Non-myeloablative transplantation of bone marrow expressing self-antigen establishes peripheral tolerance and completely prevents autoimmunity in mice.

Myeloablative transplantation of bone marrow (BM) engineered to express myelin oligodendrocyte glycoprotein (MOG) establishes central intrathymic tolerance and completely prevents MOG-induced experimental autoimmune encephalomyelitis (EAE) in mice. Here we asked whether non-myeloablative transplantation of MOG expressing BM (pMOG-bone marrow transplantation (BMT)) can also provide the same protection. Using stepwise reduction of irradiation doses, 275 cGy irradiation with pMOG-BMT protected 100% of mice from EAE development even with two subsequent re-challenge with MOG. Irradiation doses <275 cGy produced dose-dependent partial protection with significant disease protection still evident at 50 cGy. Splenocytes from 275 cGy recipients proliferated to MOG stimulation in vitro, indicating that MOG-reactive cells are present in the periphery but failed to induce disease. MOG-stimulated splenocytes produced little or no interleukin-17, interferon-γ, granulocyte-monocyte colony stimulating factor and tumor necrosis factor-α compared with EAE control. Adoptive transfer of CD4 T cells from EAE-resistant mice into Rag2(-/-) mice devoid of MOG expression resulted in MOG-induced EAE in ~74% of mice. Treatment of EAE-resistant mice with anti-programmed death 1 (PD-1) monoclonal antibody-induced EAE in 67% of mice. We conclude that non-myeloablative transplantation of self-antigen expressing BM induces robust peripheral tolerance that completely prevented EAE development. Our findings implicate clonal anergy and the PD-1 pathway in the maintenance of peripheral tolerance.

Nonmyeloablative conditioning generates autoantigen-encoding bone marrow that prevents and cures an experimental autoimmune disease.

Autoimmune diseases result from chronic targeted immune responses that lead to tissue pathology and disease. The potential of autologous hematopoietic stem cells transplantation as a treatment for autoimmunity is currently being trialled but disease relapse is an issue. We have previously shown in a mouse model of experimental autoimmune encephalomyelitis (EAE) that the transplantation of bone marrow (BM) transduced to encode the autoantigen myelin oligodendrocyte glycoprotein (MOG) can prevent disease induction. However these studies were performed using lethal irradiation to generate BM chimeras and a critical factor for translation to humans would be the ability to utilize low toxic preconditioning regimes. In this study, treosulfan was used as a nonmyeloablative agent to generate BM chimeras encoding MOG and assessed in models of EAE induction and reversal. We find that treosulfan conditioning can promote a low degree of chimerism that is sufficient to promote antigen specific tolerance and protect mice from EAE. When incorporated into a curative protocol for treating mice with established EAE, nonmyeloablative conditioning and low chimerism was equally efficient in maintaining disease resistance. These studies further underpin the potential and feasibility of utilizing a gene therapy approach to treat autoimmune disease.

Cell division autoantigen 1 plays a profibrotic role by modulating downstream signalling of TGF-beta in a murine diabetic model of atherosclerosis.

AIMS/HYPOTHESIS: Excess accumulation of vascular extracellular matrix (ECM) is an important pathological process in cardiovascular diseases including diabetes-associated atherosclerosis. We explored how a recently identified molecule, cell division autoantigen 1 (CDA1), influences the profibrotic TGF-beta pathway leading to vascular ECM accumulation. METHODS: Expression levels of genes encoding for CDA1, TGF-beta and connective tissue growth factor (CTGF) were examined in aorta from Apoe(-/-) mice with or without diabetes. We used retroviral and adenoviral constructs to knockdown or overexpress Tspyl2, the gene encoding CDA1, in mouse vascular smooth muscle cells (VSMCs) with or without TGF-beta treatment in order to demonstrate the role of CDA1 in TGF-beta signalling. RESULTS: In vivo studies indicated that the mRNA levels of CDA1-encoding gene Tspyl2 and protein levels of CDA1 were elevated in the aorta of diabetic Apoe(-/-) mice, accompanied by increased levels of Tgf-beta (also known as Tgfb1), Ctgf and ECM accumulation. In vitro studies in vascular cells showed that TGF-beta treatment rapidly increased CDA1 protein levels, which then amplified TGF-beta signalling leading to upregulation of ECM genes. Knockdown of CDA1-encoding gene Tspyl2 to reduce cellular CDA1 level markedly attenuated TGF-beta-stimulated MAD homologue 3 (drosophila; SMAD3) phosphorylation and transcriptional activities. CDA1 overproduction increased and Tspyl2 knockdown decreased expression of TGF-beta receptor type I, TbetarI (also known as Tgfbr1), but not TGF-beta receptor type II, TbetarII (also known as Tgfbr2), providing a mechanism for CDA1's action in modulating TGF-beta signalling. Knockdown of CDA1-encoding gene Tspyl2 also blocked the profibrotic effect of TGF-beta in VSMCs. CONCLUSIONS/INTERPRETATION: CDA1 plays an important role in vascular ECM accumulation by amplifying TGF-beta signalling. This is critical for the profibrotic effect of TGF-beta in the vasculature. CDA1 is therefore a potential target for attenuating vascular ECM accumulation caused by enhanced TGF-beta action, as seen in diabetic atherosclerosis.

An autoimmune disease with multiple molecular targets abrogated by the transgenic expression of a single autoantigen in the thymus.

Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.

From coiled tubules to a secretory canaliculus: a new model for membrane transformation and acid secretion by gastric parietal cells.

The acid-secreting gastric parietal cell has a unique secretory membrane system. This membrane system exists in an inactive (non-secreting) and an active (secreting) form. The current accepted model to explain the transformation events associated with the conversion of the non-secreting membrane to the secreting membrane, and vice versa, invokes membrane recycling of elongated vesicle structures. However, recent studies employing cryopreparation have shown that the non-secreting membrane in these cells is actually a complex network of helically coiled tubules. Here, we present an alternative model to explain how the membrane in parietal cells is activated to secrete HCl.

Immunoreactive beta-endorphin in a subpopulation of mouse spleen macrophages.

Using radioimmunoassay and immunofluorescence with antibodies to beta-endorphin (beta EP) and ACTH, we have shown that a subpopulation of mouse spleen cells, expressing Mac-1, a marker of macrophage differentiation, contains immunoreactive (ir)-beta EP, ir-ACTH, and smaller amounts of presumptive higher molecular weight forms of both. Neither nonadherent spleen cells, nor adherent or nonadherent cells from peripheral blood, contained detectable levels of these peptides. These findings suggest that beta EP and ACTH may be synthesized in a subpopulation of spleen macrophages, and are consistent with the possibility that these or related peptides may modulate lymphocyte function in the specific microenvironment of the spleen.

EEA1, a tethering protein of the early sorting endosome, shows a polarized distribution in hippocampal neurons, epithelial cells, and fibroblasts.

EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.

Smooth muscle-associated antigen in experimental cutaneous squamous cell carcinoma, keratoacanthoma, and papilloma.

Cryostat sections of 29 squamous cell carcinomas, 13 keratoacanthomas, and 12 papillomas, induced by 7,12-dimethylbenz(a)anthracene in the skin of rabbits and rats were examined by indirect immunofluorescence with human serum containing antibody to smooth muscle. Linear or granular staining of the cell outlines of the basal squamous cell layers was seen most extensively in the carcinomas, less in keratoacanthomas, and least in papillomas. In addition, squamous cell carcinomas showed this pattern of staining at advancing tumor margins and in invasive cords and tumor cell nests in the dermis. Four keratoacanthomas also showed prominent staining of the basement membrane area. The specificity of the staining reaction was established by its prevention on neutralization absorptions of the serum with extracts or homogenates of smooth muscle. The epidermal cells of normal rabbit and rat skin gave negative staining reactions. The presence of smooth muscle-associated antigen probably corresponds to cellular microfilaments.

Immunofluorescent characterization of rat kidney tumors according to the distribution of actin as revealed by specific antiactin antibody.

A series of 15 mesenchymal and 10 cortical epithelial tumors induced in the rat kidney by dimethylnitrosamine was investigated for immunofluorescent reactivity with a human antiactin antibody. Cells of epithelial tumors showed staining restricted to peripheral sites, corresponding to the brush border region. All various neoplastic cells forms comprising renal mesenchymal tumor were characterized by cytoplasmic staining in pattersn that varied with cell type. Epithelial profiles in the form of tubules and islands of epithelium showed staining patterns, or absence of them, consistent with their identity as sequestered segments of preexisting nephrons. It is suggested that the difference in actin distribution within the cytoplasm of cells of the two types of renal neoplasm, mesenchymal and epithelial, might reflect their difference in local invasive growth.

Increased expression of actin-like protein in human and ethylnitrosourea-induced tumors of the nervous system.

Twenty-one human intracranial tumors comprising 15 astrocytomas and 6 meningiomas and 26 ethylnitrosourea-induced rat neural tumors comprising 7 astrocytomas and 9 schwannomas were examined by indirect immunofluorescence for reactivity with a human anti-actin antibody. In cryostat sections both human and rat astrocytomas showed an increased reaction with the anti-actin antibody compared to normal astrocytes, and the reaction with astrocytomas was greater than that with meningiomas. Malignant rat schwannomas also showed prominent anti-actin staining contrasting with the negative reaction in normal Schwann cells. These in vivo observations were paralleled by concurrent studies with impression films and in vitro monolayer cultures of tumor tissue. The results, reviewed in the light pfo previous studies of anti-actin antibody reactivity with other nonneural tumors, suggest that an enhanced actin expression in vivo may be a general feature of the neoplastic state and that this increased expression may be more pronounced in malignant than in benign tumors.

Human CKIalpha(L) and CKIalpha(S) are encoded by both 2.4- and 4. 2-kb transcripts, the longer containing multiple RNA-destablising elements.

Casein kinase I (CKI) are a family of conserved second messenger-independent serine/threonine protein kinases found in all eukaryotes. The avian and mammalian CKI alpha isoform has four splice variants differing in the presence or absence of 28 amino acids ('(L)' insertion) in the catalytic domain and/or 12 amino acids ('(S)' insertion) in the regulatory domain. Here we report the isolation of cDNAs encoding human CKIalpha(L) and CKIalpha(S). We find human CKIalpha(L) has a preference to phosphorylate phosvitin over casein, with a higher K(m) for casein than phosvitin, the reverse being the case for human CKIalpha(S). Both human CKIalpha(L), and CKIalpha(S) are derived from 4.2-kb mRNA transcripts and 2.4-kb transcripts, the latter probably generated by use of an alternate polyadenylation signal identified in the longer transcripts. The 4. 2-kb transcripts contain six RNA-destabilising AU-rich element (ARE) motifs in the 3'-untranslated region (UTR), while the 2.4-kb transcripts contain a single ARE motif. In vitro analysis of CKI alpha 3'-UTR RNA sequences suggests that in HeLa cells, the longer 3'-UTR transcripts are likely to degrade approximately 13 times faster than the shorter 3'-UTR transcripts. This is the first report of a kinase mRNA containing multiple RNA-destabilising AREs in the longer of two mRNA transcripts.

Experimental autoimmune gastritis: mouse models of human organ-specific autoimmune disease.

Experimental autoimmune gastritis (EAG) is an excellent model of human autoimmune gastritis, the underlying cause of pernicious anaemia. Murine autoimmune gastritis replicates human gastritis in being characterized by a chronic inflammatory mononuclear cell infiltrate in the gastric mucosa, destruction of parietal and zymogenic cells, and autoantibodies to the alpha-and beta-subunits of the gastric H+/K+ ATPase. Disease is induced strain specifically in gastritis-susceptible BALB/c mice by methods with a greater variety than those for most other experimental autoimmune diseases. The disease is induced in the regional gastric lymph node in which pathogenic CD4+ T cells are recruited. The model provides an excellent illustration of regulation by CD4+CD25+T cells, and, indeed, the removal of such regulatory cells, e.g., by neonatal thymectomy, is thought to be a major mechanism by which disease can develop. The culprit T helper type 1 (Th1) CD4+ T cells recognize either the alpha- or beta-subunits of the gastric H+/K+ ATPase, but the beta-subunit appears to be the initiating autoantigen, while the alpha-subunit may have a role in perpetuating disease. Since no specific environmental modifiers are identifiable, the origins of the disease are intrinsic; this is illustrated by the capacity of a cytokine (GM-CSF)-dependent inflammatory stimulus in the stomach to initiate EAG, according to a transgenic model in which thymectomy is dispensible. Thus, EAG is an exquisite model for a reductionist analysis of the multiple elements that in combination induce autoimmunity in humans.

Homing and adhesion molecules in autoimmune gastritis.

The pathogenesis of autoimmune gastritis is the result of lymphocyte infiltration of the gastric mucosa, however, the events leading to the selective extravasation of autoreactive lymphocytes are unclear. Here we have examined the expression of adhesion molecules in the gastric mucosa of BALB/c mice with neonatal thymectomy-induced gastritis. The overall area of vascular endothelium was not significantly different between gastritic and non-gastritic mice. However, a significant increase in the area of mucosal endothelium expressing MAdCAM-1 in gastritic mice was observed. Treatment of neonatally thymectomized BALB/c mice with a MAdCAM-1 specific monoclonal antibody (MECA 367) reduced the incidence of autoimmune gastritis from 80 to 26%. Treatment with a monoclonal antibody (R1-2) directed to the MAdCAM-1 ligand, alpha4beta7, also resulted in a reduction in the incidence of gastritis to 40%. These findings identify the alpha4beta7/MAdCAM-I interaction as a pivotal event in the initiation of autoimmune gastritis.

Microtubules are involved in bafilomycin A1-induced tubulation and Rab5-dependent vacuolation of early endosomes.

The small GTPase Rab5 is an important regulator of membrane fusion in the early endocytic pathway. Here we have studied at the light microscopy level the morphology of early endosomes in MDCK cells stably expressing a GTPase-deficient Rab5 mutant, Rab5 Q79L, N-terminally tagged with a myc-epitope. These cells contain large vacuoles, readily visible by phase-contrast microscopy. Confocal immunofluorescence microscopy showed the presence of the epitopetagged protein on large perinuclear vacuoles, as well as on smaller peripheral structures. A subset of the perinuclear vacuoles appeared to colocalize with the late endosomal GTPase, Rab7. In addition, a population of very large Rab7-positive, Rab5 Q79L-negative structures were observed, suggesting that an increase in the size of early endosomes may be accompanied by an increased size of later or more mature endocytic structures. Using antibodies against the myc epitope and the early endosomal autoantigen EEA1 as markers, we found that endosomes in wild-type and mutant MDCK cells rapidly tubulate in the presence of bafilomycin A1, an inhibitor of vacuolar H(+)-ATPase. Elongated or tubular endosomes partially colocalized with microtubules and were redistributed upon preincubation with the microtubule depolymerizing agent nocodazole before bafilomycin A1 treatment. Treatment of the Rab5 Q79L expressing cells with nocodazole alone led to a spatial redistribution and a significant decrease in the size of EEA1-positive structures, whereas their number increased. These results implicate microtubules in the bafilomycin A1-induced tubulation of endosomes as well as in the vacuolation of endosomes caused by Rab5 Q79L.

The glycolytic enzyme enolase is present in sperm tail and displays nucleotide-dependent association with microtubules.

We examined the expression and localisation of enolase (2-phospho-D-glycerate hydrolase) in differentiating rat spermatogenic cells. We found that enolase is most abundant in mature spermatozoa and in residual cytoplasmic bodies detached from elongating spermatids with little to no enolase detected in meiotic primary spermatocytes and round spermatids. We localised enolase mostly to the tail of mature spermatozoa by immunoblotting and by immunofluorescence. RT-PCR analysis of differentiating spermatogenic cells detected only the alpha isoform of enolase. As several glycolytic enzymes are known to associate with microtubules prepared from brain, we investigated the association of enolase with brain and testis microtubules. We found that only a small fraction of testis and brain-derived cytosolic enolase (4.9% and 11.2%, respectively) co-sediments with microtubules stabilised in the presence of taxol. In the presence of certain nucleotides in excess (3 mM ATP, CTP, GTP and ITP) the association of enolase with microtubules was disrupted, however, this was not the case for UTP. This observation is consistent with the finding that in the presence of 0.5 mM AMP-PNP, a nonhydrolysable analogue of ATP, there is an increased association of enolase with microtubules. We propose that the nucleotide-dependent association of enolase with microtubules regulates enzyme activity by linking energy production to utilisation.

Novel variant of p230 trans-Golgi network protein identified by serum from Sjögren's syndrome patient.

Trans-Golgi network (TGN) protein p230 is a peripheral membrane protein associated with the cytoplasmic face of the TGN. TGNp230 is an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, associates with non-clathrin-coated vesicles arising from the TGN, and is implicated in vesicle biogenesis. Here we used an autoimmune serum from a patient with S ogren's syndrome to clone partial cDNAs from a human hepatoma HepG2 expression library. The partial cDNAs encoded a novel amino-terminal splice variant of TGNp230. Specific reactivity of the autoimmune serum for p230 is supported by immunofluorescene staining of the Golgi apparatus, immunoblotting of a > 200-kDa HeLa cell protein, and reactivity with a bacterially expressed GST-p230 fusion protein. The alternative splicing occurs within the first proline-rich domain of p230. It comprises a deletion of 30 bp followed immediately by an additional 66 bp absent in the published sequence. RT-PCR analysis indicated that the splicing occurs independently of previously reported carboxyl-terminal splicing, and that this novel splice variant is more frequent than the previously reported p230. The novel splice variant of p230 is also located at the TGN. We propose that p230 splice variants may be implicated in selection of cargo molecules for vesicles arising from the TGN.

Ovarian immunoreactive beta-endorphin and estrous cycle in the rat.

Adult female Sprague-Dawley rats were killed at different stages of a 4-day estrous cycle, and ovaries and anterior pituitaries examined for content of immunoreactive beta-endorphin by RIA and for localization by indirect immunofluorescence. Two anti-beta-endorphin antisera, both recognizing different antigenic determinants of human-beta-endorphin, showed intense immunofluorescence staining of cells localized predominantly in ovarian corpora lutea. At proestrus, both large and small luteal cells stained positively but only the large luteal cells were immunofluorescence positive at other stages of the estrous cycle. In addition, less intense staining of granulosa cells was occasionally observed in secondary and antral follicles; scattered cells in the interstitium were also weakly positive. In contrast, cells of primordial and primary follicles, and those of theca tissue were consistently negative. Ovarian levels of immunoreactive beta-endorphin were found to be lowest at estrus (2.1 +/- 0.18 ng/g; n = 8, mean +/- SE) and significantly raised in stepwise manner over metestrus and diestrus to a peak (approximately 4 X estrous levels) at proestrus; in contrast, immunoreactive beta-endorphin content of anterior pituitaries remained unaltered during the same period. Sephadex G-50 gel chromatography of ovarian extracts revealed three distinct peaks of immunoreactive beta-endorphin, a minor peak in the void volume, and two major peaks of unequal size eluting at mol wt approximately 11.5K and approximately 3.5K. The major species of low molecular weight immunoreactive beta-endorphin on reverse phase HPLC was beta-endorphin1-31. We conclude from the findings that, in adult rat ovaries, luteal, granulosa, and interstitial cells are responsible for the production of immunoreactive beta-endorphin and that this production, being related to the estrous cycle, is presumably under the direct or indirect influence of gonadotropins.

Immunoperoxidase EM localisation of cytoplasmic actin in cultured fibroblasts.

Human smooth muscle autoantibody (SMA) of defined anti-actin specificity was tested by the indirect immunoperoxidase staining method on frozen sections of tissues and on cultured rat lung fibroblasts. The serum stained tissue sections and cultured fibroblasts in a pattern identical with that obtained by indirect immunofluorescence. Ultrastructural studies carried out on the immunoperoxidase stained cells showed that the long parallel filaments spanning the long axis of cultured fibroblasts seen by light microscopy correspond with the thick bundles of microfilaments.

Flow microfluorometry detects IgM autoantibody to oligodendrocytes in multiple sclerosis.

Freshly isolated canine oligodendrocytes were reacted by indirect membrane immunofluorescence with 44 sera from patients with multiple sclerosis (MS). Using analysis by flow microfluorometry (FMF), we found significant IgM antibody binding to the surfaces of oligodendrocytes in the MS sera. The fluorescence intensity of cell surface reactions for MS sera (F.I. greater than 40 = 37.2 +/- 21.34%) was significantly different (P less than 0.001) to that for 53 sera from normal subjects (10.0 +/- 8.97%), 15 sera from patients with Murray Valley encephalitis (6.18 +/- 5.3%), 22 with brain tumours (6.18 +/- 5.3%), 25 with SLE (13.42 +/- 11.65%), 7 with GBS (9.77 +/- 3.9%) and 7 miscellaneous neurological disorders (6.87 +/- 3.04%). Cell surface binding was restricted to oligodendrocytes and was absorbed out by oligodendrocytes but not by liver cells or kidney cells. Oligodendrocytes were identified by conventional histology, phase-contrast optics, electron microscopy (EM) and by cell surface reactions with anti-galactocerebroside, a specific immunocytological marker for oligodendrocytes. A cell sort of the single 0 degree FMF scatter peak followed by EM examination confirmed that the reactive cells consisted exclusively of oligodendrocytes. Viability of oligodendrocytes before and after the staining reactions, was greater than 80% as assessed by trypan blue and fluorescein diacetate exclusion. The possibility that immune reactions mediated by the surface-reactive antibody with readily accessible cell surface autoantigens in vivo may contribute to the loss of oligodendrocytes and demyelination in MS is raised.

Cell cycle-associated autoantibodies: markers for autoimmunity and probes for molecular cell biology.

Antinuclear autoantibodies are useful diagnostic markers for systemic autoimmune diseases and as probes for the molecular cell biology of nuclear proteins. Here, we review a subset of autoantibodies to nuclear and cytoplasmic proteins involved in the cell cycle. We propose a classification of these autoantibodies into S-phase (DNA Synthesis) and M-phase (Mitosis) autoantibodies. S-phase autoantibodies are represented by autoantibodies to PCNA (Proliferating Cell Nuclear Antigen), the auxiliary protein of DNA polymerase delta. M-phase autoantibodies are represented by autoantibodies to mitotic spindle components viz. centrosomes, condensed chromosomes, centromeres, mitotic spindle proper and intercellular bridge. We have included autoantibodies to nuclear lamins as M-phase autoantibodies as lamins play a key role in reversible breakdown and reformation of nuclear membranes during mitosis. The usefulness of these autoantibodies as diagnostic markers in systemic autoimmune disease is tempered by their presence in patients with "atypical" autoimmune diseases and in normal individuals. However, as molecular probes, they have proven to be unique and invaluable tools for shedding new light on the workings of the cell cycle.