Anti-cardiolipin/beta-2 glycoprotein activities co-exist on human anti-DNA antibody light chains.

We have recently shown that the human anti-DNA antibodies B3 and 33H11 also bind cardiolipin and that the anti-autoantigen activity resides predominantly on their lambda light chains. We now show that the two auto-antibodies possess strong reactivity to the plasma-protein 2-Glycoprotein I (beta2-GPI) also. Utilizing chain shuffling experiments involving an unrelated anti-p185 antibody 4D5 with insignificant reactivity to cardiolipin or to beta2-GPI, we now demonstrate that hybrid Fabs with constituent light chain, but not the heavy chain, of B3 or 33H11, exhibit anti-cardiolipin activity. Furthermore, the constructs possessing the auto-antibody-derived light chain also exhibited significant reactivity to beta2-GPI. The results suggest that anti-DNA, anti-cardiolipin and anti-beta2-GPI activities co-exist on the light chains of the antibodies studied and, importantly, these activities could be transferred to antibody constructs by their light chains alone. Computer-generated models of the three-dimensional structures of the auto-antibodies and their hybrids, suggest predominant interaction of their light chains with domain IV of beta2-GPI.

Structure-function analysis and the molecular origins of anti-DNA antibodies in systemic lupus erythematosus.

Patients with the autoimmune rheumatic disease systemic lupus erythematosus (SLE or 'lupus') develop a wide variety of clinical and serological manifestations including the presence of antibodies to double-stranded DNA (dsDNA), which are often diagnostic and potentially pathogenic. In this review, we have examined the links between the structure and function of anti-dsDNA antibodies, emphasising their clinical associations. We have also reviewed studies involving animal models, the analysis of human antibody sequences and studies of, and using, computer modelling and crystal structure.

Apoptosis in human autoimmune diseases.

The description of apoptosis or programmed cell death nearly thirty years ago did not initially stimulate a great deal of interest. However, the ways cells die is clearly an essential part of biological homeostasis and well worth of study in its own right as the enormous literature on the subject in the past 15 years confirms. In the past decade new avenues of apoptosis research have opened up as the relationship between this form of cell death and autoimmune disease has come under increasing scrutiny. Although most research to date has been in animal study models, there are a variety of studies which have begun to explore links between apoptosis and a wider range of human autoimmune conditions. In this review we analyse briefly the background to what is known about apoptosis and focus on the increasing likelihood that abnormalities in apoptosis are contributory factors in the development of human autoimmunity.

Effect of histone and histone-RNA complexes on the disease process of murine systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is characterised by the production of a variety of autoantibodies against cell surface, nuclear and cytoplasmic antigens. The antigen or antigens responsible for the induction of this disease is/are unknown. We have analysed the antigenicity and pathogenicity of free histones and histones complexed with RNA in Balb/c, B10 Br, C57BL/6 and MRL-lpr/lpr mice by giving 1 microgram and 25 micrograms of each antigen intraperitoneally in complete and incomplete Freund's adjuvant. The same number of control animals were injected with either adjuvant or PBS. In the initial experiment we gave three doses of antigen at three weekly intervals. B10 Brown and C57BL/6 mice had no response to the antigens. Balb/c mice developed a mild transient antibody response against H1 histone, branched peptide of ubiquitinated H2A (peptide T4) and also against ssDNA. However in repeated experiments when the histone-RNA complex was injected into young MRL-lpr/lpr animals at two weekly intervals, a significantly increased antibody response was detected against H1, peptide T4 and some histone peptide residues (204-218 of H1, 1-20 and 65-85 of H2A, 1-25 of H2B, 1-21 of H3 and 1-29 of H4) compared to the control groups. Moreover, this group also showed elevated serum anti-DNA antibody levels and early impairment of renal function assessed by the urine protein levels. These experiments have demonstrated that there is a genetic variation in antibody responses against histones and histone-RNA complexes and that histone-RNA complexes exaggerate the disease in young MRL-lpr/lpr mice by inducing antibodies to basic regions of histones and other autoantigens.

Expression of a human fetal anti-DNA antibody idiotype BEG-2 beta in the families of patients with rheumatoid arthritis.

BEG-2 is a monoclonal antibody produced by the human-human hybridoma technique from a 12 weeks old human fetus. A polyclonal antiserum was raised in an (NZW x Half-lop hybrid) rabbit against BEG-2 and the anti-BEG-2 anti-idiotype was purified and characterised. Using this rabbit reagent the expression of the BEG-2 beta idiotype was analysed in 12 patients with active rheumatoid arthritis and their close family members (n = 54). Twenty five sera from healthy controls were analysed to establish a normal range. Ten of 12 patients (83%) with rheumatoid arthritis expressed the BEG-2 idiotype as well as 11 of 54 healthy unaffected relatives (20%).

Analysis of antibody reactivity in the sera of 42 patients with paraproteinaemia.

The clinical expression of disease in patients with conditions in which autoimmunity is thought to contribute to the pathogenesis of disease is the result of an unfortunate combination of predisposing and environmental factors. The presence of autoantibodies showing a variety of antigen specificities in sera from many of these patients has been closely correlated with particular spectra of organ involvement or tissue destruction. Their precise role in the disease process is as yet unclear. Sera from patients with paraproteinaemia also often contain autoantibodies to a variety of cell components, although symptoms of autoimmune disease are rarely found in this group of individuals. In this study of 42 sera from patients with paraproteinaemia we have confirmed the presence of autoantibodies in 33% (13/42) of samples. Amongst the autoantibodies detected were those to human neutrophils (3), U1RNP (8) and cardiolipin (4). In five sera, the immunoglobulin class of autoantibody did not correlate with that of the monoclonal band. This study extends previous reports of the repertoire of autoantibodies present in sera from patients with paraproteinaemia.

Antiphospholipid antibodies are induced by in vitro fertilization and correlate with paraoxonase activity and total antioxidant capacity of plasma in infertile women.

The objectives of this study were to determine whether antiphosholipid antibodies are associated with in vitro fertilization (IVF), and assess the potential biological effects of these antibodies. Sera from seventy infertile women (18 before IVF, 13 submitted to one IVF cycle and 39 after three cycles) and 28 healthy controls were collected. Anticardiolipin (anti-CL) and antiphosphatidylserine (anti-PS) antibodies, paraoxonase (PON) and Total Anti-oxidant Capacity of plasma (TAC) were measured. Anti-CL and anti-PS titres were significantly increased in treated patients compared with patients before treatment or controls (P < 0.001). There were no differences regarding anti-CL and anti-PS titres between controls and untreated patients nor when different types of infertility were considered. PON activity and TAC were significantly reduced in treated patients when compared to untreated and controls (P < 0.001; P < 0.002). PON correlated inversely with anti-CL and anti-PS IgG (r = -0.734; P < 0.001) and directly with TAC (r = 0.720, P < 0.001). In conclusion PON activity is decreased in women submitted to IVF treatment and is associated with the presence of antiphospholipid antibodies. These factors might contribute to the increased oxidative status found in these patients.

Involvement in lupus disease of idiotypes Id.F-423 and Id.IV-228 defined, respectively, upon foetal and adult MRL/Mp-lpr/lpr DNA-binding monoclonal autoantibodies.

The derivation of a monoclonal IgG3K autoantibody, designated F-423, from a foetal MRL/Mp-lpr/lpr mouse is described. It has immunochemical properties similar to DNA-binding monoclonal antibodies derived from adult mice with lupus disease in that it reacts with single-stranded DNA and, to a lesser extent, with double-stranded DNA and some forms of RNA. Its similarities to antibodies from adults extend further: it carries a public idiotype, Id.F-423, that can also be detected on antibodies from adult MRL and (NZB x NZW)F1 mice, and F-423 itself expresses other idiotypes defined originally on antibodies from adult lupus mice of both strains. Its potential involvement in pathological processes is demonstrated by two observations: (i) immunization of young MRL/Mp-+/+ mice with antibody F-423 induced the nephritic and immunological changes associated with systemic lupus erythematosus; and (ii) heterologous rabbit anti-Id.F-423 anti-idiotypic antibodies suppressed the progression of lupus disease in adult MRL/Mp-lpr/lpr mice. Similar effects were found with monoclonal antibody IV-228, an antibody derived from an adult MRL mouse and previously known to be directly nephrotoxic, and with anti-Id.IV-228 antibodies. It is concluded that even during foetal life mice of lupus-prone strains have lymphocytes capable of making pathogenic autoantibodies long before symptoms of lupus disease appear.

Transgenic models of tolerance and autoimmunity: with special reference to systemic lupus erythematosus.

Transgenic and knockout mouse carrying rearranged antigen-receptor genes have been invaluable for the elucidation of basic mechanisms in autoimmunity and have contributed new models of human autoimmune diseases. Several examples of transgenic models expressing rearranged immunoglobulin genes have been described. These models have provided a window into the events involved in this process, allowing the development and fate of self-reactive lymphocytes to be followed in vivo. In the B cell lineage, as in T cells, self-reactive cells have been found to undergo several distinct fates in vivo: they can be physically eliminated, functionally inactivated, or they can persist unchanged or become activated. Nevertheless the precise understanding of the molecular events leading to lymphocyte deletion, anergy or activation remains a challenge.

Analysis of variable region genes encoding anti-Sm and anti-cardiolipin antibodies from a systemic lupus erythematosus patient.

We have analysed the heavy and light chain variable region genes of two monoclonal antibodies, specific for the Sm antigen (RSP1; IgG kappa) and for cardiolipin (RSP4; IgM lambda), derived from a patient with active systemic lupus erythematosus (SLE). We have established that the variable region genes of the RSP1 autoantibody are somatic mutants of two germ line genes from the VH4 and V kappa 1 gene families. RSP4 antibody uses gene segments closely related to a VH3 gene member and to a V lambda 1 gene. The presence and distribution of the somatic mutations on both monoclonal autoantibodies are compatible with an antigen-driven immune process. These data suggest that in SLE a common antigenic stimulus may govern the autoantibody response against a wide spectrum of unrelated antigens, including native DNA, cardiolipin or Sm antigens, and provide further evidence that disease-associated autoantibodies are generated through antigen-selected somatic mutations.

The prevalence of antibodies to anionic phospholipids in patients with the primary antiphospholipid syndrome, systemic lupus erythematosus and their relatives and spouses.

OBJECTIVES: Antiphospholipid antibodies (aPL) have been associated with syndromes involving thrombosis, fetal loss and thrombocytopenia. Genetic and environmental conditions are among the factors attributed to the cause of autoimmune diseases such as the antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). The aim of this study was to determine whether these factors determine the prevalence of aPL. METHODS: Three groups of patients were tested for the presence of IgG, IgM and IgA anticardiolipin (aCL), antiphosphatidylinositol (aPI), antiphosphatidylglycerol (aPG) and antiphosphatidylserine (aPS) antibodies: (i) patients with primary APS (PAPS); (ii) patients with SLE and secondary APS; and (iii) patients with SLE without APS. First-degree relatives and spouses of patients with SLE/APS were also tested for circulating aPL. RESULTS: IgG aPL were particularly prevalent in patients with PAPS. IgG aPI and aCL were more prevalent in patients with PAPS than the IgM equivalents (P < 0.0001). Notably, none of the patients with PAPS had IgA aPL. A significantly higher number of relatives of patients with SLE/APS possessed IgG aPL than the normal controls. Except for aPG (P < 0.03), the prevalence of these antibodies in the relatives was not significantly different from patients with SLE/APS. The relatives also had significantly higher prevalence of IgG aPI, aPS and aCL antibodies than IgM aPL antibodies. In contrast, the prevalence of IgG aPL in the spouses was no different than in the healthy controls. CONCLUSIONS: Genetic factors, shared by patients and their relatives, seem to have some effect on the prevalence of aPL in the subjects studied, while environmental factors shared by spouses appear to have no influence.

A human fetal monoclonal DNA-binding antibody shares idiotypes with fetal and adult murine monoclonal DNA-binding antibodies.

A human DNA-binding monoclonal antibody was produced by fusing the hepatocytes from a 12-week-old human fetus with the lymphoblastoid cell line GM 4672 using polyethylene glycol. This antibody, designated BEG 2, binds to single-stranded (ss) DNA but also binds to double-stranded (ds) DNA, poly(dT), polyI and poly(ADP-ribose), but not to RNA, cardiolipin or K-30. The binding of BEG 2 to these polynucleotides can be inhibited by incubation with polynucleotides in the fluid phase. A rabbit polyclonal anti-idiotype was raised, and using this reagent it was shown that the BEG 2 idiotype is present in normal human serum (7%), systemic lupus erythematosus (SLE) sera (8%) and rheumatoid arthritis sera (23%). The extent of idiotypic sharing between BEG 2 and murine monoclonal DNA-binding antibodies, in particular monoclonal antibody (mAb) 423 (derived from a 15-day-old fetal MRL/Mp-lpr/lpr mouse) and mAb 402 (derived from an adult MRL/lpr mouse), was also investigated. Using a competition ELISA, it was shown that preincubation of BEG 2 with rabbit anti-423 and rabbit anti-402 inhibits the binding of BEG 2 to DNA, and the binding of 402 to DNA by anti-BEG 2 and anti-423. These data suggest that mAb BEG 2, 423 and 402 share common idiotypes, that autoreactivity is present in early fetal life, and that autoantibodies may be encoded for by germline genes, which have been conserved through evolution.

A human anti-dsDNA monoclonal antibody caused hyaline thrombi formation in kidneys of 'leaky' SCID mice.

There are few studies assessing the pathogenicity of human monoclonal anti-DNA antibodies. The use of SCID mice avoids the problem of rejection of the human hybridoma cells thus allowing in vivo assessment of human immunoglobulins. Using electron microscopy we have shown that the human IgG anti-dsDNA monoclonal antibody, RH14, is nephritogenic in SCID mice, causing morphological changes in the kidney due to immunoglobulin deposition. The problem with using SCID mice is that they have an abnormal immune system; normally they are used at about 2 months of age, at which time they have virtually no functional T or B cells. It is known that older SCID mice become increasingly 'leaky', that is they develop some mature lymphocyte clones. Our aim was to assess if implanting anti-DNA antibodies into older 'leaky' SCID mice would result in pathology which was observable by light microscopy. Eight-month-old SCID mice were implanted with human hybridoma cells secreting either RH14 an anti-dsDNA IgG, CL24, an antiphospholipid antibody or an irrelevant human IgG control. As previously, RH14 deposited in the kidney and caused proteinuria but unexpectedly we also observed hyaline thrombi in the kidney glomeruli and peritubular capillaries. These thrombi occurred only in the case of RH14 implanted mice and were found to stain positively for human IgG and fibrin. However, apart from the interesting thrombi, we did not observe any greater pathological damage resulting from the anti-dsDNA antibody deposition than we had seen in the younger mice; indeed, the electron microscopic findings were more limited.

An analysis of clinical disease activity and nephritis-associated serum autoantibody profiles in patients with systemic lupus erythematosus: a cross-sectional study.

OBJECTIVE: To establish the correlation between lupus nephritis-associated autoantibody levels and the presence/activity of lupus nephritis and global disease activity using cross-sectional data in patients with systemic lupus erythematosus (SLE). METHODS: Disease activity was assessed using the British Isles Lupus Assessment Group (BILAG) index. Antibody levels against single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), histones, nucleosomes and heparan sulphate (HS) were analysed by ELISA in SLE patients with (n=11) and without (n=22) nephritis and in normal controls (n=21). Antibody subclasses were also analysed. RESULTS: Higher levels of anti-dsDNA and anti-HS antibodies were found in patients with lupus nephritis, the level of anti-HS antibodies correlating with the BILAG renal score. Predominant subclasses were IgG1 and IgG3 for dsDNA antibodies, IgG2 for anti-nucleosome antibodies, and IgG2 and IgG3 for anti-HS antibodies. CONCLUSION: Correlation was demonstrated between antibodies to dsDNA, ssDNA, histones, nucleosomes and HS. There is a strong correlation between the level of anti-HS antibodies and disease activity in patients with lupus nephritis as measured by BILAG.

IgG subclasses in systemic lupus erythematosus and other autoimmune rheumatic diseases.

In this study the concentration of the different subclasses of IgG in sera from patients with a range of autoimmune rheumatic diseases (ARD) was detected by radial immunodiffusion. In the second part the IgG subclasses of autoantibodies that recognize single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), Ro, La, Sm and RNP in patients with ARD were measured by enzyme-linked immunosorbent assay. We studied 15 patients with lupus, 20 patients each with primary and secondary Sjögren's syndrome (SS) and 10 each with rheumatoid arthritis (RA), scleroderma and myositis. Twenty healthy controls were also measured. The serum concentration of IgG2 in ARD patients was generally reduced. In contrast, the concentrations of IgG1, IgG3 and IgG4 subclasses were normal or raised. A high degree of correspondence in the IgG1, IgG2 and IgG3 responses to dsDNA and ssDNA in SLE was found. Notable differences in the IgG1 anti-Ro and ssDNA responses compared to the other subclasses were seen in 1 degree and 2 degrees SS. In addition, an unexpected high level of IgG4 antibodies to ssDNA in 1 degree SS (65%) and IgG4 antibodies to Sm/RNP in RA was observed.

Phospholipid binding specificities and idiotype expression of hybridoma derived monoclonal autoantibodies from splenic cells of patients with systemic lupus erythematosus.

OBJECTIVE: To analyse the phospholipid binding specificity, functional characteristics and idiotype expression of human hybridoma derived monoclonal autoantibodies (MAb) derived from the spleens of two patients with active systemic lupus erythematosus (SLE). METHODS: The IgM MAbs binding to phospholipids were generated from spleen cells of two patients (RSP and RT) with active SLE and their specificity of binding to neutral phospholipids (phosphatidyl ethanolamine, phosphatidyl choline, platelet activating factor, sphingomyelin) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, phosphatidyl inositol and cardiolipin (CL)) analysed. Binding specificity of cross reactive antibodies (those binding to CL and DNA) was confirmed by fluid phase inhibition assays. Lupus anticoagulant activity and beta 2-glycoprotein-1 (beta 2 GP-1) requirement for the antigen binding of these MAbs were detected using the modified dilute Russell's viper venom test and modified anti-CL enzyme linked immunosorbent assay (ELISA), respectively. Expression of idiotypes (Id) Id RT-84 and Id H3 was analysed using rabbit polyclonal and murine monoclonal anti-idiotype reagents, respectively. RESULTS: Twelve clones from the patient RSP and eight clones from patient RT were reactive with phospholipids. Marked differences in phospholipid binding of these MAbs were noted, varying from truly polyreactive (RT-72 bound to most phospholipids tested) to monospecific (RT-84 bound only to CL). Furthermore, MAbs RT-84, RT-129, and RSP-57 had lupus anticoagulant activity and required beta 2 GP-1 for CL binding. It was found that 75% of phospholipid binding antibodies from RT clones expressed RT-84 Id, but none from RSP clones did so, and that Id H3 was expressed only by the RT-83 antibody. CONCLUSION: These results show that human anti-phospholipid MAbs are heterogeneous with respect to phospholipid binding, functional characteristics, and Id expression.

Human monoclonal anti-phospholipid antibodies selectively bind to membrane phospholipid and beta2-glycoprotein I (beta2-GPI) on apoptotic cells.

The ability of an anti-phospholipid (LJ1) and an anti-beta2-GPI (RSP-57) human MoAb to bind to apoptotic but not viable cells was demonstrated in this study. Both MoAbs were derived from patients with systemic lupus erythematosus and anti-phospholipid antibody syndrome. The parallel analysis of the specificity and affinity of four anti-phospholipid human MoAbs suggests that the binding of LJ1 MoAb to apoptotic cells is a specific property of this MoAb. RSP-57 MoAb recognizes apoptotic cells through beta2-GPI which becomes available for binding after the interaction with negatively charged phospholipids. This observation provides evidence that the binding of human anti-phospholipid antibodies to apoptotic cells occurs in both a beta2-GPI-dependent and independent way and involves a restricted group of epitopes. The finding that LJ1 and RSP-57 MoAbs bind apoptotic cells underlines the property of these MoAbs to act as cell membrane markers of apoptosis. Major pathological implications derive from the observation that LJ1 and RSP-57 MoAbs recognize epitopes expressed on 'early' apoptotic cells. The interference with the in vivo clearance and processing of apoptotic cells is a potential pathogenic mechanism of these antibodies.

Anti-beta2-glycoprotein I, anti-prothrombin and anticardiolipin antibodies in a longitudinal study of patients with systemic lupus erythematosus and the antiphospholipid syndrome.

OBJECTIVE: To determine anti-beta2 glycoprotein-I (anti-beta2GPI) and anti-prothrombin (anti-ProT) antibody levels, and the IgG subclass distribution of anti-beta2GPI antibodies, in serial samples from patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) having initial or recurrent thrombotic/neurological (T/N) events during the study period. To investigate the correlations between these antibodies and beta2GPI antigen, anticardiolipin antibody (aCL), anti-double-stranded (ds) DNA, C3 levels and disease activity. METHODS: Fifty serum samples were identified from seven patients with SLE who had had T/N events during the follow-up from a cohort under long-term follow-up. IgG anti-beta2GPI, anti-ProT, aCL, IgG subclasses of anti-beta2GPI and beta2GPI antigen levels were determined by ELISA. Corresponding disease activity [British Isles Lupus Assessment Group (BILAG)], anti-dsDNA and C3 levels were compared. RESULTS: IgG anti-beta2GPI antibody levels were elevated in six of the patients before and after the T/N events with less marked fluctuations than aCL antibody levels. The predominant subclass of anti-beta2GPI antibodies was IgG2 before and after the T/N events. IgG anti-ProT antibodies were negative in all cases. There was a significant but weak correlation between anti-beta2GPI and aCL antibodies. No correlation was found between disease activity and IgG anti-beta2GPI antibody and beta2GPI antigen levels. There were fluctuations in beta2GPI antigen levels and a trend to increase after T/N events was observed in some patients. CONCLUSION: Most of the patients with a T/N event during the study period had IgG anti-beta2GPI, but not IgG anti-ProT antibodies. Many IgG aCL-negative samples were found to have IgG anti-beta2GPI activity during the follow-up period. The predominant subclass of IgG anti-beta2GPI was IgG2, which may have importance in the pathogenesis of APS. beta2GPI antigen levels were found to be increased in some patients with SLE after T/N events. IgG anti-beta2GPI antibodies may be used as an adjunctive marker of future T/N events in patients with SLE and APS with aCL antibodies and lupus anticoagulant.

An analysis of apoptosis in lymphoid organs and lupus disease in murine systemic lupus erythematosus (SLE).

Apoptosis is a programmed cell death process that helps to regulate both T cell and B cell development. In this study, we have investigated the levels of apoptotic death in cells of the thymuses and spleens (white matter) of autoimmune MRL-lpr/lpr mice with progressive lymphadenopathy and SLE disease activity; we also examined the renal pathology in these animals. Fas is a cell surface receptor, which when activated initiates the sequence of events that lead to apoptosis. In MRL-lpr/lpr mice Fas is defective, so the competency for apoptosis may be reduced. In young animals of advancing age the thymuses enlarged until in 5-month-old females the average weight was three times that at 1 month, and spleen and kidney weights also increased in size disproportionately. At light microscope level apoptotic cells in tissue sections were counted using both routine eosin and haematoxylin staining (to identify them by their morphology) and in situ end-labelling of cells with DNA strand breaks; their presence was further confirmed by electron microscopy. As the mice aged, the numbers of apoptotic cells in thymic cortex, thymic medulla and spleen white pulp areas reduced significantly (P < 0.01-0.001), whereas in BALB/c normal controls they increased significantly (P < 0.05). These changes were coincident with the development of severe lupus, whose activity was assessed by measuring serum anti-ssDNA and anti-dsDNA antibody titres and urinary protein (albumin) level which were elevated significantly by 5 months of age (P < 0.001 for both ssDNA and dsDNA and P < 0.01 for urine albumin) compared with their younger counterparts. Thus, lymphoid organ enlargement, decrease in apoptotic indices, elevated serum anti-ssDNA and anti-dsDNA antibody levels, and impaired renal function coincided with the onset and severity of lupus disease in lpr mice. It seems likely that there is a causal relationship between defective deletion of autoreactive lymphoid cells, imperfect Fas-mediated apoptosis and development of murine SLE.

Molecular expression systems for anti-DNA antibodies--2.

Antibodies to double-stranded DNA are the best-known serological markers of systemic lupus erythematosus, and are closely associated with its renal pathogenesis. How these antibodies recognize DNA is not fully understood. An understanding of the relationship between the functional attributes of an antibody with the three-dimensional structure of its antigen-combining site would allow an insight into the rules that dictate auto-antibody-nucleic acid interaction and consequent pathogenicity of the autoantibody. Data from such studies could assist the development of novel drugs as an approach to specific therapies that can inhibit or disrupt protein-nucleic acid interactions. A full understanding of the binding specificities can be achieved only by experimental determination of detailed three-dimensional structure of these antibodies alone, and of their complexes with specific DNA antigens. A prerequisite of such a study is the ability to produce multimilligram quantities of the antibody protein. However, these antibodies are particularly difficult to express, probably due to their DNA-binding activity. This review attempts to focus on the recent developments on the over-expression of anti-DNA antibody fragments in heterologous cell expression systems and their purification to homogeneity that would in turn allow their structural studies via crystallization.