Ingvar Pettersson

Enzyme-linked immunosorbent assay using isolated (U) small nuclear ribonucleoprotein polypeptides as antigens to investigate the clinical significance of autoantibodies to these polypeptides

In order to investigate the clinical significance of autoantibodies to individual U small nuclear ribonucleoprotein (snRNP) polypeptides, an enzyme-linked immunosorbent assay (ELISA) using isolated 68K, A, B/B, and D polypeptides from purified U1 snRNP was developed. The ELISA levels of IgG antibodies were positively correlated with results of immunoblotting and hemagglutination. In patients positive for antibodies to ribonucleoprotein, IgG anti-68K reactivity was associated with active mixed connective tissue disease, and in particular with myositis and esophageal hypomotility. IgG B/B and D polypeptide reactivities were associated with systemic lupus erythematosus and renal disorder. Raynauds phenomenon was infrequent in patients with high IgG B/B and D polypeptide reactivities. Pleuritis/pericarditis was associated with the IgG B/B polypeptide reactivities. In longitudinal studies, ELISA levels of IgG antibodies against these polypeptides changed in parallel with disease activity.

Identification of antigenic regions of the human Ro 60 kDa protein using recombinant antigen and synthetic peptides

Autoantibodies reacting with the human Ro 60 kDa protein are present in anti-Ro/SS-A positive sera from patients with several different connective tissue diseases including Sjögrens syndrome and systemic lupus erythematosus. To investigate the humoral immune response to this protein, the pattern of antibody recognition of recombinant Ro 60 kDa proteins encoded by both full-length and deletion clones was analysed by immunoblotting. An antigenic region recognized by nearly all anti-Ro 60 kDa positive sera was found to reside in the middle part of the protein. In addition, some sera reacted with two other antigenic regions located in the amino-terminal and carboxyl-terminal part of the protein. For further mapping, overlapping peptides covering the most frequently recognized region of the protein were synthesized by solid-phase methods and used as antigens in ELISA. Three major patterns of reactivity to Ro 60 kDa peptides were found. These results not only indicate the presence of an immunodominant region but also heterogeneity in the autoimmune human response to the Ro 60 kDa antigen.

Recombinant 70-kD protein used for determination of autoantigenic epitopes recognized by anti-RNP sera.

A panel of high‐ and low‐titre anti‐RNP‐positive patient sera was tested for reactivity with human snRNP proteins. The U1 snRNP‐specific 70‐kD peptide was found to be the most prominent RNP antigen recognized by high‐titre anti‐RNP sera, mainly found in patients with mixed connective tissue disease (MCTD). The reactivity with the 70‐kD protein was further analysed with recombinant fusion proteins containing different segments of the protein. One major and two minor antigenic regions were found. Most patient sera, both with high‐ and low‐titre anti‐RNP antibodies, only recognized the major region. The most aminoterminal region, showing partial sequence homology with a mouse retroviral p30 gag protein, contained an epitope that was recognized by one serum only.

Computer analysis of the distribution of nuclear antigens: studies on the spatial and functional organization of the interphase nucleus.

The higher-order organization of DNA in chromosomes, and the degree of ordering of chromosomes in interphase chromatin and during mitosis have not yet been elucidated. The problems have, however, been debated in the cytogenetic literature since the early observations of Rabl (1885) and Boveri (1909) (for reviews, see Avivi & Feldman, 1980; Bennett, 1984). Using plant cells, Rabl noted that during anaphase the centromeres are the leading part and telomeres the trailing ends, as daughter chromosomes migrate away from each other. He also made observations on postmitotic cells, suggesting that this chromosome orientation was maintained during interphase with centromeres clustered at one pole of the nucleus and telomeres at the opposite pole in the newly formed nuclei (the ‘Rabl orientation’). This chromosome arrangement has also been observed in other plant cells (Ashley, 1979; for a review, see Avivi & Feldman, 1980) and in insect cells (Foe & Alberts, 1985). The situation in animal cells, however, is far from clear (for a review, see Comings, 1980). In recent years questions concerning the topology of chromosomes in interphase nuclei have been studied by laser-beam microirradiation techniques (Cremer et al. 1982a,b), optical sectioning and three-dimensional reconstruction of micrographs (Agard & Sedat, 1983), by in situ hybridization (Rappold et al. 1984), by analysing prematurely condensed chromosomes in fused cells (Sperling & Luedke, 1981), by statistical analysis of the distribution of specific chromosomes in metaphase spreads (Heneen & Nichols, 1972), and by studies of the frequency of chromatid and chromosome aberrations (Hager, Schroeder-Kurth & Vogel, 1982). The evidence accumulated to date suggests that chromosomes are non-randomly distributed both in interphase nuclei and during mitosis (for reviews, see Comings, 1980; Cook & Laskey, 1984). In plant cells and in certain types of insect cells, e.g. in salivary gland cells of Drosophila, homologous chromosomes are closely associated in somatic cells.

Antigenic domains on the U1 small nuclear ribonucleoprotein-associated 70K polypeptide: A comparison of regions selectively recognized by human and mouse autoantibodies and by monoclonal antibodies

Antigenic regions on the U1 small nuclear ribonucleoprotein (snRNP)-associated 70K polypeptide recognized by human and mouse autoantibodies or by monoclonal antibodies were identified and compared. Using a set of 70K fusion proteins as antigen in enzyme-linked immunosorbent assay and immunoblotting revealed that serum autoantibodies of human and of MRL/Mp mouse origin recognized a common region of the 70K polypeptide. Monoclonal anti-70K antibodies derived from a patient with mixed connective tissue disease, from an autoimmune MRL/Mp mouse, and from a BALB/c mouse immunized with purified U1 snRNP were all shown to bind to a part of the 70K polypeptide rich in charged residues and different from the region recognized by most human and MRL/Mp mouse serum autoantibodies.

Methods of epitope mapping

Concluding remarksIt is important to remember that the merits of the different approaches to epitope mapping should be judged against the purpose of the study. A peptide specifically recognized by nearly all sera containing a certain autoimmune specificity [20, 21, 26], would most likely be selected for the detection of the anti-linear/continuous epitope fraction among those autoantibodies and could be highly useful for diagnostic purposes. This is true even if a majority of the antibodies were directed against conformational/discontinuous protein epitopes. If the purpose is to study the induction or maintenance of the autoimmune response, one would also have to look at and account for the conformation-dependent autoantibodies. There is also the possibility that some pathogenic autoantibodies could constitute a small fraction requiring the complete nucleic acid-protein complex as an antigen. In that case, the ‘pathogenic’ epitope would not be identified nor mapped using the techniques discussed in this review.

Intracellular localisation of the Ro 52 kD auto-antigen in HeLa cells visualised with green fluorescent protein chimeras

Autoantibodies to the Ro/SSA and La/SSB antigens are found in patients with Sjogrens syndrome and systemic lupus erythematosus. The Ro/SSA autoantigen consists of a 52 kD and a 60 kD protein, complexed with one of four small RNA molecules. The La protein can associate with the complex. The Ro/SSA autoantigens are present in all mammalian cells, but their intracellular location is subject of controversy and their function remains unclear. To study the intracellular sorting and targeting of Ro 52 kD we have constructed expression plasmids encoding fusion proteins between the full-length Ro 52 kD protein as well as Ro 52 kD fragments and the green fluorescent protein (GFP) from the jelly fish, Aequorea Victoria. The subcellular distribution of the GFP-Ro 52 kD fusion proteins was investigated in transient expression experiments using transfected HeLa cells. The GFP-full-length Ro 52 kD fusion protein was accumulated in the cytoplasm and excluded from the nucleus. When GFP was fused with the La protein, the fluorescence was located in the nucleus. Clones coding for Ro 52 kD fragments containing the hydrophilic central part of the Ro 52 kD protein gave the same intracellular location and type of cytoplasmic speckles as the full-length Ro 52 kD protein. In contrast, both amino terminal and carboxy terminal fragments were uniformly distributed throughout the cell just like the GFP protein itself. These observations indicated a cytoplasmic location of the Ro 52 kD protein and demonstrated the crucial role of the hydrophilic domain in restricting the Ro 52 kD protein to this intracellular compartment.

Demonstration of an amino terminal La epitope recognized by human anti-La sera

In order to study the antigenic properties of the La protein we have isolated a 1650 base pair (bp)-long human cDNA encoding an anti-La reactive protein. Restriction enzyme analysis and DNA sequencing was used to compare this clone with two published but inconsistent partial sequences. Our clone extends about 220 bp further towards the 5 end than the two clones previously studied and includes a putative initiation codon. When introduced into an expression vector, stable fusion proteins were made both from the initial clone and from two deletion clones. The recombinant proteins were tested by immunoblotting against a panel of anti-La sera. All reacted with the fusion protein produced by the 1650-bp clone. About half of the anti-La sera showed reactivity against the recombinant protein from the shortest deletion clone. This indicates the presence of an epitope in the amino terminal part of the La protein, encoded by sequences not present in previously published clones.

Ro 52kD autoantibodies are detected in a subset of ANA-negative sera.

To define Ro 52kD, Ro 60kD, and La specificities of autoantibodies within ANA-negative sera, samples from 64 ANA-negative but SSA positive patients undergoing investigation due to suspected CTD were analysed, using recombinant antigens and synthetic peptides by immunoblotting and ELISA. The sera were selected from 4025 sera submitted for routine ANA analysis. Antibodies to Ro or La were detected in 42/64 sera (65%). Anti-Ro 52kD antibodies occurred most frequently and were present in 42/64 sera (65%). This was the only specificity of autoantibody detected in 18 sera. No patient had only anti-La or anti-Ro 60 antibodies. In total 18.64 patients (28%) had Ro 60 antibodies and 14/64 had anti-La antibodies (21%). Eight patients had antibodies reacting with all three antigens. We used the same set of sera to test the antigenicity of different regions of Ro 52kD represented by deletion clones and peptides derived from the Ro 52kD sequence. Out of 30 sera reacting with a recombinant deletion clone encompassing as residues 136-227, 12 sera reacted with a peptide corresponding to a 200-239. Some sera gave a low positive OD value with a peptide of a 176-196. Based on the results of this study in which we demonstrate Ro 52kD autoantibodies in 65% of selected ANA negative sera and define an autocephitope within the Ro 52kD protein composed of the leucine zipper domain, we suggest that testing for Ro 52kD antibodies could be included in an extended investigation of ANA negative patients with suspected connective tissue disease.To define Ro 52kD, Ro 60 kD, and La specificities of autoantibodies within ANA-negative sera, samples from 64 ANA-negative but SSA positive patients undergoing investigation due to suspected CTD were analysed, using recombinant antigens and synthetic peptides by immunoblotting and ELISA. The sera were selected from 4025 sera submitted for routine ANA analysis. Antibodies to Ro or La were detected in 42/64 sera (65%). Anti-Ro 52kD antibodies occurred most frequently and were present in 42/64 sera (65%). This was the only specificity of autoantibody detected in 18 sera. No patient had only anti-La or anti-Ro 60 antibodies. In total 18.64 patients (28%) had Ro 60 antibodies and 14/64 had anti-La antibodies (21%). Eight patients had antibodies reacting with all three antigens. We used the same set of sera to test the antigenicity of different regions of Ro 52kD represented by deletion clones and peptides derived from the Ro 52kD sequence. Out of 30 sera reacting with a recombinant deletion clone encompassing as residues 136-227, 12 sera reacted with a peptide corresponding to a 200-239. Some sera gave a low positive OD value with a peptide of a 176-196. Based on the results of this study in which we demonstrate Ro 52kD autoantibodies in 65% of selected ANA negative sera and define an autocephitope within the Ro 52kD protein composed of the leucine zipper domain, we suggest that testing for Ro 52kD antibodies could be included in an extended investigation of ANA negative patients with suspected connective tissue disease.

IgM and IgG Subclass Distribution of Human Anti‐Ro/SSA 60 kDa Autoantibodies

The Ro/SSA and La/SSB antigens are common targets for autoantibodies found in the sera of patients with Sjögrens syndrome and SLE. The anti‐Ro/SSA and anti‐La/SSB antibodies often appear together but are not cross‐reactive. This paper describes the humoral autoimmune response to the Ro/SSA 60 kDa protein moiety with respect to the presence of IgM and IgG1‐4 antibodies. IgM antibodies to the Ro 60 kDa protein coexisted with IgG anti‐Ro 60 kDa antibodies in nearly half of the sera. A similar fraction also contained IgM anti‐La/SSB antibodies. The frequency of sera with IgM antibodies of both specificities was that expected from random overlap.