Coronary vein leads for cardiac pacing in patients with tricuspid valve replacement.

BACKGROUND: Because after tricuspid valve replacement (TVR) the transvenous implantation of endocardial leads is contraindicated, myocardial screw-in leads were used to ventricular pacing. Recently available coronary vein (CV) leads are stimulating the left ventricle epicardially and can be implanted transvenously, too. METHOD AND RESULTS: We implanted these leads in patients (pts) with TVR (n = 7) or after valve repair (n = 1) without complications. In 7 pts we used bended CV leads with a microporous tip and only in one pt a CV lead with a stimulation via metal ring. The stimulation thresholds (ST) were stable in all pts. CONCLUSIONS: The use of CV leads offers a minimal invasive approach for permanent cardiac stimulation after TVR. Low chronic ST are resulting in an energy saving pacemaker mode. CV leads can be used after previous heart surgery as well as for difficult anatomical situations.

Splice variants of the nuclear dot-associated Sp100 protein contain homologies to HMG-1 and a human nuclear phosphoprotein-box motif.

Sp100 and PML are interferon-inducible proteins associated with a new class of nuclear domains (known as nuclear dots or PML bodies) which play a role in tumorigenesis, virus infections, and autoimmunity. While PML is extensively alternatively spliced, only two splice variants are known for Sp100. Here we describe the identification and characterization of several Sp100 splice variant proteins and support their existence by elucidation of the 3'-end of the Sp100 gene. Some of the splice variants contain a domain of significant sequence similarity with two previously described highly related interferon-inducible nuclear phosphoproteins as well as to suppressin and DEAF-1, which altogether define a novel protein motif, termed HNPP-box. One class of splice variants contains an almost complete and highly conserved copy of the DNA-binding high mobility group 1 protein sequence and thus represent novel HMG-box proteins. When expressed transiently, both major classes of Sp100 splice variant proteins localize in part to nuclear dots/PML bodies and in addition to different nuclear domains. Furthermore, PML was occasionally redistributed. These data indicate that alternatively spliced Sp100 proteins are expressed, differ in part in localization from Sp100, and might bind to chromatin via the HMG domain.

Autoantibodies against "nuclear dots" in primary biliary cirrhosis.

Autoantibodies against nuclear proteins are not always but rather frequently present in sera of patients with primary biliary cirrhosis (PBC). The specificity and diagnostic value of these autoantibodies for PBC have only recently become clear through cloning of the cDNA of some of the corresponding autoantigens which allowed the establishment of immunological assays with recombinant autoantigens expressed in E. coli and eukaryotic cells. In this report we summarize primarily the knowledge on the structure and putative function of two nuclear autoantigens, the Sp100 and PML proteins, which are present in so-called nuclear dots (NDs) and against which autoantibodies are present in a subpopulation of PBC patients. Furthermore, the type of autoimmune response (epitope specificity and immunoglobulin class) against both the Sp100 and PML proteins and the specificity of the anti-Sp100 and anti-PML autoantibodies for PBC patients and patients with other autoimmune diseases is reviewed. Current knowledge clearly indicates that determination of anti-Sp100 and anti-PML autoantibodies substantially improves diagnosis of PBC as these autoantibodies are highly specific for this disease even when autoantibodies against mitochondrial antigens, a hallmark of most PBC patients, are not found. The type of autoimmune response against the Sp100 and PML proteins also provides some clues about possible mechanisms which lead to autoantigenicity of both proteins.

A highly amplified mouse gene is homologous to the human interferon-responsive Sp100 gene encoding an autoantigen associated with nuclear dots.

In human cells, three proteins are currently known to colocalize in di screte nuclear domains (designated nuclear dots): Sp100, a transcription-activating protein autoantigenic primarily in patients with primary biliary cirrhosis; PML, a tumor suppressor protein involved in development of acute promyelocytic leukemia; and NDP52, a protein of unknown function. Here we report sequence similarities between the Sp100 protein and a putative protein encoded by a highly amplified mouse gene which is visible as an inherited homogeneously staining region (HSR) on chromosome 1 of some mouse populations. By in situ hybridization, the Sp100 gene was mapped to locus 2q37, the syntenic region of the HSR on mouse chromosome 1. Unlike the highly amplified mouse gene, Sp100 was found to be a single-copy gene and showed no restriction fragment length polymorphisms. Sequence similarities in the promoter regions and similar exon-intron organizations of the two genes were revealed. As for Sp100, steady-state levels of the mRNAs of the HSR-encoded genes could be greatly increased by interferon (IFN) treatment. As in human cells, IFN treatment led to an enlargement in both size and number of nuclear dots in mouse cells as visualized by immunofluorescence staining with autoimmune sera from patients with primary biliary cirrhosis. These data indicate that a gene located in the inherited HSR of mice, designated mSp100, is homologous to the human Sp100 gene, has a similar gene organization, and responds similarly to IFN treatment.

Two nuclear dot-associated proteins, PML and Sp100, are often co-autoimmunogenic in patients with primary biliary cirrhosis.

The nucleoproteins Sp100 and PML, the first an autoantigen predominant in patients with primary biliary cirrhosis (PBC) and the second a transformation and cell growth suppressing protein aberrantly expressed in promyelocytic leukaemia cells, were recently shown to colocalize in dot-like nuclear domains. Here we analysed whether PML, like Sp100, is also an autoantigen in patients with PBC and other autoimmune diseases, and wether both proteins interact directly. Testing sera from autoimmune patients using an immunoprecipitation assay with radiolabelled PML and an immunofluorescence assay based on a cell line overexpressing PML, autoantibodies (Aabs) against PML were found in the majority o anti-Sp100 Aab positive patients. Only very few patients with PBC or other autoimmune diseases contained anti-PML or anti-Sp100 Aabs exclusively. In contrast to Sp100, immunoreactivity of recombinant PML in immunoblots was only weak and was directed to one region. This suggests that anti-PML Aabs recognize fewer and preferentially conformation-dependent epitopes. In an immunoprecipitation assay using in vitro synthesized Sp100 and PML proteins and Abs to recombinant proteins, no direct interaction was observed. Taken together, these data indicate that Aabs against PML are as highly prevalent and specific for patients with PBC as those against Sp100. The colocalization of these autoantigens and the frequent co-occurrence of the corresponding Aabs might reflect an association of both proteins mediated by one or several other proteins.

Modification of discrete nuclear domains induced by herpes simplex virus type 1 immediate early gene 1 product (ICP0).

The outcome of herpes simplex virus type 1 (HSV-1) infection depends upon the interplay of both host and viral factors. During lytic infection, HSV-1 causes a loss of immunofluorescent staining of discrete nuclear domains (ND10). This elimination of the host's ND10 staining occurs under conditions that allow only HSV-1 immediate early viral gene expression. Western blot analysis indicates that the loss of ND10 staining is due to ND10 redistribution, rather than protein degradation or turnover. When deletion mutants of all of the HSV-1 immediate early genes were tested, only infection with an immediate early gene 1 product (ICP0) deletion mutant, d11403, was unable to eliminate ND10 antigen staining. Also, ICP0 transiently colocalized with ND10 antigens, after which ND10 antigens became undetectable. At late times during infection with d11403, the host ND10 antigens were retained in virus-induced structures which were never observed during wild-type HSV-1 infection. These results suggested that ICP0 may be directly involved in the modification of the host nuclear domain. Infection with an adenovirus recombinant that expressed ICP0 demonstrated that in the absence of other HSV-1 proteins ICP0 was sufficient for the change in nuclear distribution of host antigens located at ND10. We postulate that the trans-activation function of ICP0 during viral replication may be mediated by replacing, modifying or reorganizing nuclear host factors.

IFN enhance expression of Sp100, an autoantigen in primary biliary cirrhosis.

About 30% of patients suffering from the chronic autoimmune liver disease primary biliary cirrhosis produce autoantibodies against Sp100, a protein migrating in SDS-PAGE at a position corresponding to 100 kDa and located on discrete dot-shaped nuclear structures. The human Sp100 cDNA has recently been cloned and the deduced amino acid sequence was found to contain similarities to several transcriptional regulatory proteins; the biologic function of the Sp100 protein, however, is still unknown. In this study we present data which show that infection of HEp2 cells with influenza A virus, transformation of glial cells with SV40 DNA, and stimulation of PBL with mitogens affect the expression of the Sp100 autoantigen. These observations prompted us to investigate whether expression of the Sp100 protein is modulated by the action of IFN. Immunofluorescence staining of HEp2 and HeLa cells grown in the presence of IFN-alpha, IFN-beta, or IFN-gamma revealed an increase both in size and number of the Sp100 protein-containing nuclear dots, whereas no such effect was observed with cells treated with TNF-alpha. As measured by an immunoblot-based ELISA the amount of Sp100 protein in INF-beta-treated cells (1000 IU/ml, 18 h) was eight to nine times higher than in untreated cells. The enhanced protein expression was accompanied by an accumulation of the Sp100-specific mRNA (13-fold increase of the normal level after 10 h of INF-beta treatment of HEp2 cells). These findings characterize the Sp100 protein as a new member of IFN-modulated proteins and raise the question whether cytokine-mediated increase of Sp100 protein expression plays a role in induction of anti-Sp100 autoantibodies.

Autoantibodies to the nuclear Sp100 protein in primary biliary cirrhosis and associated diseases: epitope specificity and immunoglobulin class distribution.

Sp100, a protein with a dot-like intranuclear localization in immunofluorescence microscopy, is a major target for patient autoantibodies in primary biliary cirrhosis (PBC) and occasionally in rheumatic disorders. The human Sp100 cDNA has recently been cloned, and the deduced amino acid sequence was found to contain sequence similarities with an MHC class I domain and several transacting regulatory proteins, including HIV-1 nef proteins. In this study, recombinant Sp100 fusion proteins were used to differentiate the immunoglobulin isotypes and to map the epitopes involved in the anti-Sp100 autoimmune response. PBC patients developed IgG as well as IgM and/or IgA class anti-Sp100 autoantibodies whereas most patients with rheumatic diseases developed IgG class autoantibodies only. For epitope mapping, truncated versions of the Sp100 protein were probed for immunoreactivity in ELISA and immunoblotting. With 55 sera, 17 different reaction patterns were obtained, and at least three non-overlapping major autoantigenic domains were recognized by the majority of sera. One domain, which contains the sequence similarity with HIV nef proteins, was recognized by all anti-Sp100 sera and harbours multiple, in part discontinuous, epitopes. These data demonstrate a heterogeneous and patient-specific anti-Sp100 autoimmune response which is antigen-driven and, at least in terms of isotype composition, different in PBC and non-PBC patients.

Repetitive P68-autoantigen specific epitopes recognized by human anti-(U1) small nuclear ribonucleoprotein autoantibodies.

The major target of anti-(U1)snRNP autoantibodies, a serological marker of patients with mixed connective tissue disease and related rheumatic disorders, is a 68 kDa protein (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein particles. With recombinant p68 fusion proteins, multiple autoepitopes have been identified, and one of these has been mapped to the pentamer sequence ERKRR, which is located within antigenic domain A in the amino-terminal half of p68. The lysine residue (K) of this epitope can be replaced by isoleucine without loss of autoantibody binding. Here we have investigated whether other variants of this epitope are present on the p68 autoantigen and if these are recognized by anti-p68 autoantibodies. We identified four related motifs in the carboxy-terminal half of the p68-protein, and three of these (all containing glutamic acid instead of lysine (ERERR] mapped to the previously characterized autoantigenic domains C and D. Immunoreaction of anti-ERKRR autoantibodies, affinity-purified from domain A with recombinant fusion proteins containing either domain C or domain D of p68, revealed that anti-ERKRR autoantibodies cross-react with the ERERR-motifs. This finding, which was confirmed by competitive inhibition-ELISA with solid-phase coupled domain A-, C- and D-fusion proteins and ERKRR-containing synthetic peptides as competitors, suggests that a subset of patient autoantibodies is directed against repetitive structures on a single snRNP component.

Isolation and characterization of cDNA encoding a human nuclear antigen predominantly recognized by autoantibodies from patients with primary biliary cirrhosis.

Autoantibodies to a novel nuclear Ag, Sp100, have recently been described that recognize a nuclear protein with an apparent molecular mass of 95 to 100 kDa and a dot-like distribution within cell nuclei. By immunoscreening of a lambda gt11 cDNA expression library derived from HeLa cells with an anti-Sp100 autoimmune serum a 0.7-kb cDNA (Sp26) coding for a fragment of Sp100 was isolated. Expression of this cDNA and use of the recombinant protein in ELISA revealed that the fragment carries major Sp100 autoepitopes and that anti-Sp100 autoantibodies predominantly occur in patients suffering from primary biliary cirrhosis (50/184). The Sp26 cDNA was used as hybridization probe for isolation of longer cDNA from human liver- and placenta-derived lambda gt10 cDNA libraries. Overlapping fragments were assembled to generate a full length cDNA coding for a protein with a molecular mass of 53 kDa and an isoelectric point of 4.7. The Sp100 autoantigen expressed in vitro from this cDNA and authenticated by a capture immunoblot assay, comigrated in SDS-PAGE with the authentic HeLa autoantigen of 95 to 100 kDa and thus showed an aberrant electrophoretic mobility. Computer based protein sequence analysis of the Sp100 autoantigen revealed regions of striking sequence similarities to the alpha 1 and alpha 2 domains of various human and non-human MHC class I Ag and to several transacting transcriptional regulatory proteins.

Major autoantigenic sites of the (U1) small nuclear ribonucleoprotein-specific 68-kDa protein.

A 68-kDa protein associated with (U1)snRNP is a major target for human autoantibodies to small ribonucleoprotein particles (snRNP) prevalent in a variety of inflammatory rheumatic diseases. The epitopes recognized by these antibodies were mapped by expression of subfragments of p68 cDNA in Escherichia coli and testing of the corresponding recombinant proteins for immunoreactivity with sera of patients with autoimmune diseases. Three of four antigenic regions were analysed in detail. The immunodominant autoantigenic region was found to coincide with the RNA-binding domain of the p68 protein and was shown to contain a nested set of overlapping discontinuous epitopes. Two additional non-overlapping major antigenic domains were localized in the carboxy-terminal half of the p68 protein. Each of these two carboxy-terminal domains was shown to contain more than one conformation-dependent epitope. Taking into account previous mapping studies, the data demonstrate that p68 contains at least four antigenic regions, each of which harbours multiple epitopes which are recognized in a patient-specific manner.

Human anti-p68 autoantibodies recognize a common epitope of U1 RNA containing small nuclear ribonucleoprotein and influenza B virus.

Autoantibodies from patients with systemic rheumatic diseases were used to map antigenic sites on the 68-kD autoantigen (p68) associated with (U1)RNA-containing small nuclear ribonucleoprotein (snRNP) particles. With truncated recombinant fusion proteins and synthetic peptides, a subset of anti-p68 autoantibodies was found to recognize the amino acid sequence motif Glu-Arg-Lys-Arg-Arg (ERKRR). To investigate the possible involvement of epitopes shared by microbial antigens and host self-components in initiation of autoimmunity (molecular mimicry), a sequence data bank was screened for proteins containing an amino acid motif identical or related to ERKRR. The identical motif was found on the M1 matrix protein of influenza B viruses, and affinity-purified human anti-ERKRR autoantibodies recognized this epitope also in the viral amino acid sequence context. The common epitope recognized by human autoantibodies suggests that influenza B virus infection may play a role in initiation of the anti-p68 and anti-(U1)RNP autoimmune response.

Epitope mapping with a recombinant human 68-kDa (U1) ribonucleoprotein antigen reveals heterogeneous autoantibody profiles in human autoimmune sera.

Several cDNA fragments encoding parts of the (U1)RNP specific 68-kDa autoantigen were expressed in Escherichia coli and the fusion proteins were used as substrate for localization of the autoreactive epitopes. We have identified a region of approximately 30 amino acids reacting with more than 90% (16 of 17) of all human anti-p68 sera tested, regions which carry only a few and a region with no autoepitopes. Comparative analysis of epitopes recognized on partially degraded fusion proteins indicated that the anti-p68 autoimmune response is polyclonal. It involves generation of antibodies to several epitopes including one in a region with retroviral gag protein homology speculated to play a role in the initiation of the autoimmune response. Each of the 17 sera tested contained a different set of autoantibody specificities. These data are not consistent with random mutation as a sole mechanism of anti-p68 autoantibody induction and argue for an Ag-driven autoimmune response.

A recombinant autoantigen derived from the human (U1) small nuclear RNP-specific 68-kd protein. Expression in Escherichia coli and serodiagnostic application.

A human liver complementary DNA expression library was screened using sera from patients with high titers of autoantibodies, to search for clones expressing major autoantigens that are relevant in connective tissue diseases. One of the clones isolated expressed a major epitope(s) that was immunoreactive with anti-U1 RNP sera, as shown by several techniques. Affinity-purified autoantibodies from the cloned RNP protein specifically recognized the 68-kd U1 RNP protein of HeLa cell nuclear extracts. All sera containing anti-U1 RNP antibodies detected by immunodiffusion, counterimmunoelectrophoresis, or immunoblotting also recognized the cloned RNP protein. The RNP antigen-expressing bacterial colonies and the partially purified cloned RNP fusion protein have been applied to fast and sensitive immunologic assays for the detection and quantification of anti-U1 RNP antibodies.

[Nuclear antibodies as serologic markers in progressive systemic scleroderma]. / Kernantikörper als serologische Marker der progressiv systemischen Sklerodermie (PSS).

In all, 36 patients with progressive systemic sclerosis (29 women, 7 men) were studied clinically and immunologically; 15 patients had acrosclerosis (type I) and 21, sclerosis extending beyond the wrist (type II). The sera of all patients were evaluated for ANA (HEp-2-cells), Scl-70, centromere and other ENA antibodies. The centromere antigen was characterized by immunoblotting. All patients had high-titer ANA antibodies (100%); 36% of patients had the Scl-70 antibody (a marker antibody for PSS); and in 22% of our patients a centromere antibody was detected. In all cases the anti-centromere sera reacted with a 19.5-kd polypeptide and in 2 cases they reacted with 23- and 25.5-kd proteins in addition. In patients with centromere antibodies there was increased organ involvement (heart, lung, kidney) compared with patients who had anti-Scl-70 or other nuclear antibodies.

Anti-(U1)RNP and anti-Sm autoantibody profiles in patients with systemic rheumatic diseases: differential detection of immunoglobulin G and M by immunoblotting.

Autoimmune sera from patients with systemic lupus erythematosus, scleroderma, or both disorders reactive with the (U1)RNP and Sm antigens were analyzed according to their IgG- and IgM-autoantibody profiles by immunoblotting with HeLa nuclear extracts. Anti-(U1)RNP-specific autoantibodies directed against the 68-kDa polypeptide were found to be predominantly of the IgG type, whereas for the other (U1)RNP-specific protein, 33 kDa, a concomitant occurrence of IgG and IgM class autoantibodies was observed for most patients. In contrast, Sm-specific anti-29/28-kDa autoantibodies were found to be more frequently of the IgM than of the IgG type, while Sm-specific anti-16-kDa antibodies of both classes were present simultaneously in most sera. Of the serum collection reported here only one (U1)RNP-specific serum has been found which lacks anti-Sm antibodies of either class. In general, preclassification of sera by immunodiffusion and counterimmunoelectrophoresis corresponds to the IgG but not to the IgM profile as determined by immunoblotting.

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I.

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.

Human anti-centromere sera recognise a 19.5 kD non-histone chromosomal protein from HeLa cells.

Autoimmune sera from 18 scleroderma patients were found to give a centromere positive immunofluorescence response on formaldehyde fixed HeLa cells and on chromosome spreads. Immunoblotting experiments with a protein fraction enriched in HeLa chromosomal proteins revealed that the antigenic target common to all 18 sera is a polypeptide of 19.5 kD. This polypeptide, which is not one of the core histones, is not soluble under conditions which favour the release of nuclear ribonucleoprotein particles. Antigen specific purification of autoantibodies with subsequent immunofluorescence studies confirmed that the 19.5 kD antigen is restricted to cell cycle-dependent single or double spheres at the centromere of HeLa chromosomes. Two additional polypeptides of 23 kD and 25.5 kD immunoreactive with five of the 18 centromere positive sera are not located at the centromere region, suggesting that other autoantibody systems are present in these sera.