Binding of SLE autoantibodies to native poly(I), ROS-poly(I) and native DNA: a comparative study.

Reactive oxygen species (ROS) are implicated in a variety of human diseases. The formation of pathogenic anti-DNA antibodies in systemic lupus erythematosus (SLE) has been extensively investigated. ROS-modified DNA has been found to be a better antigen for anti-DNA antibodies found in SLE sera. A comparative binding of SLE autoantibodies with native poly(I), ROS-poly(I) and nDNA has been studied. Affinity-purified SLE IgG exhibited a high degree of specificity towards the ROS-modified poly(I) in comparison to native DNA and native poly(I), reiterated visually by gel retardation assay. The data suggested that hydroxyl radical-modified nucleic acids like RNA and DNA might be agent for the induction of circulating SLE anti-DNA autoantibodies.

Antigenicity of poly(I) and ROS-poly(I) and their recognition of human anti-DNA autoantibodies.

The effect of hydroxyl radicals on polyinosinic acid [poly(I)] was studied. Strand breaks, base alteration and a decrease in absorbance at 248 nm (lambda max) were observed upon *OH modification of poly(I). The broad antigen specificity of the induced anti-poly(I) and anti-ROS-poly(I) antibodies showed diverse antigen binding characteristics similar to those of SLE autoantibodies. Recognition of both poly(I) and ROS-poly(I) by human SLE anti-DNA autoantibodies was observed. The possible significance of these findings in the etiology of SLE has been discussed.

A multidimensional autoantibody analysis specifying systemic lupus erythematosus patients with neuropsychiatric symptomatology.

Utilizing a newly perfected multiple-response permutation procedure, we analyzed the autoantibody titer of systemic lupus erythematosus (SLE) patients during active and convalescent periods of disease, SLE patients without neurological involvement, and three other comparison groups (patients with active tuberculosis, patients with multiple sclerosis, and healthy normal controls). The multidimensional analysis we used distinguished those SLE patients with neurological involvement from the other SLE patients. Differences were noted by a univariate analysis measuring antibodies to single- and double-stranded DNA, poly (G), Sm, RNP, Ro, La, and gangliosides. Elevated concentrations of the common anti-DNA idiotype 16/6 were also noted among SLE patients with neuropsychiatric illness. This report stresses that increased disease activity in SLE patients with neuropsychiatric phenomena is reflected by their autoantibody profile.

Autoantibodies in the sera of patients with lymphoma.

Sera of 84 patients with Hodgkin's disease (HD) and 55 patients with non-Hodgkin's lymphoma (NHL) were examined for the presence of autoantibodies to ssDNA, dsDNA, Poly (I), Poly (G), cardiolipin, histones, RNP. Sm, Ro (SS/A), La (SS/B) and the common anti-DNA idiotype (16/6) using an enzyme-linked immunosorbent assay (ELISA). Anti-ssDNA antibodies were detected in the sera of 20 patients with lymphoma (23.8%), more among those with NHL than HD (16 vs. 4 patients p < 0.01). Anti-RNP and anti-Sm antibodies were found in 16 (21.7%) and 14 lymphoma patients (20%) respectively, significantly more than in the controls (p < 0.05) in both antibodies). These findings remained valid following subgrouping of the patients into those with HD and NHL. With all the other autoantibodies examined no significant difference could be observed in the incidence between lymphoma patients and controls. These results differ from our previous survey carried out on sera of patients with solid tumors in whom no increased frequency of any of the autoantibodies could be determined. In view of the evidence suggesting an increased risk of lymphoma in a number of autoimmune diseases our results extend this relationship to an increased incidence of autoantibodies among patients with lymphoma.

Amino acid sequence of the FV region of a human monoclonal IgM (NOV) with specificity for the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1, which cross-reacts with polynucleotides and with denatured DNA.

The complete amino acid sequences of the VH and VL regions of a biologically significant Ig, IgMNOV, were determined. IgMNOV is reactive with the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1. As reported earlier, it cross-reacts completely with polynucleotides poly(A) and poly(I) and to a lesser extent with denatured DNA and protects newborn rats against infection with E. coli K1, and is equal in potency to the standard horse anti-group B meningococcal serum. The reduced and alkylated chains were sequenced directly, identifying the L chain as lambda-subgroup II and the mu-H chain as subgroup III. The complete sequence of the VL region was determined by sequencing peptides generated by cleavage with Staphylococcus aureus protease, chymotrypsin, and trypsin. The H chain was cleaved with cyanogen bromide followed by enzymatic cleavages to obtain a large part of the VH region sequence. The structure was completed by sequencing tryptic peptides of the Fab fragment and by mass-spectrometric analysis.

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.

Nucleic acid-binding specificity and idiotypic expression of canine anti-DNA antibodies.

Serum samples collected from eleven lupus dogs during an active phase of the disease all bound native and denatured DNA, poly(dT), poly(I) and poly(G). Nine bound poly(C); 10 bound poly(U); and 3 bound poly(A). Sera from 22 normal dogs were negative with all of these antigens. The canine sera were also probed for the presence of three idiotypic markers, one related to human lupus anti-DNA antibodies and two related to murine lupus antibodies. One canine lupus serum expressed idiotopes related to murine anti-DNA idiotype (Id) termed H130: (a) the canine serum bound to anti-H130 anti-Id; (b) it inhibited the binding of anti-H130 Id to its homologous Id; and (c) the anti-H130 Id inhibited the binding of the canine serum to DNA. These findings suggest that anti-DNA variable regions exhibit interspecies similarities, probably reflecting the conservation of the encoding gene segments through evolution.

Naturally-occurring tumor-reactive autoantibodies: a monoclonal antibody from normal mice reacts with tumor cells and with DNA.

A natural IgM monoclonal antibody, 1.67, was generated from apparently healthy unstimulated BALB/c mice. This antibody reacted with L5178Y murine T cell lymphoma, with human Raji cells, and with several normal cells. Further analysis of its ligand binding capacity disclosed strong binding to single-stranded DNA (ssDNA). However, this naturally-occurring monoclonal antibody binds to different epitopes on cell membranes and on DNA than another anti-DNA monoclonal antibody (18/103/1) from human origin. This conclusion was based on competition assays. Furthermore, NOA 1.67 lacks the 16/6 idiotype expressed on the 18/103/1 antibody. The 16/6 idiotype is shared by human and mouse lupus monoclonal autoantibodies that bind simultaneously to lymphoid cells and DNA. This is a first report on a natural autoantibody that binds to malignant and to normal cell membrane(s) as well as to ssDNA. It may have regulatory functions controlling malignancy and or autoimmunity.

Affinity of human carcinoma cell nuclei for polyinosinic acid demonstrated by monoclonal antibodies.

The anti-nuclear cross reactivity of the monoclonal anti-actin antibodies M 372/809 was studied in some detail. The reactivity against a number of nuclear constituents was examined in the ELISA test and the capacities of these constituents to block the M 372/809 anti-nuclear and anti-actin reactions were evaluated in indirect immunofluorescence tests against tissue sections and monolayer cultures of fibroblasts and Vero cells. The repetitive polynucleotides polyinosinic and polyguanylic acid and their deoxyanalogues, actin and vimentin, were found to have the antigenic epitope. The epitope was covered or otherwise inactivated in the presence of polycytidylic acid. Using the M 372/809 antibodies as a reagent, carcinoma cell nuclei were found commonly to have an affinity for polyinosinic and polyguanylic acid. This was seldom noted with non-neoplastic cells.

Multiple serologic reactions and their relationship to clinical activity in systemic lupus erythematosus.

Analysis of the binding of serum from 56 systemic lupus erythematosus patients to native DNA (nDNA), denatured DNA (dDNA), poly I, poly(dT), RNA, and cardiolipin revealed multiple antigen binding in many of the sera. Raised levels of antibodies (IgG and/or IgM) to denatured DNA were found in the highest percentage (68%) of patients. A small subset of patients with multiple raised IgM antibodies, renal disease, and vasculitic skin rash was identified. No correlation between multiple serologic activity and clinical disease was found.

Supplement-induced cytotoxic cells (SICC) generated from mouse thymus or spleen cells cultured in the presence of interleukin 2 and/or polyinosinic acid.

Mouse thymocytes and spleen cells from unprimed C57BL/6 donors generate broadly reactive cytotoxic cells during 5 days of culture in vitro with polyinosinic acid (5') (poly(I] and/or supernatant from PMA-treated EL4 leukemia cells which contains interleukin 2 (IL-2) activity. We refer here to such cytotoxic cells as "supplement-induced cytotoxic cells" or SICC. Thymocytes are dependent on the supernatant factor(s), whereas spleen cells are usually stimulated by poly(I) alone. Polyinosinic acid acts synergistically with supernatant factor(s) to stimulate generation of SICC by both thymocytes (SICC-T) and spleen cells (SICC-S) when the IL-2 activity of the supernatant is inadequate alone. SICC can be generated by both splenocytes and thymocytes in medium supplemented with fetal calf serum or syngeneic plasma. SICC are active in 4 hr 51Cr-release tests against syngeneic, allogeneic, and xenogeneic tumors but not against lipopolysaccharide-induced lymphoblasts. Embryonic fibroblasts, too, are sensitive to SICC generated by thymocytes. In complement-dependent depletion tests, cytotoxic activity is partially sensitive (SICC-T) or fully sensitive (SICC-S) to anti-Thy-1 and -H-2 but not to anti-Lyt-1, -Lyt-2, or -asialo GM1.

Polynucleotide helix geometry and stability. Spectroscopic, antigenic and interferon-inducing properties of deoxyribose-, ribose-, or 2'-deoxy-2'-fluororibose-containing duplexes of poly(inosinic acid) . poly(cytidylic acid).

Circular dichroism, absorbance temperature profiles, antigenic properties, and interferon-inducing capacity of the nine double-stranded complexes between poly(inosinic acid) and poly(cytidylic acid) [(rI)n . (rC)n] and their deoxy- and 2'-deoxy-2'-fluoro-analogues were studied. The complexes containing only ribose or 2'-fluororibose chains showed similar CD spectra and thermal stabilities. Anti-(rI)n . (rC)n antibodies were well recognized by the duplexes containing 2'-fluororibose in either one strand. These two duplexes were also efficient interferon inducers. Presence of fluororibose in both strands decreased the affinity of anti-(rI)n . (rC)n antibodies slightly and abolished interferon-inducing activity. All polydeoxyriboside-containing complexes showed CD spectra significantly different from the previous group; they showed also 40- to 100-times lower affinity for the anti-(rI)n . (rC)n antibodies, and did not induce interferon. It is concluded that the structure of (rI)n . (rC)n, an A-type helix, is little perturbed by substitution of one or both strands by 2'-fluororibose. Substitution by deoxyribose in either strand considerably changes the structure of the helices. The hybrids containing poly(deoxycytidylic acid) retain at least some of the structural features of the B-type helix poly(deoxyinosinic acid) . poly(deoxycytidylic acid).

Demonstrations of the production of specific antibodies to poly(I).poly(C) in rabbits.

The rabbit antiserum against poly(I).poly(C) purified by hydroxyapatite column chromatography contained three distinct antibodies. They were fractionated into three antibody populations by a series of precipitations (with poly(A).poly(U), poly(I), and poly(I).poly(C)) and their specificities were examined by quantitative complement fixation, double diffusion tests and radioimmunoassay. The first population was common to the double helical structure of double-stranded RNAs. The second was specific for poly(I) and the third was specific for poly(I).poly(C). These studies demonstrated that specific antibodies exclusively reactive with poly(I).poly(C) existed in the rabbit antiserum against poly(I).poly(C).

Antibodies to natural double-stranded RNA and to synthetic polyribonucleotides in the sera of patients with systemic lupus erythematosus.

A parallel testing of antibodies to double-stranded ribonucleic acid in 80 sera from patients with systemic lupus erythematosus by the membrane binding method using natural 3H-dsRNA preparation and synthetic 125I-poly I. poly C preparation revealed a good correlation (r = +0.81). In a selected set of patients with SLE we observed a slight tendency to preferential binding of the natural preparation and a lower frequency of anti-dsRNA antibodies when compared to that reported by other investigators. The presence of anti-dsRNA did not correlate with the presence of anti-dsDNA.

Immunochemical measurement of conformational heterogeneity of poly(inosinic acid).

Several pure poly(I) preparations differed in: (a) their complement fixation reactivity with anti-poly(I) antiserum; (b) their ability to bind to a solid-phase anti-poly(I) antibody-Sepharose column; (c) their ability to inactivate serum complement; and (d) their reactivity with purified antibodies to double-stranded RNA. In particular, poly(I) samples that could induce interferon production differed from non-inducer poly(I)s; the inducers reacted weakly with anti-poly(I) antiserum and were the only ones that reacted with antibodies to double-stranded RNA. One inducer poly(I) did not inactivate complement, and differed from non-inducer poly(I) in quantitative aspects of poly(I) . poly(C) formation with varying amounts of poly(C). An additional type of poly(I) preparation reacted poorly with anti-poly(I) antiserum, did not react with anti-double-stranded-RNA antibodies and failed to induce interferon production. The varying forms of poly(I) were not interconvertible by boiling and rapid chilling. These results indicate that several different stable structural forms of poly(I) may result from a standardized synthetic procedure.

Immunogenicity of rice dwarf virus-ribonucleic acid.

Using rice dwarf virus (RDV)-RNA which was extracted from RDV and further purified by MAK-column chromatography, anti-RNA antibodies were produced in rabbits immunized with RDV-RNA antibodies were prods immunized with RDV-RNA-methylated bovine serum albumin complexes. The antisera, as analzyed by complement fixation, cross-reacted with synthetic double stranded RNAs (poly (A)-poly (U), poly (I)-poly (C)), but not with native or denatured DNA, rRNA, tRNA, 5 S RNA and nucleic acids from rice plants. RDV-RNA treated with heat or dimethylsulfoxide was markedly reduced in reactivity to the antisera. When RDV-RNA was digested with RNase A at low salt concentration, its complement fixation activity was abolished. In double diffusion tests, two different precipitation lines were demonstrated between the antiserum and RDV-RNA. One of the precipitation lines connected with those was formed between the antisera, poly (A)-poly (U) and poly (I)-poly (C). As regards immunoglobulin classes of the antibodies, one of the two rabbits employed had antibody activity only in IgG.

Study of thymic factors. II. Failure of thymosin to alter the natural history of NZB and NZB/NZW mice.

The effects of daily injections of thymosin, bovine fraction V, on the natural history of NZB and NZB/NZW F1 mice were investigated. With the use of several dose schedules, no significant differences were discovered in treated versus control groups when survival, autoantibodies, and mitogen responsiveness were compared. These results provide further evidence that thymosin may have little or no role in the treatment of the autoimmune disease of New Zealand mice. More encouraging research in thymic extracts and their measurement is necessary before clinical trials in SLE are considered.