Coexistence of anti-RNA polymerase III and anti-U1RNP antibodies in patients with systemic lupus erythematosus: two cases without features of scleroderma.

Anti-RNA polymerase III (RNAP III) antibodies are highly specific for scleroderma (SSc) and associated with diffuse SSc and renal crisis. Coexistence of anti-RNAP III and other SSc autoantibodies is rarely documented. We report three cases with coexisting anti-RNAP III and anti-U1RNP. Autoantibodies in 3829 sera from rheumatology clinics were screened by immunoprecipitation. Anti-RNAP III-positive sera were also examined by immunofluorescence and anti-RNAP III ELISA. In total, 35 anti-RNAP III-positive sera were identified by immunoprecipitation, in which three had coexisting anti-U1RNP. All three were anti-RNAP III ELISA positive. Two had anti-RNAP I dominant (vs. RNAP III) reactivity and showed strong nucleolar staining. A case with anti-U1/U2RNP (U2RNP dominant) had systemic lupus erythematosus (SLE)-SSc overlap syndrome; however, the remaining two cases had SLE without signs of SSc. All three cases of anti-RNAP III + U1RNP fulfilled ACR SLE criteria but none in the group with anti-RNAP III alone (p = 0.0002). In contrast, only one case in the former group had sclerodermatous skin changes and Raynaud's phenomenon, vs. 92% with scleroderma in the latter (p < 0.05). Although anti-RNAP III is highly specific for SSc, cases with coexisting anti-U1RNP are not so uncommon among anti-RNAP III positives (8%, 3/35) and may be SLE without features of SSc.

Use of monoclonal antibodies for the characterization of novel DNA-binding proteins recognized by human autoimmune sera.

Autoantibodies to a DNA-binding heterodimer consisting of 70,000 and 80,000 dalton subunits were identified in 30-50% of human autoimmune sera from patients with systemic lupus erythematosus (SLE), mixed connective tissue disease (MCTD), and scleroderma. Three murine monoclonal antibodies (mAb) against the heterodimer were produced in BALB/c mice by immunizing with isolated human B cell nuclei. By immunofluorescence, the mAb and autoimmune sera demonstrated both speckled nucleoplasmic staining and diffuse nucleolar staining in all human cell types examined. The nucleoplasmic staining was sensitive to DNase but not RNase pretreatment, while the nucleolar staining was sensitive to RNase but not DNase pretreatment. Biochemical characterization of the 70,000 and 80,000 dalton proteins using the mAb indicated that two forms of the antigen, with different mobilities on sucrose gradients, are present in human B cells. A 10 S form consists of the physically associated 70,000 and 80,000 dalton proteins, while a larger, 10-20 S form probably represents the same two proteins bound to DNA. Binding of the proteins to nucleolar RNA could not be confirmed in biochemical studies. These studies indicate that non-histone, DNA-binding proteins may be more frequently recognized by autoantibodies in SLE, MCTD, and scleroderma than has been previously recognized. Along with previous studies on RNA-binding proteins such as Sm, RNP, Ro, and La, the present findings suggest that nucleic acid-binding proteins, as a class, may be particularly frequent targets of autoimmunity in SLE and related disorders.

Description and partial characterization of a nucleolar RNA-associated autoantigen defined by a human monoclonal antibody.

B lymphocytes from a patient with systemic lupus erythematosus (SLE) and several circulating autoantibodies (including antinucleolar antibodies) were immortalized by fusion with a hypoxanthine/guanine phosphoribosyl transferase (HGPRT)-deficient human B cell line. Multiple human monoclonal antibodies (mAb) were obtained which, in solid-phase enzyme immunoassay, were reactive with DNA. One mAb was of special interest because it reacted strongly with both single-stranded DNA and an extractable nuclear antigen found in rabbit thymus extract (RTE). In an immunofluorescent assay using fixed human cells, the latter mAb also bound predominantly to cell nucleoli. A combination of enzyme digestion and metabolic inhibitor studies of the target cells in this immunofluorescent assay suggested that the antigen(s) bound by the mAb was an RNA-associated protein or a ribonucleoprotein that is distinct from intact RNA polymerase I and not associated with the transcriptional units of the nucleolus. In other experiments, using fractions of RTE isolated by ion-exchange chromatography, the antigens bound by the mAb were shown to be highly negatively charged molecules. Immunoprecipitation and SDS-PAGE analyses of labeled cell extracts bound by the mAb revealed a doublet of 17 and 18 kD. Since the original patient's serum autoantibodies also bound to both an RNase-sensitive, acidic, extractable nuclear antigen and to nucleoli, and immunoprecipitated proteins of similar molecular masses in SDS-PAGE, it appears that the described mAb is a product of an immortalized autoantibody-producing B cell clone from the SLE patient's peripheral blood. This mAb probably defines a novel RNA-associated autoantigen residing predominantly in the nucleolus or, less likely, a variant of either RNA polymerase I or the ribosomal autoantigens (P proteins).

Interaction between anti-DNA and anti-DNA-binding protein autoantibodies in cryoglobulins from sera of patients with systemic lupus erythematosus.

We have previously shown that sera from some patients with SLE and related disorders contain autoantibodies to a DNA-binding protein complex designated p70/p80. The present study shows that anti-p70/p80 autoantibodies are frequently accompanied by anti-DNA antibodies and cryoglobulins. When the cryoglobulins were isolated, they were found to be specifically enriched in both anti-p70/p80 and anti-DNA activities. The anti-p70/p80 and anti-DNA antibodies were found to be distinct populations of autoantibodies rather than a single crossreactive species, since they could be separated from one another by chromatography on DNA-cellulose. Certain human anti-DNA mAbs could inhibit the binding of autoimmune polyclonal anti-p70/p80 antibodies to p70/p80, suggesting that anti-DNA antibodies might also associate with the variable regions of some anti-p70/p80 antibodies in the cryoglobulins. Binding of one murine anti-p70/p80 mAb (111-12) also was inhibited by certain human anti-DNA mAbs, but the binding of another murine mAb (162-11) to a different epitope of p70/p80 was not. These studies suggest that certain anti-DNA antibodies may interact with the variable regions of a population of anti-p70/p80 antibodies. The cryoglobulins found in the sera containing both anti-p70/p80 and anti-DNA antibodies may represent immune complexes consisting, in part, of idiotype and antiidiotype.

Induction of lupus-associated autoantibodies in BALB/c mice by intraperitoneal injection of pristane.

Intraperitoneal injection of pristane (2,6,10,14 tetramethylpentadecane) is a standard technique for obtaining monoclonal antibody-enriched ascitic fluid. However, pristane also induces plasmacytomas and an erosive arthritis resembling rheumatoid arthritis in BALB/c mice, probably as a consequence of enhanced interleukin 6 production. We report here that the production of autoantibodies characteristic of systemic lupus erythematosus (SLE) is a further consequence of injecting pristane in BALB/c mice. Anti-Su antibodies appeared as early as 1-2 mo after a single injection of 0.5 ml pristane, followed by anti-U1RNP and anti-Sm antibodies after 2-4 mo. Within 6 mo of pristane injection, 9 of 11 BALB/c mice had developed anti-Su, anti-U1RNP, anti-U2RNP, anti-Sm, and possibly anti-U5RNP antibodies. Autoantibodies were not produced by 20 BALB/c mice of the same age and sex that were not injected with pristane. Thus, autoantibodies characteristic of lupus were induced in mice that are not usually considered to be genetically susceptible to the disease. The induction of autoantibodies associated with SLE by pristane may be relevant to understanding the role of abnormal cytokine production in autoantibody production and the pathogenesis of autoimmune disease. Furthermore, the induction of high titer autoantibodies by pristane dictates caution in the use of ascitic fluid as a source of monoclonal antibodies, since the polyclonal antibodies induced by pristane may copurify with the monoclonal antibody secreted by an injected hybridoma.

Lamin B autoantibodies in sera of certain patients with systemic lupus erythematosus.

Sera from four patients with systemic lupus erythematosus containing antibodies that yield nuclear rim staining of HEp-2 cells by indirect immunofluorescence were identified and characterized. Each serum contained autoantibodies reacting strongly with lamin B on western blots. One of the four sera displayed weaker reactivity with lamins A and C, while the other three displayed only minimal reactivity with lamins A and C. Titers of antilamin antibodies ranged from 1:1,250 to 1:36,250. Two of the sera also reacted at a dilution of 1:20 with cytoplasmic filaments of PTK-2 cells, suggesting that a small fraction of the autoantibodies in these sera may bind to alpha-helical domains of the lamins that are homologous to those of intermediate filaments. The majority of the antilamin antibodies in these patients' sera are specific for portions of the lamin B molecule that are not homologous to lamins A and C, however. The findings suggest that autoantibodies to the nuclear lamina may, in some instances, be responsible for a rim pattern in the fluorescent antinuclear antibody assay. In addition, autoantibodies to the nuclear lamina in sera of certain patients with systemic lupus erythematosus may be useful for defining the molecular structure and biological functions of lamin B, as well as for studying mechanisms of autoimmunity.

Role of a major autoepitope in forming the DNA binding site of the p70 (Ku) antigen.

The Ku antigen is a heterodimer consisting of 70- and 80-kD protein subunits that binds to termini of double-stranded DNA. DNA binding appears to be mediated partly by the 70-kD (p70) subunit, but the precise mechanism of its association with DNA is unclear. High-titer autoantibodies in sera from certain patients with systemic lupus erythematosus recognize at least eight distinct epitopes of Ku, and inhibit DNA binding. In the present studies, the binding of DNA to truncated p70 fusion proteins was determined in Southwestern blots and DNA immunoprecipitation assays. Appropriate folding of the p70 protein was crucial for efficient DNA binding. The minimal DNA binding site, amino acids 536-609, contains a major conformational autoepitope of p70 (amino acids 560-609). Deletion of amino acids 601-609, or substitution of ala-ala-ala for lys-ser-gly at positions 591-593, eliminated DNA binding as well as autoantibody binding, suggesting that the same secondary or supersecondary structure is involved in both DNA binding and autoantibody recognition. Residues within the DNA binding site/autoepitope closely resemble the helix-turn-helix motif in bacteriophage lambda Cro protein and certain other DNA binding proteins, and mutations predicted to destabilize this structure eliminated DNA binding. Adjacent to the helix-turn-helix is a highly basic domain (positions 539-559) that was also required for DNA binding. The findings suggest that the DNA binding site of p70 consists of a basic domain adjacent to a helix-turn-helix structure that also forms a major autoepitope.

Absence of autoantigen Ku in mature human neutrophils and human promyelocytic leukemia line (HL-60) cells and lymphocytes undergoing apoptosis.

The Ku autoantigen is a heterodimer of 70- and 80-kD proteins recognized by autoantibodies from patients with systemic lupus erythematosus and related diseases that is the DNA-binding component of a DNA-dependent protein kinase. The catalytic activity of DNA-dependent protein kinase is carried by a 350-kD subunit (p350). In light of the recently described role of Ku in repairing double-strand DNA breaks, we investigated the regulation of Ku and p350 levels in neutrophils, a terminally differentiated cell type destined to undergo apoptosis. Since the appearance of double-strand DNA breaks is characteristic of apoptosis, we were interested in the possibility that Ku might oppose programmed cell death. Analysis of peripheral blood cells by flow cytometry using anti-Ku and anti-p350 monoclonal antibodies revealed that neutrophils were unstained, whereas resting (G0) lymphocytes were positive. The absence of Ku in mature neutrophils was confirmed by Western blotting and enzyme-linked immunosorbent assay for Ku antigen. In contrast, the human promyelocytic leukemia line, HL-60, which undergoes differentiation toward neutrophils after dimethylsulfoxide treatment, was positive for Ku and p350. In view of the short lifespan of neutrophils and the prolonged half-life of Ku and p350 (> 5 d), these data suggested that Ku was actively degraded during myeloid differentiation. Analysis of HL-60 cells by flow cytometry revealed that Ku staining was bimodal. Cells in G1/G0, S, or G2/M were all stained positively, whereas cells with a subdiploid DNA content characteristic of apoptosis were Ku negative. Similar results were obtained with phytohemagglutin-stimulated human lymphocytes. These data suggest that the Ku antigen is actively degraded in both myeloid cells destined to undergo apoptosis and apoptotic lymphocytes, raising the possibility that degradation of Ku may help to prevent the inappropriate repair of fragmented nuclear DNA during apoptosis.

Initiation of autoimmunity to the p53 tumor suppressor protein by complexes of p53 and SV40 large T antigen.

Antinuclear antibodies (ANAs) reactive with a limited spectrum of nuclear antigens are characteristic of systemic lupus erythematosus (SLE) and other collagen vascular diseases, and are also associated with certain viral infections. The factors that initiate ANA production and determine ANA specificity are not well understood. In this study, high titer ANAs specific for the p53 tumor suppressor protein were induced in mice immunized with purified complexes of murine p53 and the Simian virus 40 large T antigen (SVT), but not in mice immunized with either protein separately. The autoantibodies to p53 in these mice were primarily of the IgG1 isotype, were not cross-reactive with SVT, and were produced at titers up to 1:25,000, without the appearance of other autoantibodies. The high levels of autoantibodies to p53 in mice immunized with p53/SVT complexes were transient, but low levels of the autoantibodies persisted. The latter may have been maintained by self antigen, since the anti-p53, but not the SVT, response in these mice could be boosted by immunizing with murine p53. Thus, once autoimmunity to p53 was established by immunizing with p53/SVT complexes, it could be maintained without a requirement for SVT. These data may be explained in at least two ways. First, altered antigen processing resulting from the formation of p53/SVT complexes might activate autoreactive T helper cells specific for cryptic epitopes of murine p53, driving anti-p53 autoantibody production. Alternatively, SVT-responsive T cells may provide intermolecular-intrastructural help to B cells specific for murine p53. In a second stage, these activated B cells might themselves process self p53, generating p53-responsive autoreactive T cells. The induction of autoantibodies during the course of an immune response directed against this naturally occurring complex of self and nonself antigens may be relevant to the generation of specific autoantibodies in viral infections, and may also have implications for understanding the pathogenesis of ANAs in SLE. In particular, our results imply that autoimmunity can be initiated by a "hit and run" mechanism in which the binding of a viral antigen to a self protein triggers an immune response that subsequently can be perpetuated by self antigen.

Interleukin 6 dependence of anti-DNA antibody production: evidence for two pathways of autoantibody formation in pristane-induced lupus.

Pristane induces a lupus-like syndrome in nonautoimmune mice characterized by the development of glomerulonephritis and lupus-associated autoantibodies. This is accompanied by overproduction of interleukin (IL)-6, a cytokine linked with autoimmune phenomena. The goal of this study was to evaluate the role of IL-6 in autoantibody production in pristane-induced lupus. BALB/cAn IL-6-deficient (-/-) and -intact (+/+) mice were treated with pristane or phosphate-buffered saline, and autoantibody production was evaluated. Pristane induced high levels of immunoglobulin (Ig)G anti-single-stranded DNA, -double-stranded (ds)DNA, and -chromatin antibodies in IL-6(+/+), but not IL-6(-/-) mice by enzyme-linked immunosorbent assay. High titer IgG anti-dsDNA antibodies also were detected in sera from +/+, but not -/-, mice by Crithidia luciliae kinetoplast staining. The onset of IgG anti-dsDNA antibody production in +/+ mice occurred >5 mo after pristane treatment, well after the onset of nephritis, suggesting that these antibodies are not directly responsible for inducing renal disease. In contrast to anti-DNA, the frequencies of anti-nRNP/Sm and anti-Su antibodies were similar in pristane-treated IL-6(-/-) and IL-6(+/+) mice. However, levels were higher in the +/+ group. These results suggest that IgG anti-DNA and chromatin antibodies in pristane-treated mice are strictly IL-6 dependent, whereas induction of anti-nRNP/Sm and Su autoantibodies is IL-6 independent. The IL-6 dependence of anti-DNA, but not anti-nRNP/Sm, may have implications for understanding the patterns of autoantibody production in lupus. Anti-DNA antibodies are produced transiently, mainly during periods of disease activity, whereas anti-nRNP/Sm antibody levels are relatively insensitive to disease activity. This may reflect the differential IL-6 dependence of the two responses.

Transformation and control of ultra-short pulses in dispersion-engineered photonic crystal fibres.

Photonic crystal fibres (PCFs) offer greatly enhanced design freedom compared to standard optical fibres. For example, they allow precise control of the chromatic dispersion (CD) profile--the frequency dependence of propagation speed--over a broad wavelength range. This permits studies of nonlinear pulse propagation in previously inaccessible parameter regimes. Here we report on spectral broadening of 100-fs pulses in PCFs with anomalously flat CD profiles. Maps of the spectral and spatio-temporal behaviour as a function of power show that dramatic conversion (to both longer and shorter wavelengths) can occur in remarkably short lengths of fibre, depending on the magnitude and shape of the CD profile. Because the PCFs used are single-mode at all wavelengths, the light always emerges in a fundamental guided mode. Excellent agreement is obtained between the experimental results and numerical solutions of the nonlinear wave equation, indicating that the underlying processes can be reliably modelled. These results show how, through appropriate choice of CD, nonlinearities can be efficiently harnessed to generate laser light at new wavelengths.

Assembly of the glomerular filtration surface. Differentiation of anionic sites in glomerular capillaries of newborn rat kidney.

Glomerular development was studied in the newborn rat kidney by electron microscopy and cytochemistry. Glomerular structure at different developmental stages was related to the permeability properties of its components and to the differentiation of anionic sites in the glomerular basement membrane (GBM) and on endothelial and epithelia cell surfaces. Cationic probes (cationized ferritin, ruthenium red, colloidal iron) were used to determine the time of appearance and distribution of anionic sites, and digestion with specific enzymes (neuraminidase, heparinase, chondroitinases, hyaluronidases) was used to determine their nature. Native (anionic) ferritin was used to investigate glomerular permeability. The main findings were: (a) The first endothelial fenestrae (which appear before the GBM is fully assembled) possess transient, negatively charged diaphragms that bind cationized ferritin and are impermeable to native ferritin. (b). Two types of glycosaminoglycan particles can be identified by staining with ruthenium red. Large (30-nm) granules are seen only in the cleft of the S-shaped body at the time of mesenchymal migration into the renal vesicle. They consist of hyaluronic acid and possibly also chondroitin sulfate. Smaller (10-15-nm) particles are seen in the earliest endothelial and epithelial basement membranes (S-shaped body stage), become concentrated in the laminae rarae after fusion of these two membranes to form the GBM, and contain heparan sulfate. They are assumed to be precursors of the heparan sulfate-rich granules present in the mature GBM. (c) Distinctive sialic acid-rich, and sialic acid-poor plasmalemmal domains have been delineated on both the epithelial and endothelial cell surfaces. (d) The appearance of sialoglycoproteins on the epithelial cell surface concides with the development of foot processes and filtration slits. (e) Initially the GBM is loosely organized and quite permeable to native ferritin ;it becomes increasinly impermeable to ferritin as the lamina densa becomes more compact. (f) The number of endothelial fenestrae and open epithelial slits increases as the GBM matures and becomes organized into an effective barrier to the passage of native ferritin.

Role of antigen selectivity in autoimmune responses to the Ku (p70/p80) antigen.

Levels of anti-Ku (p70/p80) antibodies were measured longitudinally in sera from four individuals with systemic lupus erythematosus or related disorders. Antibodies to the native Ku antigen (p70/p80 complex) varied over a range of up to 577-fold. Large fluctuations were also observed in the levels of autoantibodies to several distinct epitopes of the Ku (p70/p80) antigen. Levels of these individual autoantibody populations generally paralleled one another, suggesting that they are coordinately regulated. A similar pattern of anti-DNA antibody fluctuation was seen in some sera. To examine the possibility that these autoantibodies were generated by polyclonal B cell activation, the levels of anti-Ku (p70/p80) and anti-DNA antibodies were compared to the levels of antibodies to Escherichia coli proteins, tetanus toxoid, and bovine insulin, transferrin, cytochrome c, serum albumin, and thyroglobulin. In sera from the same individual, anti-Ku (p70/p80) antibodies were sometimes produced in the complete absence of polyclonal activation, and at other times were accompanied by increased polyclonal activation. Anti-DNA antibody levels more closely paralleled the level of polyclonal activation than did the anti-Ku (p70/p80) levels. These studies suggest that anti-Ku (p70/p80) antibodies are generated by an antigen-selective mechanism, but that polyclonal activation frequently, although not invariably, accompanies autoantibody production. This observation is consistent with the possibility that polyclonal activation might be secondary to autoantibody production.

Autoimmune sera reactive with Sm antigen contain high levels of RNP-like antibodies.

Ribonucleoprotein particles containing Sm antigen were separated from particles containing both Sm and RNP antigens by ion-exchange chromatography to study the recognition of these antigens by autoimmune sera. By using the separated antigens, anti-Sm and/or anti-RNP antibodies were detected in approximately 60% of sera from systemic lupus erythematosus patients by both enzyme-linked immunosorbent assay and immunoprecipitation of radiolabeled antigens followed by analysis on sodium dodecyl sulfate-polyacrylamide gels. These antibodies were detected in 30% of the same sera using the standard passive hemagglutination technique. Competition experiments demonstrated that all of the sera tested that contained anti-Sm antibodies also had anti-RNP-like reactivity. This latter reactivity usually represented 80% or more of the total Sm and RNP binding activity in lupus sera. The binding to RNP-like determinants by several of the sera was uniquely resistant to treatment of the antigen with snake venom exonuclease. These studies indicate that humoral immunity against Sm and RNP antigens in systemic lupus erythematosus is directed primarily against a single type of ribonucleoprotein particle in which the two antigens are physically associated. The specific binding to a single type of ribonucleoprotein particle suggests that this particle may be especially immunogenic and that it might play an important role in induction of the humoral immune response to Sm and RNP.

Distinctive immune response patterns of human and murine autoimmune sera to U1 small nuclear ribonucleoprotein C protein.

The Ul small nuclear ribonucleoprotein (snRNP), a complex of nine proteins with Ul RNA, is a frequent target of autoantibodies in human and murine systemic lupus erythematosus (SLE). Anti-Sm antibodies recognizing the B'/B, D, E, F, and G proteins of Ul snRNPs are highly specific for SLE, and are nearly always accompanied by anti-nRNP antibodies recognizing the Ul snRNP-specific 70K, A, and/or C proteins. Previous studies suggest that human anti-nRNP antibodies recognize primarily the U1-70K and Ul-A proteins, whereas recognition of Ul-C is less frequent. We report here that autoantibodies to U1-C are more common in human autoimmune sera than believed previously. Using a novel immunoprecipitation technique to detect autoantibodies to native Ul-C, 75/78 human sera with anti-nRNP/ Sm antibodies were anti-Ul-C (+). In striking contrast, only 1/65 anti-nRNP/Sm (+) MRL mouse sera of various Igh allotypes was positive. Two of ten anti-nRNP/Sm (+) sera from BALB/c mice with a lupus-like syndrome induced by pristane recognized Ul-C. Thus, lupus in MRL mice was characterized by a markedly lower frequency of anti-U1-C antibodies than seen in human SLE or pristane-induced lupus. The results may indicate different pathways of intermolecular-intrastructural diversification of autoantibody responses to the components of Ul snRNPs in human and murine lupus, possibly mediated by alterations in antigen processing induced by the autoantibodies themselves.

Autoantibodies to RNA polymerase II are common in systemic lupus erythematosus and overlap syndrome. Specific recognition of the phosphorylated (IIO) form by a subset of human sera.

Autoantibodies to RNA polymerases (RNAP) I, II, and III are reported to be highly specific for the diagnosis of scleroderma (systemic sclerosis, SSc). In the present study, the specificity of autoantibodies to RNAP I and III for SSc was confirmed by immunoprecipitation of 35S-labeled proteins. However, we report here the previously unrecognized production of anti-RNAP II autoantibodies by 9-14% of patients with SLE and mixed connective tissue disease/overlap syndrome. 12 out of 32 anti-RNAP II positive sera (group 1) immunoprecipitated a diffuse 220-240-kD band identified as the largest subunit of RNAP II whereas the remaining 20 (group 2) immunoprecipitated preferentially the 240-kD phosphorylated (IIo) form of the large subunit. After pulse labeling, group 1 sera immunoprecipitated only the 220-kD (IIa) RNAP II subunit, whereas the diffuse IIa/IIo band plus the 145-kD second largest RNAP II subunit (IIc) were immunoprecipitated after several hours of cold chase, suggesting that these sera recognized primarily the largest subunit of RNAP II. Group 2 sera recognized the IIc subunit after pulse labeling, and immunoprecipitated the IIc and IIo, but not the IIa, subunits after cold chase. Although it has been suggested that autoantibodies to RNAP II are usually accompanied by anti-RNAP I/III in SSc, all but one of the anti-RNAP II positive sera from SLE or mixed connective tissue disease/overlap syndrome patients, as well as most of the SSc sera, were negative for anti-RNAP I/III. Moreover, in contrast to previous reports suggesting that anti-RNAP antibodies rarely coexist with other SSc subset marker antibodies, anti-RNAP II antibodies were often accompanied by anti-Ku, anti-nRNP, or anti-topoisomerase I autoantibodies in the present study. We conclude that autoantibodies to RNAP II are not a specific marker for SSc, whereas autoantibodies to RNAP I/III are associated primarily with SSc. In addition, we have identified two distinctive patterns of RNAP II antigen recognition by autoantibodies, one of them characterized by specific recognition of the transcriptionally active (phosphorylated) form of RNAP II. The clinical significance of these different patterns remains to be determined.

Acquired congenital heart block. Pattern of maternal antibody response to biochemically defined antigens of the SSA/Ro-SSB/La system in neonatal lupus.

The molecular basis of autoantibody reactivity with components of the SSA/Ro-SSB/La particle exhibited by sera of mothers of infants with severe and permanent manifestations of neonatal lupus (NLE) was investigated using immunoblotting and immunoprecipitation. The characteristics of NLE that were studied included congenital complete heart block (CCHB), second degree heart block, and hepatic fibrosis. Antibodies specific for one or more components of the SSA/Ro-SSB/La particle were found in sera from all 20 mothers of permanently affected infants. However, no antibody specific for a single peptide of this particle was common to all sera. Using tissue extracts from a human cell substrate, 80% of these sera had antibodies to one or more components of the SSA/Ro particle demonstrable by immunoblotting. The predominant antibody response in the NLE group was to the newly recognized 52-kD SSA/Ro peptide component. In contrast, antibodies to the 60-kD SSA/Ro component although present, were the least represented and not significantly increased in frequency among mothers of these infants, compared with a group of 31 mothers with autoimmune diseases such as systemic lupus erythromatosus (SLE) but who had healthy offspring. Antibodies directed to the 48-kD SSB/La antigen were demonstrated in 90% of the NLE mothers often accompanying antibodies against the 52-kD SSA/Ro component. The combination of antibodies to 48- and 52-kD structures was significantly increased in the NLE group, with an odds ratio of 35. The type of cell or tissue substrate was shown to influence detectability of antibodies. The 52-kD SSA/Ro peptide and the 48-kD SSB/La peptide were abundant in cardiac tissues from fetuses aged 18-24 wk, further supporting the possible relevance of these peptides to heart block.

p95vav associates with the nuclear protein Ku-70.

The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed.

Recognition of multiple epitopes in the coiled-coil domain of lamin B by human autoantibodies.

The nuclear lamina of mammalian cells consists of three major proteins, lamins A, B and C, which form a fibrous meshwork interposed between the inner nuclear membrane and the chromatin. Sera from certain patients with systemic lupus erythematosus (SLE) and autoimmune liver disease contain high titers of autoantibodies against lamin B. We have shown previously that anti-lamin B autoantibodies in SLE recognize epitopes highly specific for lamin B, even though lamin B and lamins A/C are highly homologous proteins. To further characterize the specificities of these autoantibodies, fusion proteins carrying fragments of lamins B and C were tested for reactivity with SLE sera by immunoblotting. Five distinct epitopes of lamin B were identified, at least four of which were located in the highly conserved coiled-coil rod domain. Epitopes located on amino acids (AA) 80-193 and 245-303 were recognized by 4/10 and 8/10 anti-lamin B positive sera, respectively. Affinity purified anti-lamin B autoantibodies reacted preferentially with lamin B, indicating that they recognized mainly portions of lamin B that differ from lamins A and C. On the contrary, most of the affinity-purified anti-lamin C autoantibodies from SLE sera cross-reacted with lamin B, suggesting that the anti-nuclear lamina immune response in these patients is directed primarily against lamin B. The preferential reactivity of these sera with multiple epitopes specific to lamin B, and the finding that the autoantibodies to lamins A and C present in some of these sera cross-react with lamin B suggest that autoantibodies to lamin B are generated in response to the authentic lamin B protein rather than a cross-reactive foreign protein.

Role of free p70 (Ku) subunit in posttranslational stabilization of newly synthesized p80 during DNA-dependent protein kinase assembly.

The Ku antigen (p70/p80 heterodimer) is the DNA binding component of a DNA-dependent serine/threonine kinase (DNA-PK), the catalytic activity of which is carried by a 350 kDa polypeptide (p350). In the present studies, the assembly of p70, p80, and p350 was investigated in human K562 (erythroleukemia) cells, and rabbit (RK13) or murine (L-929) cells infected with recombinant vaccinia viruses directing the synthesis of human p70 and p80. Pulse-chase analysis and density gradient centrifugation revealed a pool of free p70 subunits in K562 cells that dimerized within minutes with newly synthesized p80, whereas Ku became associated with newly synthesized p350 1 to 4 h after the onset of p70/p80 heterodimer assembly. A stable pool of free p80 subunits was not detected, and newly synthesized p80 was degraded rapidly (t1/2 < 1.5 h) unless it became incorporated into a p70/p80 dimer. The explanation for the absence of unassembled p80 subunits in K562 cells was investigated further by expressing human p70 and p80 individually or together in Ku-deficient RK13 or L-929 cells infected with recombinant vaccinia viruses p70-vacc and/or p80-vacc. As in uninfected K562 cells, the t1/2 of the free recombinant human p80 subunit expressed in RK13 cells was < 1.5 h unless it was "rescued" by dimerization with p70. The t1/2 of human p70 as well as p70/p80 heterodimers was > 16 h in RK13 cells.(ABSTRACT TRUNCATED AT 250 WORDS)