Metastatic malignant melanoma arising in a mature ovarian cystic teratoma: a case report and literature review.

Primary malignant melanoma arising inform the ovary is rare, with only 30 cases described in the literature to date. The case reported here occurred in a 19-year-old woman and was rapidly progressive, resulting in death only 37 days following initial presentation. Management of this case is discussed in the context of the other reported cases. Surgery, ranging from an ovarian cystectomy to a radical debulking procedure, has been the main treatment with adjuvant chemotherapy utilized in only 4 of the previous cases. Key problems in management relate to the ability to make the diagnosis at the time of surgery and the overall poor response rates of melanoma to adjuvant chemotherapy.

Human autoantibodies directed against the RNA recognition motif of La (SS-B) bind to a conformational epitope present on the intact La (SS-B)/Ro (SS-A) ribonucleoprotein particle.

In systemic autoimmunity, the human B cell response to the La (SS-B) autoantigen is polyclonal and directed to both conserved and human-specific epitopes. This study has further characterized the B cell epitope(s) present within the conserved central region of the La protein, LaC (amino acids 111-242) containing the RNA recognition motif (RRM, aa 111-187). Ten overlapping and non-overlapping protein fragments spanning LaC were expressed in bacteria as NH2-terminal fusions with glutathione-S-transferase. The fusion proteins were tested by ELISA for reactivity with a panel of human anti-La sera in order to define the nature of the epitopes. Ninety-two percent of patient sera containing anti-La antibodies reacted with the region of La containing the RRM. Fine mapping of this reactivity using deletion mutants indicated that the deletion of 19 amino acids from either the NH2-terminal or COOH-terminal region of the RRM was associated with loss of antibody reactivity, suggesting that the immunodominant epitope expressed in this region is discontinuous. Autoantibodies affinity-purified from the La RRM fragment to remove other specificities immunoprecipitated newly synthesized native La (SS-B)/Ro (SS-A) complexes, providing additional evidence that autoantibodies were recognizing a conformational epitope. The findings indicate that the human autoantibody response to La involves recognition of a conformational determinant involving the conserved RRM region without necessarily interfering with the RNA-dependent association of the La/Ro ribonucleoprotein.

Antibody specificities of Thai and Australian scleroderma sera with topoisomerase I recombinant fusion proteins.

Autoantibodies that react with the nuclear enzyme topoisomerase I (Topo I) are used as a diagnostic marker of diffuse scleroderma. To better define immune reactivity to Topo I, antibody epitopes in two patient populations were analyzed using recombinant Topo I proteins. Two overlapping partial cDNA clones encoding the complete amino acid sequence of Topo I were isolated from human placenta. Using the polymerase chain reaction, specific regions of Topo I were amplified and cloned into the pGEX expression vectors. To map Topo I epitopes, recombinant fusion proteins were analyzed by immunoblotting with 66 anti-Topo I sera from Thai and Australian patients with diffuse scleroderma. Six distinct epitope regions were identified along the length of the 765 amino acid enzyme. Almost all sera contained antibodies that recognized the midregion of Topo I (amino acids 453-560), as well as antibodies to one of more of the other epitope regions. Sixty percent of the sera contained antibodies that recognized a COOH-terminal epitope region (amino acids 658-765) encompassing the active site of the enzyme. This subset of Topo I antibodies could be responsible for the inhibition of enzymatic activity previously reported in vitro. Heterogeneous patterns of reactivity with the six Topo I epitope regions were observed, although over half the sera could be assigned to one of six distinct patterns. In general, antibodies in the Thai sera reacted more strongly with the six epitope regions. Furthermore, two of the epitope regions reacted exclusively with Thai sera, suggesting a degree of racial or geographical specificity in the autoantibody response to Topo I. The identification of multiple epitopes in Topo I conforms with the polyclonal autoantibody response to intracellular Ag found in other multisystem autoimmune diseases and is presumed to be driven by the presentation of multiple peptides from Topo I itself.

Antinuclear antibodies in patients with scleroderma (systemic sclerosis) and in their blood relatives and spouses.

OBJECTIVE: To test the postulate that there is a higher prevalence of antinuclear antibodies (ANAs) in serum samples from blood relatives and from spouses of patients with scleroderma than in control samples, and that this provides evidence for both genetic and environmental factors influencing autoimmunity in scleroderma. METHOD: Testing for ANAs was performed on 58 patients with scleroderma, 30 of their spouses, 74 first degree relatives, and 66 control subjects broadly age matched to the patients, their spouses, and about half of the relatives (siblings and parents). RESULTS: On the basis of an ANA titre of > 40 as positive, 12 (18%) of the controls, 55 (95%) of the patients, one (3%) of the spouses and five (7%) of the relatives would be classified as positive. Thirty seven (64%) of the patients had defined specificities (ACA, Scl 70, U1 (RNP)) but none of the controls, spouses, or relatives had antibodies of these specificities. CONCLUSION: These findings give no support to the postulate that environmental or genetic factors contribute to the ANAs in scleroderma.

Definition of a discontinuous immunodominant epitope at the NH2 terminus of the La/SS-B ribonucleoprotein autoantigen.

High-titer IgG autoantibodies to the La/SS-B ribonucleoprotein (RNP) are a hallmark of patients with primary Sjogren's syndrome. Anti-La/SS-B-positive human sera bind to multiple epitopes on recombinant La/SS-B, although the initial response is against an immunodominant epitope within the first 107 NH2-terminal amino acids (aa). Sequence analysis has identified a striking homology between aa 88-101 in this NH2-terminal region of La/SS-B and a feline retroviral gag polypeptide suggesting the anti-La/SS-B response may be initiated by cross-reactivity with an exogenous agent. In the present study, detailed mapping of this NH2-terminal epitope, using recombinant La/SS-B purified from the expression of overlapping DNA fragments spanning aa 1-107, has shown that this immunodominant epitope is a complex conformational or discontinuous epitope dependent upon both aa 12-28 and 82-99 for expression, even though these regions share no homology with each other. This requirement questions the significance of the homology between La/SS-B and a retroviral gag polypeptide in the generation of the B cell response to La/SS-B and is in accord with the general concept that B cells recognize conformational epitopes on antigens rather than small linear peptide sequences. The finding also reinforces the notion that native autoantigen could be the initiator of the autoimmune response.

Inhibitory autoantibody to a conformational epitope of the pyruvate dehydrogenase complex, the major autoantigen in primary biliary cirrhosis.

The mitochondrial autoantibodies present in primary biliary cirrhosis (PBC) react with the 2-oxoacid dehydrogenase enzymes that include the pyruvate dehydrogenase complex (PDC). All epitopes so far demonstrable, including the inner lipoyl domain of PDC-E2, have been revealed by immunoblotting. To identify other epitopes, advantage was taken of the capacity of PBC sera to inhibit in vitro the catalytic function of the PDC enzyme. PBC sera were analyzed by affinity chromatography, using columns containing either recombinant PDC-E2 or intact PDC. Fractions that bound to the column (B) and nonbinding effluent fractions (NB) were tested by immunoblotting and ELISA and for their capacity to inhibit enzyme function. After separation on the PDC-E2 column the B fractions were reactive with PDC-E2 and intact PDC, whereas the NB fractions did not react by immunoblotting or ELISA with PDC-E2 but did react strongly by ELISA with PDC and did strongly inhibit the enzyme function. After separation of sera on the PDC column, the B fractions reacted more strongly with PDC than PDC-E2 by ELISA and strongly inhibited the enzyme function, whereas the NB fractions were nonreactive. Thus we describe a hitherto undetected population of autoantibodies in PBC sera that react only with intact PDC but not with the recombinant PDC-E2 subunit that contains the lipoyl epitope, are demonstrable by ELISA but not by immunoblotting, and notably, inhibit enzyme function. These nonblotting inhibitory autoantibodies in PBC are presumed to react with an exclusively conformational determinant perhaps presented by the tertiary structure of the entire enzyme complex.

Estimation of amounts of anti-La(SS-B) antibody directed against immunodominant epitopes of the La(SS-B) autoantigen.

The contribution of circulating anti-La(SS-B) antibody to the hypergammaglobulinaemia seen in primary Sjögren's syndrome is unknown. In this study levels of anti-La(SS-B) antibody directed against three immunodominant epitopes of the anti-La(SS-B) autoantigen were measured by ELISA in 84 anti-La(SS-B)+ sera using purified recombinant protein and antibody affinity-purified against the three anti-La(SS-B) fusion proteins. There was marked variation in the amounts of IgG anti-La(SS-B) antibody detected, with levels ranging from 0.02 mg/ml to 11 mg/ml. The anti-La(SS-B) levels were greater than 1 mg/ml in 61% of patients; in 18% of sera the anti-La(SS-B) level constituted 10% or more of the total serum IgG. However, other patients were seen with marked hypergammaglobulinaemia and low anti-La(SS-B) concentrations. These results support an antigen-driven mechanism for the anti-La(SS-B) response and suggest that anti-La(SS-B) antibody production is regulated independently of other immunoglobulins.

Mapping of epitopes on the La(SS-B) autoantigen of primary Sjögren's syndrome: identification of a cross-reactive epitope.

Autoepitopes on the ribonucleoprotein La(SS-B) were identified by using recombinant La(SS-B) polypeptides and sera from 166 patients with the antinuclear autoantibody anti-La(SS-B). The La(SS-B) polypeptides were encoded by polymerase chain reaction-derived overlapping or nonoverlapping fragments of the La(SS-B) gene, which encodes a protein of 408 amino acids (aa). Of the 166 sera tested, 99% reacted with a fusion protein comprising the first 107 N-terminal aa (LaA); 91% reacted with a fusion protein comprising aa 111 to 242 (LaC), and 91% reacted with a fusion protein comprising aa 346 to 408 (LaL2/3) at the C terminus of La(SS-B). The order of immunodominance as assessed by the number of sera reacting with each epitope and the strength of the reactivity was LaA (aa 1 to 107) greater than LaC (aa) 111 to 242) much greater than LaL2/3 (aa 346 to 408). Cross-reactivity was observed between antibodies eluted from LaC (aa 111 to 242) and LaL2/3 (aa 346 to 408), but there was no significant primary sequence homology between the two regions. The LaC region contained at least two epitopes, one encompassing a putative RNA-binding motif (aa 112 to 187) which was recognized by 83% of patient sera. Serial serum samples from three patients showed that the antibody response to La(SS-B) was initially directed to the N terminus (LaA, aa 1 to 107), but over a period of time all three major epitopes, including that encompassing the putative RNA-binding motif, were recognized. This result suggests that the primary immune response to La(SS-B) is restricted to an immunodominant epitope. As the specificity of the autoantibody response broadens, it includes the RNA-binding motif, which may have important implications for the expression of disease.

The F1 hybrid effect in allogeneic lymphocyte cytotoxicity. Points of similarity between hybrid resistance and ALC.

Allogeneic lymphocyte cytotoxicity (ALC) is characterized by the rapid destruction of intravenously injected allogeneic lymphocytes by unsensitized hosts. While ALC has been reported in several mammalian species, it has been most extensively studied in rats. All the available in vivo and in vitro evidence points to NK cells as the effectors of ALC. The experiments described in this communication show that when donor and host share common ALC determinants, the extent to which allogeneic lymphocytes are killed is greatly reduced, and sometimes even abolished, relative to the killing that would have occurred in the absence of shared determinants. Thus, allogeneic lymphocyte transfers in inbred rat strain combinations having the general pattern A----B are associated with significantly higher levels of ALC than are the corresponding (A x B)F1----B, A----(A x B)F1 or (C x A)F1----(C x B)F1 lymphocyte transfers. The reduced ALC is not due to inability of the F1 hybrid to respond with the full vigor of the parental strains. Nor is it due to an absolute requirement for homozygous presentation of the donor ALC determinants. It is concluded that impaired self-recognition may be an important determinant of killing in ALC, as in some other NK cell-mediated phenomena. Although ostensibly differing immunogenetically from hybrid resistance in mice, ALC includes a range of patterns of reactivity, some of which are similar to those that characterize hybrid resistance. It is suggested that hybrid resistance and ALC may represent quantitative variants of a similar process.

Autoantibodies to the major nucleolar phosphoprotein B23 define a novel subset of patients with anticardiolipin antibodies.

Of 164 sera with antinucleolar antibodies, 7 (4.3%) were shown by Western blotting to react with a 37-kd polypeptide in a nuclear extract of HeLa cells and with the recombinant protein expressed by a complementary DNA clone encoding the major nucleolar protein B23. Six of the 7 sera (86%) had antibodies to cardiolipin (aCL), and the sample that was negative for aCL had had lupus anticoagulant on previous testing. All 7 patients had either systemic lupus erythematosus (SLE) or a variant of SLE, suggesting that anti-B23 identifies a subset of patients with SLE associated with a high frequency of aCL.

Autoepitopes reactive with anti-SS-B/La.

Sera from 120 patients with suspected autoimmune rheumatic disease and antinuclear antibodies of anti-SS-B/La specificity were examined by Western blotting for reactivity with the SS-B/La polypeptide of HeLa cells and recombinant SS-B/La derived from a 1.4 kilobase (kb) cDNA encoding approximately 90% of the SS-B/La molecule. All sera reacted with the HeLa cell and the recombinant SS-B/La. One hundred and fourteen (95%) reacted with a set of three Staph. aureus V8 protease-resistant peptides of Mr 30,000, 29,00 and 28,000 from a methionine-rich region of HeLa cell SS-B/La designated the X domain, and 98 (82%) reacted with another set of two protease-resistant peptides of Mr 24,000 and 23,000 from a phosphorylated region of HeLa cell La designated the Y domain. One reacted weakly with the Y domain only. All sera that reacted with X and Y reacted more strongly with X, suggesting that X was the major epitope. Antibodies affinity purified from the X domain reacted strongly with the X peptides but not with the Y peptides and conversely, antibodies affinity purified from the Y domain reacted with the Y peptides but not with the X peptides. Both antibodies reacted with a fusion protein comprising 102 amino acids at the carboxyl terminus of the SS-B/La molecule. This protein contained no methionine, demonstrating that methionines were not involved in the antibody-binding site. Over 80% of patients whose only criteria for selection was the presence of anti-SS-B/La had the clinical, histologic, serologic and phenotypic features of Sjögren's syndrome whilst the remaining 20% had at least two of the features.(ABSTRACT TRUNCATED AT 250 WORDS)

The nucleotide sequence of a human cDNA encoding the highly conserved nucleolar phosphoprotein B23.

A cDNA clone containing the complete coding sequence for the human nucleolar phosphoprotein B23 was isolated from a Burkitt's lymphoma cDNA library by immunoscreening with human autoantibodies. The B23 clone contained a 1.3 kb cDNA insert encoding a polypeptide of 294 amino acids with a predicted molecular mass of 32,539 daltons. The deduced B23 amino acid sequence contained 2 acidic domains rich in aspartic and glutamic acid, a feature shared by a number of nuclear and nucleolar proteins. The human B23 amino acid sequence showed 98% homology with rat B23 and 68% homology with the Xenopus laevis nucleolar phosphoprotein, NO38 showing that the primary structure of B23 is highly conserved among these species.

Racial differences in antinuclear antibody patterns and clinical manifestations of scleroderma.

The profile of antinuclear antibodies (ANA) in 49 Thais with scleroderma (systemic sclerosis) was compared with that in 68 white Australians with scleroderma. Forty-eight (98%) of the Thais and all (100%) of the white Australians were positive for ANA, with the majority (100% and 97%, respectively) showing a diffuse speckled pattern of nuclear fluorescence. The distribution of the patterns was different in the 2 races; 35 (71%) of the Thais and 17 (25%) of the Australians showed staining of the nucleolus, and 1 (2%) of the Thais and 35 (51%) of the Australians showed staining of the centromeres. The frequency of precipitating antibodies to extractable nuclear antigens was also strikingly different: 86% in Thais and 26% in Australians (P less than 0.001). Precipitating antibodies to Scl-70 (topoisomerase I), the predominant extractable nuclear antigen in patients with scleroderma, were detected in 37 (76%) of the Thais and 18 (26%) of the Australians, and these were shown by Western blotting to react with the Scl-70 (topoisomerase I)-associated polypeptides. Differences in the frequencies of the ANA specificities in the 2 races were consistent with differences in the clinical manifestations of scleroderma; all of the Thai patients, in contrast to 15% of the Australian patients, had diffuse scleroderma with widespread skin involvement. This suggest that environmental or genetic factors may influence the expression of scleroderma.

Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase.

Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency (greater than 95%) of autoantibodies to a Mr 70,000 mitochondrial antigen, a component of the M2 antigen complex. We have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase (EC 2.3.1.12) of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.

Antinuclear antibodies as molecular and diagnostic probes.

Much progress has been made in the past decade in defining the specificities of antinuclear antibodies (ANA) that are present in the blood of patients with multi-system autoimmune rheumatic diseases, and ANA now have an important place in diagnostic immunology. Disease-specific ANA have been defined and the intracellular autoantigens against which they react have been characterized. ANA have also established an important place in cell biology. Their use as probes has enabled molecular biologists to isolate, purify and assay the function of the highly conserved molecules with which they react, revealing new insights into the role these molecules play in gene transcription and translation. Cloning of the genes encoding these molecules in addition to providing information on primary structure has also provided human recombinant proteins for use as pure substrates for the development of simple and highly sensitive diagnostic assays in rheumatology. Studies on the epitopes with binding sites for ANA should provide new knowledge on how the immune response to these molecules evolves, how it is maintained and what role ANA play in the pathogenesis of disease.

Characteristics and epitope mapping of a cloned human autoantigen La.

The La (SS-B) polypeptide is a ribonucleoprotein against which high titer antinuclear antibodies (ANA) react in the human autoimmune disease primary Sjögren's syndrome. To identify the autoepitopes with which the ANA anti-La (anti-SS-B) reacts, we isolated a 1.4-kb cDNA clone for La from a lambda gt10 library made from a human Burkitt's cell line. This clone contained an open reading frame of 1065 bp, encoding a 40.1-kDa polypeptide that corresponded to the carboxyl-terminal end of the La protein. The predicted polypeptide sequence of the recombinant protein was highly charged and unrelated to any previously published sequence. We also compared this clone to a previously published cDNA sequence for La and demonstrated significant differences, particularly that the open reading frame in our cDNA continued for 926 additional bases 3' to a putative termination codon in the previously reported sequence. The recombinant La protein was expressed in Escherichia coli and tested for reactivity with 200 sera containing ANA of various specificities. Only the sera containing anti-La antibodies reacted with the cloned La. By expressing subclones of the La cDNA as fusion proteins with beta-galactosidase, we have localized at least one epitope for the binding of anti-La antibodies to the carboxyl-terminal 103 amino acids of the La protein. No anti-La binding could be demonstrated to the region of the La protein that had previously been predicted to contain an autoepitope for the binding of anti-La (SS-B) antibodies. Studies of cloned autoepitopes could provide important clues to the role ANA play in disease and lead to targeted intervention in the treatment of primary Sjögren's syndrome.

Autoantibodies to mitochondria in systemic sclerosis. Frequency and characterization using recombinant cloned autoantigen.

Mitochondrial autoantibodies, a hallmark of primary biliary cirrhosis (PBC), have been widely described for many years in patients with systemic sclerosis, and there have been several reports of the concurrence of systemic sclerosis and PBC. However, there is very little information with respect to the significance of these autoantibodies or any definitive evidence that the antigens involved represent the mitochondrial autoantigens (M2 complex) described in PBC. We have cloned and sequenced a rat complementary DNA which encodes for all the epitopes recognized by autoantibodies to the major, or 70-kd, mitochondrial autoantigen in patients with PBC. Using this recombinant fused autoantigen, as well as by immunoblotting with human placental mitochondria, we tested for antimitochondrial antibody specificity in sera from 250 patients with systemic sclerosis. Nineteen sera (7.6%), including those from patients with CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) and diffuse scleroderma, had reactivity with human placental mitochondria proteins by immunoblot testing. All 19 sera reacted with the M2 complex. All sera that reacted with the 70-kd protein likewise reacted with the recombinant cloned autoantigen. The predominant autoantibody isotype to the 70-kd protein was IgG3. Interestingly, the 70-kd protein is 11% proline, an amino acid which is frequently preceded by hydrophobic amino acids.

Serological diagnosis of primary Sjögren's syndrome by means of human recombinant La (SS-B) as nuclear antigen.

Human recombinant La nucleoprotein was purified from cultures of Escherichia coli containing a vector with a 1.4 kilobase cDNA encoding La; the nucleoprotein was used to test for antinuclear antibodies (ANA) to La. Serum samples from 260 patients with autoimmune diseases associated with ANA and 100 healthy subjects were tested by an enzyme-linked immunosorbent assay (ELISA). Samples from 47 (94%) of 50 patients with primary Sjögren's syndrome and 1 (7%) of 14 patients with secondary Sjögren's syndrome reacted with the recombinant La. No reactivity was demonstrated in 196 patients with other ANA-associated autoimmune diseases or in 100 healthy subjects. The study confirms the high correlation between ANA, anti-La, and primary Sjögren's syndrome and shows how gene cloning can provide large quantities of human autoantigens for use in highly specific and sensitive diagnostic assays.