Antibody to the mitotic spindle apparatus: immunologic characteristics and cytologic studies

A naturally occurring autoantibody directed at the mitotic spindle polar apparatus (MSA) was detected in sera from 18 patients with defined or evolving connective tissue diseases by routine indirect immunofluorescence on a tissue culture cell. This IgG antibody stained the mitotic spindle poles of dividing tissue culture and tissue section substrates, with staining most prominent on substrates of human origin. With some sera, and cell lines, interphase cells showed isolated nuclear staining. Cytoplasmic staining was not apparent in any sera on any substrate. This specificity was shown to be distinct from tubulin by double fluorescence labeling studies. Patient sera exhibiting only this specificity did not precipitate antigens in rabbit or calf thymus nuclear extracts. However, several patients with systemic lupus erythematosus exhibited anti-MSA in combination with other autoantibodies. This autoantibody may be a useful probe for spindle pole-related structures.     

Autoantibody responses of individuals in an oil sands development community

The authors studied autoantibodies in sera from 214 individuals in an oil sands community by indirect immunofluorescence, an addressable laser bead immunoassay, and the Crithidia luciliae assay. They compared results with exposure data that included urinary metabolites, health questionnaires, and overt disease as recorded by visits to a physician or hospitalization. The prevalence of autoantibodies was 13%, compared with 10% in 30 controls from a distant community (p > 0.05). The study group had higher autoantibody titers and some disease-specific autoantibodies, but these were not associated with reported autoimmune diagnoses. There was no evidence of increased autoimmune disease or response in this petroleum development population with low levels of exposure.     

Identification of the B-cell epitopes of the early endosome antigen 1 (EEA1)

Early endosome antigen 1 (EEA1) is a target autoantigen in patients diagnosed with neurological and other autoimmune conditions. Eighteen of 65 sera (28%) that displayed a vesicular cytoplasmic staining pattern also immunoprecipitated the recombinant EEA1. These 18 sera were selected for further clinical, serological and epitope mapping studies. Thirty-six percent of the 18 patients had neurological diseases. Seventeen sera (94%) reacted with the partial length EEA1 constructs that included the C-terminal zinc finger (+FYVE) and the methyl accepting domain (LeuMA: amino acids 82-1411) in an addressable laser bead assay suggesting that the assay may be used for rapid laboratory detection of anti-EEA1 antibodies. Three of seven sera selected for epitope mapping studies bound to EEA1 peptides represented by amino acids 1096-1125, and two reacted with peptides represented by amino acids 1296-1320. One serum reacted only with the C-terminal peptide 1096-1125. The remaining serum reacted with other EEA1 epitopes. This data was supported by the observations that all the sera immunoprecipitated the C-terminal +FYVE (EEA1 1064-1411) construct, a peptide that also contained the linear epitopes 1096-1140. The limited epitope mapping studies suggest that the sera from patients with non-neurological diseases recognized epitopes in the central and C-terminal EEA1 domains, whereas the patients with neurological disease recognized a more restricted set of epitopes in the C-terminal.     

Identification of GRASP-1 as a novel 97 kDa autoantigen localized to endosomes

We have identified an autoantigen that is recognized by antibodies from an 18-year-old female with a history of recurrent infections who later in her clinical course developed Raynaud's phenomenon and telangiectasias. By indirect immunofluorescence (IIF), the index serum produced a unique cytoplasmic discrete speckled (CDS) staining pattern that partially colocalized with early endosome antigen 1 (EEA1) but not Golgi complex or other cytoplasmic organelles in HEp-2 cells. When HEp-2 cells were treated with 0.1 N HCl, the cytoplasmic speckled staining of the index serum was markedly decreased, suggesting that the reactive antigen was soluble. Western blot analysis showed a reactive approximately 97 kDa protein in a saline soluble protein preparation from HeLa cells. Mass spectrometric analysis of the excised 97 kDa band that was immunoprecipitated from HeLa cell extracts identified GRASP-1 as a possible target. The index serum and anti-GRASP-1 antibodies colocalized to structures in the cytoplasm of HEp-2 cells. Synthetic peptides representing the full-length GRASP-1 protein were used to identify reactive epitopes. Like many other cytoplasmic autoantigens, GRASP-1 has numerous coiled-coil domains throughout the protein with the exception of short segments at the amino and carboxyl terminus.     

Autoantibodies in lupus nephritis patients requiring renal transplantation

The goal of this nested case-control study was to compare autoantibody profiles in systemic lupus erythematosus (SLE) patients with lupus nephritis (LN), lupus nephritis patients requiring renal transplantation (LNTP) and a SLE control group without nephritis (CON). Sera were assayed for a variety of autoantibodies by addressable laser bead immunoassay (ALBIA) and enzyme-linked immunoassay (ELISA) and to dsDNA by Crithidia luciliae assay. The frequency of nucleosome autoantibodies was significantly greater in the LNTP group (79%) compared to the LN (18%) and CON (9%) groups (P < 0.0005). The frequency of other autoantibodies, including anti-dsDNA, did not differ significantly between groups. Among patients with LN, the odds of progressing to renal transplantation was 16-fold higher (OR 16.5 [95% CI 2.5, 125.7], P = 0.0005) in patients testing positive for anti-nucleosome antibodies compared to those who tested negative. Furthermore, the level of anti-nucleosome antibodies was significantly ( P < 0.00005) higher in the LNTP group (3.69 +/- 2.79) than the LN (0.51 +/- 0.51) and CON (0.34 +/- 0.44) groups. Review of 48 renal biopsies from 29 patients indicated that there was no difference in renal histological classification among patients with anti-nucleosome antibodies compared to those who tested negative. Our observations suggest that nucleosome autoantibodies are a biomarker for more severe SLE renal disease requiring transplantation.     

The role of the thymus-hypothalamus-pituitary-gonadal axis in normal immune processes and autoimmunity

Gonadal steroids play an important role in the development and regulation of the immune system. Their effects may be mediated through a thymus-hypothalamus-pituitary-gonadal axis. The thymus gland secretes factor(s), including thymosin beta 4, that affect the release of gonadotropin releasing hormone (GnRH). GnRH regulates the subsequent release of luteinizing hormone, thereby affecting early ovarian development. Thymic factors may be modulated by gonadal steroids. Studies indicate that levels of thymosin beta 4 decrease in postmenopausal and ovariectomized women. In diseases such as systemic lupus erythematosus, abnormal patterns of estrogen metabolism may affect thymic function and contribute to the etiology of the disease.     

Prolonged improvement of Raynaud's phenomenon and scleroderma after recombinant tissue plasminogen activator therapy

We describe a patient with systemic sclerosis (SSc; scleroderma) characterized by severe Raynaud's phenomenon, cutaneous sclerosis, and digital ulceration and subsequent amputation who was treated with recombinant tissue plasminogen activator (rt-PA) after acute myocardial infarction. She showed prompt improvement of the Raynaud's phenomenon and healing of the digital ulceration. After 18 months of followup, the Raynaud's phenomenon has remained mild, and there has been improvement in the cutaneous sclerosis. Since the pathophysiology of SSc has been associated with disorders of fibrinolysis and coagulation, this patient represents an interesting index case that might prompt further evaluation of rt-PA therapy in carefully selected patients.     

Primary biliary cirrhosis and autoantibodies.

Fifty years have passed since anti-mitochondrial antibodies were found in patients with primary biliary cirrhosis (PBC). PBC is an autoimmune hepatic disease in which 85-90% of patient antibodies bind to mitochondrial antigens that include pyruvate dehydrogenase complex (PDC)-E2 and other members of the oxaloacid dehydrogenase family. In addition, indirect immunofluorescence (IIF) assays utilizing HEp-2 cell substrates have been used to identify anti-centromere antibodies in 20-30% of PBC sera. These antibodies are generally easily recognized, however, anti-nuclear envelope and anti-multiple nuclear dot antibodies are occasionally more difficult to recognize with certainty by IIF. The use of enzyme linked immunosorbent assays that utilize recombinant gp210 (an autoantigen of the nuclear envelope) and/or sp100 (a protein target represented by multiple nuclear dots) should be particularly considered in anti-mitochondrial antibody negative PBC sera. Although the clinical significance of these antibodies still remains to be determined, there is evidence that the existence of anti-gp210 antibodies are related to poorer prognosis and more aggressive disease progression.