and -(dT)n specificities associated with anti-DNA antibodies from autoimmune MRL mice

The specificity of anti-DNA antibodies in the sera of unimmunized autoimmune MRL mice was initially assessed via an enzyme-linked immunosorbent assay (ELISA). Antibody binding profiles to a panel of immobilized antigens (AMP-, GMP-, CMP-, UMP-, and TMP-BSA, ss- and dsDNA) demonstrated high levels of immunoglobulins reacting with GMP and ssDNA and intermediate levels with AMP, TMP, and dsDNA. Fractionation of serum anti-DNA antibodies into subsets on the basis of their binding to GMP- and TMP-agarose indicated that the resulting GMP- or TMP-reactive antibodies bound to their homologous nucleotides and ssDNA. Competition-inhibition studies with soluble mono-, oligo-, and polynucleotides revealed that GMP- and TMP-reactive antibodies were highly specific for oligo(dG)n and -(dT)n sequences, respectively. Whereas the relative affinity of TMP-reactive autoantibodies to oligo(dT)n increased with oligonucleotide length (n = 2, 4, 6, 8, 10, 15), GMP-reactive antibodies preferentially recognized oligo(dG)10 (Ka congruent to 1 x 10(7) M-1). While neither antibody recognized oligo(dA)8 and -(dC)8 competitors, mixed-base oligonucleotides were inhibitory at concentrations approximately 10-fold greater than similarly sized oligo(dG)n and -(dT)n sequences. Similar characterizations of both pooled and individual MRL sera indicated that anti-DNA antibodies represent 8-10% of the total serum IgG. More importantly, GMP-reactive autoantibodies predominated and accounted for 60-70% of the entire unbound anti-DNA antibody population.     

Dual Specificity and the Formation of Stable Autoimmune Complexes

Since dual specificity at the antibody active-site level involves new principles relative to monospecific antigen-antibody interactions and may be a general property of autoantibodies, it was important to further characterize such antibodies. Four lupus derived autoantibodies were studied to understand parameters and mechanisms involved in the participation of dual-specific antibody molecules in the formation of highly stable immune complexes. Because the dual-specific binding properties of selected lupus-related murine autoantibodies had been previously described using a solid-phase polystyrene-based ELISA, a conformational sensitive membrane based assay (CSI) was used on a comparative basis to further characterize NZB/NZW F₁ murine monoclonal anti-DNA autoantibodies BV 04–01 (anti-ssDNA), BV 16–19 (anti-ssDNA), BV 17–45 (anti-dsDNA), and BV 16–13 (anti-dsDNA). All four monoclonal autoantibodies exhibited anti-IgG binding in the solid-phase ELISA. However in the CSI assay, only anti-dsDNA monoclonal autoantibodies BV 17-45 and BV 16-13 demonstrated anti-IgG binding, while anti-ssDNA autoantibodies BV 04–01 and BV 16–19 did not. Upon subjection to time-dependent thermal denaturation, with and without thiol reduction at 100°C in the CSI, the self-binding activities of BV 17–45 and BV 16–13 were abrogated demonstrating that the recognized IgG autoepitope(s) possessed conformational or discontinuous three-dimensional properties. The immunological implications of dual specificity are discussed on a structure–function basis and its correlation with formation of pathogenic immune complexes. © 1997 John Wiley & Sons, Ltd.     

Model for helicase translocating along single-stranded DNA and unwinding double-stranded DNA

A model is proposed for non-hexameric helicases translocating along single-stranded (ss) DNA and unwinding double-stranded (ds) DNA. The translocation of a monomeric helicase along ssDNA in weakly-ssDNA-bound state is driven by the Stokes force that is resulted from the conformational change following the transition of the nucleotide state. The unwinding of dsDNA is resulted mainly from the bending of ssDNA induced by the strong binding force of helicase with dsDNA. The interaction force between ssDNA and helicases in weakly-ssDNA-bound state determines whether monomeric helicases such as PcrA can unwind dsDNA or dimeric helicases such as Rep are required to unwind dsDNA.     

Monoclonal anti-deoxyribonucleic antibodies. I. Isotype and specificity studies

Ten monoclonal anti-DNA antibodies generated in three separate fusion experiments performed using nonimmunized B/W spleen cells were studied. Their antigenic specificities were demonstrated to be identical and directed against a conformational determinant of the B helical form of double-stranded DNA (dsDNA). These data suggest that autoantibodies to dsDNA in B/W mice could constitute a homogeneous population.     

The in vitro production of anti-DNA antibody by cultured peripheral blood or tonsillar lymphoid cells from normal donors and SLE patients

Anti-DNA antibody responses by cultured circulating lymphocytes from SLE patients and by the tonsillar lymphoid cells of normal donors were detected and enumerated by a sensitive specific ELISA of culture supernatants, or by a hemolytic anti-DNA PFC assay. Although spontaneous IgM and IgG anti-DNA and anti-ssDNA responses were characteristic of SLE lymphocytes and spontaneous IgM anti-ssDNA responses were characteristic of tonsillar lymphocytes, the circulating lymphocytes of normal controls never produced anti-DNA antibodies spontaneously, and rarely after PWM stimulation. The anti-DNA antibody PFC response of tonsil lymphocytes correlated directly with the total number of immunoglobulin-producing cells measured by a reverse hemolytic PFC assay. Mixing experiments in which we employed cultures of comparable numbers of separately enriched autologous circulating and tonsillar B and T cells revealed that tonsillar tissue contained an enriched population of anti-DNA antibody precursor B cells and/or helper T cells.     

Clonotypes of anti-DNA antibodies expressing specific idiotypes in immune complexes of patients with active lupus nephritis

IEF, using 6 M urea, provides a unique opportunity to analyze the spectrotypes of antibodies in immune complexes (IC) in vivo. Using this technique, we have analyzed the clonotypes of anti-DNA antibodies expressing specific Id in the circulating IC of patients with active lupus nephritis. Serum anti-ssDNA and anti-dsDNA antibodies showed heterogeneous spectrotypes. The antibodies isolated from circulating IC had a restricted clonotype and a neutral charge and were directed mainly to ssDNA and, to a lesser extent, to dsDNA. These samples failed to form complexes with DNA when they were subjected to absorption to a DNA-coupled Sepharose column. Anti-DNA antibodies expressed specific Id, termed O-81 or NE-1, which were detected only in the IC of patients with active lupus nephritis. Anti-DNA clonotypes, including O-81 and NE-1 idiotypes, were also found in the eluates of renal glomeruli of lupus patients. These results indicate that subpopulations of anti-DNA antibodies in circulating IC are limited, and may play an important role in the pathogenesis of lupus nephritis.     

Human and murine anti-DNA antibodies induce the production of anti-idiotypic antibodies with autoantigen-binding properties (epibodies) through immune-network interactions

To examine the potential role of immune-network interactions in the production of lupus autoantibodies, normal NZW rabbit antibody responses were analyzed after immunization with one of the following Ig preparations: human lupus serum anti-dsDNA antibodies, human lupus serum anti-ssDNA antibodies, a mixture of human lupus serum anti-dsDNA and anti-ssDNA antibodies, the MRL-lpr/lpr anti-dsDNA mAb H241, and the MRL-lpr/lpr anti-ssDNA mAb H130. Four of five rabbits produced Ig typical of lupus autoantibodies: individual rabbit Ig cross-reacted with multiple autoantigens including nucleic acids, cardiolipin, SmRNP, glomerular extract, laminin, and exogenous Ag. Rabbit anti-Id against human anti-dsDNA antibodies were highly specific for dsDNA. Notably, in each serum the autoantibody activity was confined to the anti-Id Ig fraction. A similar spontaneously occurring Id-anti-Id interaction was also found between anti-ssDNA and anti-dsDNA antibodies isolated from an individual lupus patient. These results indicate that lupus autoantibodies which share Ag binding properties with pathogenic Ig, including both cross-reactive and anti-dsDNA antibodies, can induce the production of Ig with similar autoantigen binding properties through immune-network interactions. This phenomenon, if unregulated, could lead to the amplification of pathogenic autoantibody production in individuals with systemic lupus.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

In vitro manipulation of human anti-DNA antibody production by anti-idiotypic antibodies conjugated with neocarzinostatin

Anti-DNA Id, 0-81, consist of 5 to 51% of Id in human anti-ssDNA antibodies; NE-1-Id shares 2 to 20% of those in anti-dsDNA antibodies. Thus, both 0-81-Id and NE-1-Id are of the cross-reactive Id that are commonly present among anti-DNA antibodies. In order to manipulate the production of anti-DNA antibodies by human PBL, we used mouse antiidiotypic mAb or those conjugated with a cytotoxic agent, neocarzinostatin. Treatment with the conjugates caused profound suppression of anti-ssDNA and anti-dsDNA antibody synthesis related to 0-81- and NE-1-Id. This was attributed to the specific killing of the clones bearing anti-DNA Id among the lymphocytes, evidenced by the indirect rosette formation tests. The Id-mediated suppression was not solely due to selective elimination of Id-positive B cells, because 50 to 92% of anti-DNA antibodies were suppressed by treatment with the conjugates. This was supported by flow cytometry analysis that showed a decrease of anti-Id-reactive cells when T cells were treated with the conjugates. This method, then, will permit an analysis of the question as to whether T cells reactive to anti-idiotypic antibodies might participate in the regulatory mechanism for anti-DNA production and, in addition, may lead to a new therapy for SLE.     

TMP-reactive autoantibodies in human SLE sera demonstrate thymine-dependent oligonucleotide specificity

Autoantibodies present in the sera of lupus patients and specific for single-stranded (ss) DNA were fractionated into subsets based upon their reactivity towards 5' nucleotide haptens. As evaluated by ELISA testing, antibodies retained by TMP-agarose bound to TMP-BSA and ssDNA but not to other nucleotide-BSA conjugates or to double-stranded (ds) DNA. Competition-inhibition studies further revealed that TMP-enriched oligo- and polynucleotides were the preferred antigens for these affinity purified antibodies. Similar assays with sequence- or size- defined oligonucleotides further implied that those oligonucleotides comprised entirely of TMP residues were most antigenic and that antigenicity increased with size (length). These results document the existence of a TMP-dependent oligonucleotide specificity among a diverse population of autoanti-ssDNA antibodies.     

Evaluation of auto-antibodies in chronic mucocutaneous candidiasis without endocrinopathy

Six patients with chronic mucocutaneous candidiasis (CMCC) were investigated for the presence of auto-antibodies during the course of the infection. Sera were tested for antibodies to native DNA (dsDNA) and denatured DNA (ssDNA), mitochondrial and microsomal antigens, smooth muscle, gastric parietal cells, basal membrane and skin intercellular substance, parathyroid glands, thyroglobulin and microsomal antigen, immunoglobulins and for anti-nuclear antibodies.Auto-antibodies were detected by radioimmunoassay, immunofluorescence, hemagglutination and other routine methods. Tests were performed at the end of the observation period, with the same batches of antigens and at the same time for all patients. Organ-specific antibodies (gastric parietal cells and intercellular substance) were found at low titers in five patients. Anti-smooth muscle antibodies were increased in two patients. In four patients antibodies to ssDNA were elevated. Moreover high titers of anti-ssDNA antibodies correlated well with disease activity after treatment with Ketoconazole in four tested patients. The possibility that C. albicans infection may induce auto-antibodies should be considered in assessing their disease activity significance in other chronic infected patients. The mechanisms of appearance of auto-antibodies and their immunopathological significance in CMCC are discussed.     

Characterization of mouse and human monoclonal antibodies cross-reactive with SLE serum antibodies to guanosine

Two new monoclonal antibodies, one a mouse IgM and the other a human IgM that reacted with guanosine, were compared to human serum antibodies from patients with systemic lupus erythematosus (SLE). The human monoclonal antibody was polyspecific in its binding to the nucleoside bases, whereas the mouse monoclonal antibody was relatively specific for guanosine when compared by using an enzyme-linked immunosorbent assay (ELISA). Neither antibody bound polyguanylic acid or denatured single-stranded (ss) DNA, however. Serum IgG antibodies from seven patients with SLE cross-reacted with the mouse monoclonal antibody and showed considerable specificity for guanosine. In contrast, the human serum IgG antiguanosine antibodies also bound ssDNA but not dsDNA or polyguanylic acid. Serum IgG antibodies to guanosine measured by ELISA from the seven SLE patients had a decreased response when compared to the total serum IgG response to ssDNA, and most of the antibodies that bound guanosine also bound ssDNA. These studies provide new evidence that there are specific IgG antibodies to guanosine in SLE sera that are a small fraction of the antibodies to ssDNA. Further efforts to define the role of these guanosine antibodies in SLE may provide a better understanding of the basic mechanisms responsible for the development of SLE in man.     

Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.     

Generation of polyclonal antibodies against nisin: immunization strategies and immunoassay development.

Murine polyclonal antibodies reactive to the lantibiotic bacteriocin nisin A (nisA) have been produced by immunization with nisA-cholera toxin and nisA-keyhole limpet hemocyanin (nisA-KLH) conjugates. Mice immunized with nisA-cholera toxin developed nisA-specific antibodies with low relative affinities and poor sensitivities, while the immunization of mice with nisA-KLH conjugates resulted in the production of nisA-specific antibodies with high relative affinities and much-increased sensitivities. nisA antibodies could also be readily mass produced in less than 8 weeks in ascites fluid by using the nisA-KLH conjugate. A competitive direct enzyme-linked immunosorbent assay (ELISA) whereby nisA-horseradish peroxidase and free nisA competed for antibody binding was devised. The detection limit for nisA in the competitive direct ELISA with the nisA-KLH-generated antibodies was from 5 to 100 ng/ml, while the amount of free nisA required for 50% antibody binding inhibition ranged from 0.3 to 5 micrograms /ml. Both antisera and ascites polyclonal antibodies cross-reacted with nisZ either in the supernatant of a producer strain or with the pure lantibiotic but did not cross-react at all with non-lantibiotic-type bacteriocins. These polyclonal antibodies should find a wide usage from nisA ELISA analysis in foods and other matrices.     

Mutational analysis of a sequence-specific ssDNA binding lupus autoantibody

11F8 is a murine anti-ssDNA monoclonal autoantibody isolated from a lupus prone autoimmune mouse. This mAb binds sequence specifically, and prior studies have defined the thermodynamic and kinetic basis for sequence-specific recognition of ssDNA (Ackroyd, P. C., et al. (2001) Biochemistry 40, 2911-2922; Beckingham, J. A. and Glick, G. D. (2001) Bioorg. Med. Chem. 9, 2243-2252). Here we present experiments designed to identify the residues on 11F8 that mediate sequence-specific, noncognate, and nonspecific recognition of ssDNA and their contribution to the overall binding thermodynamics. Site-directed mutagenesis of an 11F8 single-chain construct reveals that six residues within the complementarity determining regions of 11F8 account for ca. 80% of the binding free energy and that there is little cooperativity between these residues. Germline-encoded aromatic and hydrophobic side chains provides the basis for nonspecific recognition of single-stranded thymine nucleobases. Sequence-specific recognition is controlled by a tyrosine in the heavy chain along with a somatically mutated arginine residue. Our data show that the manner in which 11F8 achieves sequence-specific recognition more closely resembles RNA-binding proteins such as U1A than other types of nucleic acid binding proteins. In addition, comparing the primary sequence of 11F8 with clonally related antibodies that differ by less than five amino acids suggests that somatic mutations which confer sequence specificity may be a feature that distinguishes glomerulotrophic pathogenic anti-DNA from those that are benign.     

DNA recognition of a 24-mer peptide derived from RecA protein

A novel 24-residue peptide (L2-G), Ile-Arg-Met-Lys-Ile-Gly-Val-Met-Phe-Gly-Asn-Pro-Glu-Thr-Thr-Thr-Gly-Gly-Asn-Ala-Leu-Lys-Phe-Tyr, derived from RecA can discriminate a single-stranded DNA (ssDNA) from a double-stranded DNA (dsDNA) and a new developed support with this peptide recognizes not dsDNA but ssDNA. The 24-mer peptide with L2 and helix G amino acids of Escherichia coli RecA protein showed the ssDNA binding property with more than 1000 times affinity difference for the dsDNA. However, truncated 15-mer peptide showed no ssDNA binding activity. In the ssDNA binding, L2-G changed its conformation with the perturbation of an alpha-helix structure. The ssDNA binding and the DNA discrimination property of this peptide were due to almost all L2 and helix G amino acids, respectively. This result is useful to design synthetic peptides as functional materials for DNA recognition.     

High-affinity antibodies to ssDNA are produced by CD-B cells in systemic lupus erythematosus patients

It was previously shown that human CD5+B cells are committed to the production of polyreactive "autoantibodies" that bind different self-Ag, including ssDNA, IgG Fc fragment, thyroglobulin, and insulin, as well as exogenous Ag, e.g., tetanus toxoid and other bacterial components. To define the contribution of the CD5+B cell subset to the production of antibodies to ssDNA in SLE, we quantitated, purified, and functionally characterized circulating CD5+ and CD5-B cells in patients and in healthy subjects. We found that in SLE patients circulating CD5+B cells were not increased in number, and both CD5+ and C5-B cells spontaneously secreted antibodies to ssDNA. Studies of the mAb we generated using CD5+ and CD5-B cells revealed that two types of antibody to ssDNA are produced in SLE. The first type, of the IgM class, is produced mainly by CD5+B cells and displays a moderate affinity for ssDNA (Kd, 10(-6) to 10(-7) mol/liter) and is polyreactive, not dissimilar to the autoantibodies inducible in the normal human B cell repertoire by polyclonal activation. The second type, mostly of the IgG and IgA class, is produced mainly by CD5-B cells and displays a higher affinity (Kd, 10(-8) mol/liter) for and reacts only with ssDNA. The functional features of the high affinity anti-ssDNA antibodies and the phenotype of their producing B cells mimic those characteristic of a "mature" Ag-driven response, such as that to tetanus toxoid in tetanus toxoid-vaccinated subjects.