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N F Rothfield

Publications and source records attributed to N F Rothfield.

At least 19 recordsLinked to original sources
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

Migration of centromere proteins in rabbit spermatids.

Human autoantibodies were used to localize centromere proteins by immunoelectron microscopy, immunofluorescence, and confocal microscopy in isolated cells and in cryosections of rabbit testis. A computer-assisted three-dimensional reconstruction of the positions and sizes of fluorescent spots allowed us to follow their movements during the different phases of spermiogenesis. In very young spermatids, the centromeres were distributed within a space separated from both the external nuclear limits and the nuclear core. They moved towards the nuclear center in cap phase spermatids, where they clustered into a few large centromeric masses. In preelongating spermatids, the immunolabeled proteins were dispersed within an equatorial area, where they formed one large mass. In late spermatids, the mass moved towards the posterior part of the nucleus, and, in the spermatozoon, the two basal knobs located at the base of the nuclei were the only strongly immunolabeled structures, while no labeling of the main part of the nucleus was observed. Since the number of centromeres remained close to the number of chromosomes until the cap phase of spermatid differentiation, we hypothesize that the labeling of young spermatids corresponds to centrometric proteins associated with their specific DNA counterparts, while the centromere proteins, possibly detached from their DNA loci, were released from nuclei of old spermatids in the same way as are histones and transition proteins.

Animals↗
PubMed1992

Autoantibodies in scleroderma.

The antinuclear and antinucleolar antibodies found in patients with scleroderma are discussed. Many of the autoantigens have been characterized, the cDNA cloned and the epitopes defined. Many of the more common autoantigens are DNA-binding proteins, which are very important in transcription and in cell division. The clinical significance of the autoantibodies is discussed: their prevalence and their relation to disease manifestations and prognosis is examined. The unique exclusiveness of the major autoantibodies is discussed. The data on the immunoglobulin and IgG subclass is reviewed in relation to the overall concept that most autoantibodies in scleroderma patients represent the result of an antigen-driven response.

Antigens↗
PubMed1991

Disruption of centromere assembly during interphase inhibits kinetochore morphogenesis and function in mitosis.

The relationship between the kinetochore and the centromeric heterochromatin that surrounds it is unknown. Anti-centromere autoantibodies (ACAs) that recognize antigens found in the heterochromatin beneath the kinetochore disrupt mitotic events when microinjected into human cells. We show here that ACAs interfere with two different stages of centromere assembly during interphase, resulting in abnormal kinetochore structures during mitosis. Antibody injection prior to late G2 results in the subsequent failure to assemble a trilaminar kinetochore. Such chromosomes bind microtubules but are incapable of movement. Antibody disruption of events during G2 produces unstable kinetochores that prevent the normal transition into anaphase. These experiments present a novel way to examine events in the pathway of kinetochore assembly that occur during interphase, at a time when this structure cannot be visualized directly.

Autoantibodies↗
PubMed1991

Prognostic significance of anticentromere antibodies and anti-topoisomerase I antibodies in Raynaud's disease. A prospective study.

Seventy-seven patients with Raynaud's disease were studied for a mean of 4 years (range 1-11 years) to determine the relationship between autoantibodies and long-term clinical outcome. Anticentromere antibodies (ACA) were assayed by indirect immunofluorescence and by immunoblotting of HeLa cell chromosome extracts. Antibodies to topoisomerase I (anti-topo I) were assayed by immunodiffusion and immunoblotting. Antibodies to the major centromeric protein, CENP-B, and anti-topo I were studied by enzyme-linked immunosorbent assay (ELISA). Eight patients developed telangiectasias, 4 developed skin tightening, and 4 developed a connective tissue disease other than scleroderma. The presence of ACA at the start of the study was associated with the development of telangiectasias (P less than 0.003). An initial 100-kd band on immunoblot in conjunction with a positive anti-topo I ELISA result was associated with the development of tight skin (P less than 0.0025), while a 100-kd band with a negative anti-topo I ELISA result was associated with the subsequent development of a connective tissue disease other than scleroderma (P less than 0.0073). Patients who were initially ACA positive, had the 100-kd band on immunoblot, or had positive ELISA results for anti-topo I or for anti-CENP-B were 63-fold more likely to develop signs of connective tissue disease by the end of the study (P less than 0.000009). The presence of any of these autoantibodies was more sensitive (100%), although less specific (75%), than were findings from nailfold capillaroscopy (sensitivity 67% and specificity 95%) in predicting subsequent clinical progression. We conclude that findings of assays for anti-topo I and ACA complement the findings from nailfold capillaroscopy in providing useful prognostic information in Raynaud's disease.

Antibodies↗
PubMed1991

Idiotypic analysis of human anti-topoisomerase I autoantibodies.

Anti-topoisomerase I autoantibodies (anti-topo I) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's phenomenon. Polyclonal anti-idiotypic sera were raised against affinity-purified anti-topo I from 2 patients with scleroderma (EM, SG) and 1 healthy individual (NM). All 3 anti-topo I preparations expressed immunodominant private Ids in or near the antigen binding site of the autoantibody. Further analysis of Id-EM showed isotypic restriction to IgG and a stable Id-expression over the course of 9 years. Id-SG and Id-NM were expressed on IgG and on IgA. The idiotypic character of anti-topo I closely resembles that of anti-centromere autoantibodies which are associated with the CREST syndrome of scleroderma. The data suggest an antigen-driven process in the origin of autoantibodies in scleroderma.

Antigens↗
PubMed1991

Idiotypic analysis of human anticentromere autoantibodies.

The idiotypes (Ids) of anticentromere antibodies (ACA) have been studied using a fusion protein obtained from cloned cDNA of the major centromere antigen, CENP-B, for isolation of the autoantibodies. IgG-ACA were affinity purified from 4 patient sera and anti-Ids prepared in rabbits. Analysis revealed the existence of two distinct types of immunodominant Ids. One Id is near the antibody combining site and one is framework associated. A longterm longitudinal study of Id expression in a patient who seroconverted from ACA (-) to ACA (+) when she developed Raynaud's phenomenon showed a close correlation between Id expression and ACA titers (r = 0.94). These results may be interpreted as evidence for an autoantigen driven process in the anticentromere immune response.

Adult↗
PubMed1990

The IgG, IgM, and IgA isotypes of anti-topoisomerase I and anticentromere autoantibodies.

We studied the expression of IgG, IgM, and IgA autoantibodies in the anti-topoisomerase I and anticentromere immune responses by enzyme-linked immunosorbent assay, immunoblotting, and immunofluorescence. While IgG autoantibodies were most common, IgA autoantibodies were also frequently found, but IgM autoantibodies were rare. This is the first report of IgA autoantibodies in scleroderma.

Autoantibodies↗
PubMed1990

Use of molecular cloning methods to map the distribution of epitopes on topoisomerase I (Scl-70) recognized by sera of scleroderma patients.

We report the initial molecular characterization of the autoimmune response against DNA topoisomerase I (topo I; Scl-70). Sera from 36 patients with scleroderma and 4 healthy control subjects were studied using 6 subcloned portions of topo I. Twenty-three sera recognized at least 2 independent epitopes on the molecule. Therefore, anti-topo I, like other non-organ-specific autoantibodies characterized to date, is polyclonal and multifocal. The cloned protein should prove suitable for sensitive early detection of anti-topo I in the clinical setting.

Autoantibodies↗
PubMed1990

Autoantibodies to topoisomerase I (Scl-70): analysis by gel diffusion, immunoblot, and enzyme-linked immunosorbent assay.

Anti-topoisomerase I autoantibodies (anti-topo I, anti-Scl-70) are associated with proximal scleroderma and are of prognostic significance in patients with Raynaud's disease. To establish a highly sensitive and specific system for the detection of anti-topo I, we have investigated sera from 409 patients and controls by Ouchterlony gel diffusion, Western immunoblot on chromosome proteins, and solid-phase enzyme-linked immunosorbent assay (ELISA) with purified topoisomerase I as antigen. The ELISA was more sensitive than the gel diffusion technique and was more specific than the Western immunoblot, while the immunoblot may identify additional autoantibodies.

Autoantibodies↗
PubMed1990

Injection of anticentromere antibodies in interphase disrupts events required for chromosome movement at mitosis.

We have used autoantibodies to probe the function of three human centromere proteins in mitosis. These antibodies recognize three human polypeptides in immunoblots: CENP-A (17 kD), CENP-B (80 kD), and CENP-C (140 kD). Purified anticentromere antibodies (ACA-IgG) disrupt mitosis when introduced into tissue culture cells during interphase. We have identified two execution points for antibody inhibition. Antibodies injected into the nucleus greater than or equal to 3 h before mitosis prevent the chromosomes from undergoing normal prometaphase movements in the subsequent mitosis. Antibodies injected in the nucleus during late G2 cause cells to arrest in metaphase. Surprisingly, antibodies introduced subsequent to the beginning of prophase do not block mitosis. These results suggest that the CENP antigens are involved in two essential interphase events that are required for centromere action in mitosis. These may include centromere assembly coordinate with the replication of alpha-satellite DNA at the end of S phase and the structural maturation of the kinetochore that begins at prophase.

Anaphase↗
PubMed1990

Extensive calcinosis cutis with systemic lupus erythematosus.

Calcinosis cutis is a common clinical feature of dermatomyositis and scleroderma but is only rarely reported in association with systemic lupus erythematosus (SLE). We describe three patients with long-standing systemic lupus erythematosus in whom extensive calcinosis cutis developed. We identify characteristics our patients share in common with 23 previously described patients.

Adult↗
PubMed1989

Correlation of anti-Ro antibody with photosensitivity rash in systemic lupus erythematosus patients.

We studied 131 patients with systemic lupus erythematosus (SLE) to determine the relationship between the presence of anti-Ro antibody in these patients and specific manifestations of SLE. There was a significant correlation between anti-Ro and photosensitivity rash in SLE patients, regardless of the type of rash. No significant association was found between anti-Ro and other SLE criteria or between anti-Ro and demographic variables.

Adult↗
PubMed1989

Isotype analysis of the anti-CENP-B anticentromere autoantibody: evidence for restricted clonality.

Utilizing the centromere B fusion protein (CENP-B) and specific, matched monoclonal antiisotype reagents in an enzyme-linked immunosorbent assay, we found that anti-CENP-B autoantibodies were skewed to the IgG1 isotype. The overall kappa:lambda light chain ratio was 2:1, although several individual sera showed a strong predominance of one of the light chains. Isoelectric focusing of light chain-skewed sera showed polyclonal patterns. Our findings are consistent with the anti-CENP-B autoantibody response being a chronic, antigen-driven response.

Autoantibodies↗
PubMed1989

Human autoantibody to topoisomerase II.

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.

Adult↗