The kinetochore is part of the metaphase chromosome scaffold.

We used antisera from patients with the CREST syndrome of scleroderma (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) to show that an antigenic component of the kinetochore present in metaphase chromosomes is also present in nonhistone chromosome scaffolds isolated following extensive digestion of the DNA and extraction of the bulk of chromosomal protein. All sera from 12 scleroderma CREST patients previously shown by immunofluorescence microscopy to have circulating antikinetochore antibodies recognise a protein of Mr 77,000 (CREST-77) in an immunoblotting assay. 9 of the 12 sera also recognise an antigen of Mr 110,000 (CREST-110). These proteins are present in isolated chromosomes and nonhistone scaffolds derived from them by two different procedures. Sera of five scleroderma CREST patients who are antikinetochore negative (by immunofluorescence) bind to neither protein in immunoblots. These data suggest that CREST-77 (and possibly CREST-110) is a component of the human kinetochore, and that the kinetochore is an integral part of the mitotic chromosome scaffolding.

A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

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.

Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen.

We have isolated a series of overlapping cDNA clones for approximately 95% of the mRNA that encodes CENP-B, the 80-kD human centromere autoantigen recognized by patients with anticentromere antibodies. The cloned sequences encode a polypeptide with an apparent molecular mass appropriate for CENP-B. This polypeptide and CENP-B share three non-overlapping epitopes. The first two are defined by monoclonal antibodies elicited by injection of cloned fusion protein. Epitope 1 corresponds to a major antigenic site recognized by the anticentromere autoantibody used to obtain the original clone. Epitope 2 is a novel one not recognized by the autoantibody. These epitopes were shown to be distinct both by competitive binding experiments and by their presence or absence on different subcloned portions of the fusion protein. The third independent epitope, recognized by a subset of anticentromere-positive patient sera, maps to a region substantially closer to the amino terminus of the fusion protein. DNA and RNA blot analyses indicate that CENP-B is unrelated to CENP-C, a 140-kD centromere antigen also recognized by these antisera. CENP-B is the product of a 2.9-kb mRNA that is encoded by a single genetic locus. This mRNA is far too short to encode a polypeptide the size of CENP-C. The carboxy terminus of CENP-B contains two long domains comprised almost entirely of glutamic and aspartic acid residues. These domains may be responsible for anomalous migration of CENP-B on SDS-polyacrylamide gels, since the true molecular mass of CENP-B is approximately 65 kD, 15 kD less than the apparent molecular mass deduced from gel electrophoresis. Quite unexpectedly, immunofluorescence analysis using antibodies specific for CENP-B reveals that the levels of antigen vary widely between chromosomes.

High titers of autoantibodies to topoisomerase I (Scl-70) in sera from scleroderma patients.

Patients with rheumatic diseases often have circulating autoantibodies to nuclear components. The clinical significance of the antibodies is controversial, although in some cases they are valuable in the diagnosis of the disease. This report presents results of a study of Scl-70, an autoantigen recognized by sera of many patients with the most severe form of progressive systemic sclerosis. It was possible to show, by three independent criteria, that Scl-70 is the abundant nuclear enzyme DNA topoisomerase I. Therefore, antibody probes of high titer and high affinity are now available for the study of this important nuclear enzyme.

The relation of immunoglobulin class, pattern of anti-nuclear antibody, and complement-fixing antibodies to DNA in sera from patients with systemic lupus erythematosus.

Sera from 55 patients with systemic lupus erythematosus were studied to clarify the significance of the patterns of nuclear fluorescence observed. The sera in which the IgG fraction produced a peripheral pattern of nuclear fluorescence were found to contain complement-fixing antibodies to native DNA and to DNA-histone complexes. This correlation did not exist when complement-fixing activity was compared to the IgM nuclear patterns. Sera which contained only complement-fixing antibodies to denatured DNA and which did not react with native DNA or nucleoprotein did not produce the peripheral pattern of nuclear fluorescence. The data suggest that single strands of DNA were not the reactive groups in the nucleus responsible for the peripheral pattern. The results support the conclusion that DNA within a DNA-protein complex may be the nuclear antigen responsible for the peripheral pattern of nuclear fluorescence. Analysis of the clinical data revealed that a close correlation existed between the presence of IgG peripheral pattern, complement-fixing antibodies to DNA and histone-DNA complexes, and clinical manifestation of active disease.

Clinical significance of serum properdin levels and properdin deposition in the dermal-epidermal junction in systemic lupus erythematosus.

61 biopsies of normal skin from the deltoid area and lesional skin from various sites from 48 patients with systemic lupus erythematosus (SLE) were studied for the presence of properdin, C3, C4, and immunoglobulins (IgG, IgM, and IgA) in the dermal-epidermal junction (DEJ) using direct and indirect immunofluorescence. Properdin was present in 50% of normal and 40% of lesional skins. Properdin was present without C4 in only 2 of 38 nonlesional skin biopsies and in only 2 of 20 lesions. There was no significant difference in incidence of deposition of any of the six proteins studied between nonlesional and lesional skin. The frequency of deposition of each of the proteins correlated with clinical disease activity. The presence of proteins in the DEJ did not correlate with the presence of active renal disease at the time of biopsy nor with previously documented active nephritis. In addition, no other single clinical manifestation correlated with the presence of DEJ deposition of any protein studied. IgA was not demonstrated in the DEJ of nonlesional skin of 16 patients in remission and was present in 7 of 23 patients with active disease (P less than 0.05). Deposition of properdin in lesional skin correlated with the presence of extracutaneous disease activity (P less than 0.05). Analysis of serologic studies on serum obtained at the time of biopsy revealed a statistically significant correlation between C4 and C3 (r = 0.67). This correlation was stronger than that between properdin and C4 (r = 0.37). Titer of antinuclear antibody and percent of DNA binding correlated better with C4 levels than with properdin levels. Serum properdin levels were significantly lower in patients with active disease than in those in remission (P less than 0.05). Serum properdin levels were significantly lower in patients with properdin deposits in lesional skin than in those without properdin deposits. The data suggest that both alternative and classical pathways are activated in patients with clinically active SLE.

Three human chromosomal autoantigens are recognized by sera from patients with anti-centromere antibodies.

We have identified 39 individuals with anti-centromere antibodies (ACA) in our patient population, all of whom have Raynaud's syndrome or disease. We have used sera from the ACA-positive patients and from 123 controls (22 normal individuals and 101 additional patients with either Raynaud's disease or Raynaud's syndrome plus an associated connective tissue disease) to screen the proteins of highly purified human (HeLa) mitotic chromosomes by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotting. Three antigens were recognized by the sera from the ACA-positive patients. These were centromere protein (CENP)-B (80,000 mol wt--recognized by all ACA-positive sera), CENP-A (17,000 mol wt--recognized by 38 of 39 ACA-positive sera), and CENP-C (140,000 mol wt--recognized by 37 of 39 ACA-positive sera). None of these antigens were recognized by any of the 123 control sera, although binding was occasionally seen to other chromosomal antigens. Therefore the ACA response is highly uniform in our patient population. Antibody to CENP-B shows a 100% correlation with anti-centromere staining by indirect immunofluorescence.

Immunoglobulin M autoantibody to vimentin intermediate filaments.

Serum from a patient with the CREST Syndrome and systemic lupus erythematosus contained an IgM antibody that reacted at dilutions up to 1:800 with a fibrous cytoplasmic network in several epithelioid and fibroblastic cell lines. The antibody was shown by immunofluorescence microscopy to label a specific subset of cytoskeletal polymers, the intermediate filaments. The reactive antigen from this biochemically heterogeneous group of filaments was established as the 58,000-mol wt protein, vimentin: (a) the patient's serum reacts with a range of cell lines that contain intermediate filaments composed of vimentin, but not with cells whose intermediate filaments are composed of different protein subunits; (b) in PTK2 epithelioid cells the serum reacts with the class of filaments that coils around the nucleus after colchicine treatment (vimentin) and not with the filaments that remain dispersed after colchicine (prekeratin); and (c) the component of reactive cells that combines with the serum is shown by immunoelectrophoresis to be a 58,000-mol wt protein antigen. A similar antibody that binds intermediate filaments of PTK2 cells was encountered at lower titer in some sera from other patients with connective tissue diseases and in control sera. Previous routine antinuclear antibody assays using mouse liver or commercially prepared HEp-2 cells have failed to reveal anticytoskeletal antibodies in patient sera, perhaps due to inadequate presentation or preservation of cytoplasmic antigens.

Analysis of human antitopoisomerase-I idiotypes.

Antibodies to topoisomerase-I are present in approximately 26% of patients with scleroderma and are rarely found in patients with other diseases. In the current study, the expression of the antitopoisomerase-I (antitopo-I) idiotype from two scleroderma patients (E.M. and S.G.) and from a healthy individual (N.M.) were studied. Idiotype EM-SCL was restricted to the three classes of antitopo-I, whereas idiotypes SG-SCL and NM were found in all classes of antitopo-I as well as in their non-antitopo-I Igs. Sera from 9 of 10 antitopo-I-positive unrelated scleroderma patients expressed idiotype SG-SCL and some also expressed idiotype NM. Sera from N.M.'s 3 daughters and from 7 of 18 nonrelated normals expressed idiotype NM in the three immunoglobulin classes of non-antitopo-I. Two of the antitopo-I antibodies expressed a cross-reacting idiotype (CRI) that is present in non-antitopo-I antibodies from the same donor. Contrary to the natural CRI, SG-SCL's CRI is closely associated with the antigen binding site. Antitopo-I idiotypes are on the heavy chains. Like many other autoantibodies, Id-SG-SCL use VH4.2-1, DXP1, and JH4 in germline configuration.

Autoantibodies in scleroderma and tightskin mice.

There is much evidence to suggest that scleroderma in human patients is caused by a fundamental defect in the immune system. In tightskin mice, the scleroderma syndrome is associated with autoimmunity, particularly autoantibodies interacting with scleroderma target antigens.

Aseptic necrosis of bone in systemic lupus erythematosus. Relationship to corticosteroid therapy.

The relationship of corticosteroid therapy to the development of aseptic necrosis (AN) in 365 patients with systemic lupus erythematosus (SLE) was investigated. Seventeen patients (4.7%) were identified as having AN. The dosage of corticosteroids ingested during the initial period of therapy in patients with AN was tabulated and compared with that of 25 SLE control patients. There was a substantially greater dose of costicosteroids ingested in the first one, three, and six months of therapy in the patients with AN than in the control SLE group. Severity of disease and duration of therapy were not found to correlate with AN. Total corticosteroid dose was virtually identical in both groups. Thus, high initial corticosteroid dosages in patients with SLE seem to be associated with the development of AN.

Immune-complex nephritis in bacterial endocarditis.

Glomerulonephritis developed in a 42-year-old man with subacute bacterial endocarditis caused by an alpha-hemolytic Streptococcus. The patient showed hypocomplementemia and an elevated serum rheumatoid factor titer. Immunofluorescence microscopy of a renal biopsy specimen demonstrated granular deposits of IgM and C3 in all glomeruli studied. With the indirect immunofluores(ence technique and specific antiserum, the antigen in glomerular deposits was observed to correspond to the organism found in the blood cultures. These findings can be taken as further evidence that the glomerulonephritis of subacute bacterial enoocarditis represent an immune-complex disease.

Efficacy of antimalarials in systemic lupus erythematosus.

No recent study has been published on the efficacy of antimalarial drugs in the treatment of systemic lupus erythematosus (SLE). However, antimalarial drug therapy was reported in several early studies to control SLE more effectively than corticosteroids alone and to permit lower doses of steroids to be used. To confirm these findings, we conducted a retrospective study of 43 SLE patients who had been taking antimalarials but in whom such therapy had been discontinued after two to 13 years due to the development of macular changes. Each patient served as his own control by the matching of each year during which antimalarial drugs were taken with one year without antimalarial medication. There was a total of 76 matched years for the 43 patients. Overall, no significant correlation was shown between antimalarial drug therapy and the presence or absence of general symptoms (fever, fatigue, weight loss); skin, cardiac, pulmonary, or central nervous system manifestations, arthritis, or other symptoms or signs that were measured. Although antimalarials reduced the required dosage of corticosteroids significantly (p less than 0.05), the amount of reduction was not considered clinically meaningful. Treatment with antimalarial drugs significantly reduced the number of disease flare-ups (p less than 0.05). There were also significant differences between the years with and without chloroquine therapy 500 mg per day (the only regimen for which adequate data were available for analysis) in terms of general symptoms and skin manifestations (p less than 0.05). A prospective, double-blind study of large numbers of patients would be important to confirm the findings of this retrospective study--and the observations of many rheumatologists that antimalarials are helpful in the treatment of SLE.

Antibodies to native DNA and serum complement (C3) levels. Application to diagnosis and classification of systemic lupus erythematosus.

The sensitivity and specificity of the presence of antibodies to native DNA and low serum C3 levels were investigated in a prospective study in 98 patients with systemic lupus erythematosus who were followed for a mean of 38.4 months. Hospitalized patients, patients with other connective tissue diseases, and subjects without any disease served as the control group. Seventy-two percent of the patients with systemic lupus erythematosus had a high DNA-binding value (more than 33 percent) initially, and an additional 20 percent had a high DNA-binding value later in the course of the illness. Similarly, C3 levels were low (less than 81 mg/100 ml) in 38 percent of the patients with systemic lupus erythematosus initially and in 66 percent of the patients at any time during the study. High DNA-binding and low C3 levels each showed extremely high predictive value (94 percent) for the diagnosis of systemic lupus erythematosus when applied in a patient population in which that diagnosis was considered. The presence of both abnormalities was 100 percent correct in predicting the diagnosis os systemic lupus erythematosus. Both tests should be included in future criteria for the diagnosis and classification of systemic lupus erythematosus.

Familial partial deficiency of the third component of complement (C3) and the hypocomplementemic cutaneous vasculitis syndrome.

Familial hypocomplementemia of the third component of complement (C3) was found in four members of a family. The prospositus had cutaneous vasculitis, hypocomplementemia, arthralgia, proteinuria and thrombocytopenia. The combination of clinical, laboratory and pathologic findings resembled the "hypocomplementemic cutaneous vasculitis syndrome" (HCVS) or the "SLE-like syndrome" but serum C3 concentration was 35 to 57 per cent of normal in the propositus and in three relatives. Results of Clq precipitins, cryoglobulins and serologic tests for systemic lupus erythematosus were negative. Proteinuria (815 mg/day) but no hematuria was present. Analysis of the C3 phenotypes in this family showed that three hypocomplementemic members were apparent homozygous C3 slow but one was heterozygous C3 fast-slow. Metabolic studies with 125-Iodinated C3 in the clinically normal mother showed a 50 per cent reduction in C3 synthesis which was consistent with hypocomplementemia documented by serum protein assay. The occurrence of an immune complex-like disease (with characteristics of the HCVS) in a patient with a familial deficiency of C3 suggests that the preexisting C3 deficiency may predispose such persons to certain diseases.

Autoantibodies in patients with primary pulmonary hypertension: association with anti-Ku.

PURPOSE: Patients with primary pulmonary hypertension (PPH) frequently have Raynaud's phenomenon, serum antinuclear antibodies (ANAs), and/or pulmonary vascular lesions similar to those seen in certain connective tissue diseases, especially scleroderma. A number of relatively disease-specific autoantibodies have been described in connective tissue diseases but have not been studied in patients with PPH. Therefore, sera from PPH patients were studied for a variety of autoantibodies, seeking a possible link between this pulmonary disorder and connective tissue diseases. PATIENTS AND METHODS: Sera from 31 patients with PPH and 24 with secondary pulmonary hypertension (SPH) were studied for the following autoantibodies: anti-centromere (indirect immunofluorescence of Hep-2 cells), anti-CENP-B by immunoblotting and enzyme immunoassay (EIA) using cloned CENP-B fusion protein, anti-topoisomerase I (Scl-70), anti-Ku using immunoblotting of affinity purified antigens, anti-cardiolipin using EIA, and anti-Ro (SS-A), La (SS-B), Sm, nRNP, Jo-1, PM-Scl, and Mi-2 by counter-current immunoelectrophoresis. RESULTS: Anti-Ku antibodies were found in 23% of patients with PPH, 4% with SPH, and none of 24 normal controls (PPH versus SPH, p = 0.06: PPH versus controls, p = 0.01). Antibodies to CENP-B were found in one patient each with PPH and SPH, anti-topoisomerase I in one with SPH, and anti-Ro (SS-A) and La (SS-B) in one with PPH. Overall, 12 patients (39%) with PPH had Raynaud's phenomenon or positive ANA results, with 9 (29%) having more specific autoantibodies associated with connective tissue diseases. CONCLUSIONS: These results further suggest a link between at least a subgroup of patients with PPH and autoimmune connective tissue diseases, with anti-Ku antibodies being a possible new serologic marker.

Autoantibodies in systemic sclerosis.

There are 3 major autoantibodies in sera from patients with scleroderma: 1) anticentromere antibodies (ACA), 2) anti-topoisomerase I (anti-topo I), and 3) anti-RNA polymerases. Each is present in about 25% of patients and are mutually exclusive. ACA are found in patients with primary and secondary Raynaud's disease and in patients with primary biliary cirrhosis. Anti-topo I and anti-RNA polymerases are found exclusively in scleroderma. Each autoantibody is present in specific subsets of scleroderma patients. ACA and anti-topo I have been well studied and their presence and titer are stable over time. The anti-topo I and ACA are of all three isotypes, recognize multiple epitopes on the antigens and have stable cross reactive or private idiotypes. The antigen, topoisomerase I, has domains which have homology to viral proteins. Other autoantibodies predominantly recognize nucleolar antigens, are found in less than 15% of patients, and are not specific for scleroderma.

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.