Neonatal lupus erythematosus. Report of serological and immunogenetic studies in twins discordant for congenital heart block.

Autoantibody, HLA studies and C4 phenotypes were performed on twins discordant for isolated congenital heart block. Serum from the mother and cord blood from the infants revealed Ro(SSA) and La(SSB) antibodies in all three sera. No significant difference in Ro(SSA) antibody titre was noted in the cord blood of either twin when compared with maternal titres, as detected by a sensitive ELISA assay. The infants' mother was HLA-DR3 positive. Both infants had identical HLA and C4 phenotypes. Immunoblot analysis revealed that sera from both mother and infants reacted with the 52-kDa Ro(SSA) macromolecule. Quantitative cord blood IgM levels were not elevated in either twin. This study indicates that placental transfer of anti-Ro(SSA) or anti-La(SSB) alone to the fetus is not sufficient for the expression of congenital complete heart block. We conclude from this experiment of Nature that there must be a second event determining which infant develops complete heart block, but this is unknown at present.

CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate.

We have isolated and characterized a set of overlapping cDNA clones that encode the human centromere autoantigen centromere protein C (CENP-C). The identity of these clones has been established using several criteria. First, they were shown to encode a polypeptide that migrates at the expected position for CENP-C on SDS-polyacrylamide gel electrophoresis. Second, we have demonstrated that this polypeptide shares at least two epitopes with human CENP-C. Polyclonal antibodies were raised to fusion proteins encoded by nonoverlapping regions of the cDNA clones. These antibodies were shown to recognize a protein at a position appropriate for CENP-C on immunoblots of human chromosomal proteins. In addition, we used indirect immunofluorescence to demonstrate that these antibodies recognize centromeres of HeLa chromosomes in the expected pattern for CENP-C. Localization of CENP-C by immunoelectron microscopy reveals that this protein is a component of the inner kinetochore plate.

Detection of anti-Ro(SSA) antibodies by gel double diffusion and a 'sandwich' ELISA in systemic and subacute cutaneous lupus erythematosus and Sjögren's syndrome.

A newly described Ro 'sandwich' ELISA was compared to the gel double diffusion technique to detect anti-Ro(SSA) antibodies in Sjögren's syndrome, systemic and subacute cutaneous lupus erythematosus patients. This study demonstrates that the ELISA assay increased the frequency of detection of anti-Ro(SSA) antibodies in these well defined connective tissue disease patients by approximately 5-10% compared to the gel double diffusion anti-Ro(SSA) antibody assay. The study also confirms that some patients make anti-Ro(SSA) antibodies directed solely at unique human Ro(SSA) antigen epitopes. We also detected the existence of a significant Sjögren's syndrome patient population failing to make significant anti-Ro(SSA) antibodies. We conclude from our study that the gel double-diffusion technique employing human spleen extract as a source of the Ro(SSA) antigen is, at present, the most cost-effective test to detect anti-Ro(SSA) antibodies.

Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.

BACKGROUND AND METHODS: We describe five patients with underlying neoplasms in whom painful mucosal ulcerations and polymorphous skin lesions developed, usually with progression to blistering eruptions on the trunk and extremities. Histologic examination showed vacuolization of epidermal basal cells, keratinocyte necrosis, and acantholysis. Immunofluorescence testing revealed atypical pemphigus-like autoantibodies in perilesional epithelium and serum from all five patients. We studied the antigenic specificities of the autoantibodies by indirect immunofluorescence and immunoprecipitation, using extracts of 14C-labeled human keratinocytes. IgG purified from the serum of one patient was passively transferred to four neonatal mice to test for pathogenicity. RESULTS: Immunofluorescence testing showed that the autoantibodies bound to the surface of tissues containing desmosomes, including complex and simple epithelia, and myocardium. An identical and unique complex of four polypeptides with molecular weights of 250, 230, 210, and 190 was immunoprecipitated by all serum samples. The 250-kd polypeptide comigrated with desmoplakin I (a protein found in the desmosomes of all epithelia), and the 230-kd antigen comigrated with the antigen of bullous pemphigoid. Cutaneous blisters, a positive Nikolsky's sign, and epidermal and esophageal acantholysis developed in all mice into which the autoantibody was injected. Electron microscopy showed epidermal acantholysis similar to lesions of experimentally induced pemphigus vulgaris. CONCLUSION: These five patients with cancer had a novel acantholytic mucocutaneous disease characterized by autoantibodies that were pathogenic after passive transfer. The autoantibodies from these patients reacted with an antigen complex composed of desmoplakin I and the 230-kd antigen of bullous pemphigoid and two as yet unidentified epithelial antigens. We suggest the term "paraneoplastic pemphigus" for this disease.

Isolation of a human epidermal cDNA corresponding to the 180-kD autoantigen recognized by bullous pemphigoid and herpes gestationis sera. Immunolocalization of this protein to the hemidesmosome.

Autoantibodies present in the sera of patients with bullous pemphigoid (BP) bind to the basement membrane zone of normal human skin and commonly recognize two epidermal proteins, the BP240 and BP180 antigens. Two BP antigen cDNA clones from a lambda gt11 human keratinocyte library have been identified on the basis of reactivity with a BP serum. The fusion protein (FP) produced by one clone immunoadsorbed autoantibodies, which specifically recognized the BP180 by antigen, showing no cross-reactivity with BP240 by immunoblot analysis. The FP produced by the second clone immunoadsorbed autoantibodies which specifically reacted with the BP240 epidermal antigen. Northern blot analysis demonstrated that the BP180 and BP240 antigens are encoded by distinct RNA transcripts with lengths of 6.0 and 8.5 kb, respectively. Immunoblot analysis of the BP180 lysogen extract identified a 135-kD FP which was recognized by 7 of 16 BP sera and 7 of 8 herpes gestationis sera. A rabbit antiserum prepared against the lysogenic BP180 FP specifically recognized the BP180 antigen from human epidermal extracts by immunoblotting, labeled the BMZ by indirect immunofluorescence, and bound to human epidermal hemidesmosomes by immuno-electron microscopy. These results indicate that the BP180 antigen recognized by BP and herpes gestationis autoantibodies is a unique hemidesmosomal polypeptide, distinguishable from the BP240 antigen.

Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads.

We have screened for the presence of two centromere autoantigens, CENP-B (80 kDa) and CENP-C (140 kDa) at the inactive centromere of a naturally occurring stable dicentric chromosome using specific antibodies that do not cross-react with any other chromosomal proteins. In order to discriminate between the active and inactive centromeres on this chromosome we have developed a modification of the standard methanol/acetic acid fixation procedure that allows us to obtain high-quality cytological spreads that retain antigenicity with the anti-centromere antibodies. We have noted three differences in the immunostaining patterns with specific anti-CENP-B and CENP-C antibodies. (1) The amount of detectable CENP-B varies from chromosome to chromosome. The amount of CENP-C appears to be more or less the same on all chromosomes. (2) CENP-B is present at both active and inactive centromeres of stable dicentric autosomes. CENP-C is not detectable at the inactive centromeres. (3) While immunofluorescence with anti-CENP-C antibodies typically gives two discrete spots, staining with anti-CENP-B often appears as a single bright bar connecting both sister centromeres. This suggests that while CENP-C may be confined to the outer centromere in the kinetochore region, CENP-B may be distributed throughout the entire centromere. Our data suggest that CENP-C is likely to be a component of some invariant chromosomal substructure, such as the kinetochore. CENP-B may be involved in some other aspect of centromere function, such as chromosome movement or DNA packaging.

cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment.

cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.

The sequence and expression of the divergent beta-tubulin in chicken erythrocytes.

We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.

Sequence and expression of the chicken beta 3 tubulin gene. A vertebrate testis beta-tubulin isotype.

We report the determination of the complete DNA sequence for c beta 3, a chicken beta-tubulin gene which we show to be the dominant beta-tubulin expressed in testis. Like all previously studied vertebrate beta-tubulin genes, the gene is divided into four exon sequences interrupted by three intervening sequences (located between amino acids 19 and 20, within codon 56, and within codon 93). Analysis of the program of expression of this gene indicates that it encodes the dominant chicken testis beta-tubulin, although it is also expressed at lower levels in a wide variety of cell and tissue types. Comparison of the predicted polypeptide sequence for c beta 3 with four other available chicken beta-tubulin genes confirms our earlier suggestion that within an otherwise conserved framework, sequences within two variable region domains serve to define specific beta-tubulin polypeptide isotypes. The data indicate that the c beta 3 gene encodes a unique beta-tubulin isotype which diverges from the dominant neuronal beta-tubulin isotype in 18 of 445 residues (4%). Although the protein coding regions of the c beta 3 gene are highly homologous to the chicken c beta 1, c beta 2, c beta 4, and c beta 5 genes previously reported by us, no significant sequence homology with these previously analyzed genes is discernible in the 5'- or 3'-untranslated region sequences, in the intervening sequences, or in the presumptive transcriptional promoter sequences.

Localization of the L1Md family of repeated sequences in the mouse albumin-alpha-fetoprotein gene complex.

Studies on the beta-globin gene complex in the mouse have demonstrated the existence of repeated DNA sequences interspersed throughout the intergenic regions (1,2). These sequences are members of families of middle repetitive sequences and have been mapped to specific intergenic sites in the 60 kbp beta-globin complex. In this study we present evidence that members of this middle repetitive family of DNA sequences, the L1Md family, are interspersed throughout the mouse albumin and alpha-fetoprotein gene complex. Unlike those of the beta-globin complex, all of which are found in the intergenic regions, these sequences are localized within intron 12 of the albumin gene and intron 3 of the AFP gene as well as twice in the 13.5 kbp intergenic region that links the albumin gene to the AFP gene.

Molecular mechanism of extinction of liver-specific functions in mouse hepatoma x rat fibroblast hybrids: extinction of the albumin gene.

Hybrids formed by the fusion of mouse hepatoma (BWTG3) and rat fibroblast (JF1) cells exhibit the extinction of mouse albumin and alpha-fetoprotein synthesis. Karyotype analyses suggest that all parental chromosomes are present in the hybrids. The extinction, therefore, of mouse hepatocyte genes is attributed to the inhibitory action of the rat genome. In these studies, we show that these hybrids possess and express the mouse beta-glucuronidase gene (which is encoded on the same chromosome as the mouse albumin and alpha-fetoprotein gene), and we present data of Southern blot analysis which demonstrate that such hybrids have indeed retained both mouse and rat albumin DNA sequences. In addition, using mouse albumin cDNA, we have shown by cDNA-RNA reassociation kinetics that albumin mRNA is virtually absent in these hybrids. We conclude from these studies that the extinction of albumin synthesis involves a mechanism which results in the loss of cytoplasmic albumin mRNA.