New data on the in situ position of the inactive X chromosome in the interphase nucleus of human fibroblasts

Summary The in situ spatial distribution of nucleolus-organizing-region (NOR) bearing chromosomes in relation to the inactive X chromosome was studied during interphase in human fibroblasts. The respective positions of these chromosomes were examined in 30 growing and 32 resting fibroblasts from reconstructed nuclei, using nucleoli and the Barr body as ultrastructural markers. Experimental values for the distance between the nucleoli and the Barr body were estimated by their coefficient of closeness and compared to the uniform distribution. The following results were obtained: (1) the distribution patterns for the two populations of nuclei were similar, (2) the distribution of the NOR-bearing chromosomes in relation to the inactive X chromosome varied and differed significantly from a uniform distribution, and (3) in many cases the Barr body was observed to be in a juxta-nucleolar position. The internal distribution revealed by this study is compared with the data in the literature, especially with the conflicting data obtained by other methods used to determine the interphase arrangement of chromosomes. The relationship between interphase and metaphase arrangements such as can be deduced with these methods, is discussed in relation to the mechanisms of the formation of metaphase plates or chromatid translocations.     

Cytogenetic evidence for the absence of an inactivated X chromosome in a human female (XX) breast carcinoma cell line

Summary Barr body staining procedures were applied directly to the chamber slide cultures of female amniotic cells, WI38 fibroblasts, normal female kidney cells, and a human breast carcinoma cell line, Elco. A high frequency of Barr bodies was found in all the normal female control cells; however, no Barr bodies were observed in the Elco cells. By trypsin G-banding analysis, two normal X chromosomes were identified in all Elco cells. The late DNA replication pattern of the cell line was then studied with the terminal BrdU pulse method. Both X chromosomes in the Elco cell line were found to be euchromatic with a characteristic R-banding pattern; no late-replicating X chromosome was observed. The absence of both a Barr body and a late-replicating heterocyclic X chromosome provides strong cytogenetic evidence that an inactivated X chromosome is absent in the human breast carcinoma cells bearing two X chromosomes.     

Cytochrome P450 enzymes and UDP-Glucuronosyltransferases as hepatocellular autoantigens

Cytochromes P450 and UDP-Glucuronosyltransferases (UGT) are targets of microsomal autoantibodies in liver and kidney (LKM). LKM autoantibodies are observed in autoimmune hepatitis, in some patients with viral hepatitis, drug-induced hepatitis and autoimmune hepatitis as disease component of the autoimmune polyglandulars syndrome type 1 (APS-1). In autoimmune hepatitis LKM antibodies are markers of autoimmune hepatitis type 2. The major target of LKM-1 antibodies is cytochrome P450 2D6; a second less frequent target was the described UGTs of family 1. In autoimmune hepatitis LKM-1 autoantibodies are usually directed against small linear epitopes. LKM autoantibodies are also associated with infection with hepatitis viruses C and D. In hepatitis C about 1–2% of patients develop LKM-1 autoantibodies. About 60% of these autoantibodies are conformation dependent. The presence of LKM autoantibodies in hepatitis C may be associated with an increased risk in interferon treatment. LKM-3 autoantibodies are found in about 8% of patients with hepatitis D and are directed against conformational epitopes. Patients treated with certain drugs may develop drug induced hepatitis. In hepatitis induced by tienilic acid, tienilic acid is activated by and covalently bound to cytochrome P450 2C9. Activation of the immune system results in the formation of autoantibodies against cytochrome P450 2C9 (LKM-2) and infiltration of the liver with immune cells. A similar mechanism has been described for dihydralazine induced hepatitis, where autoantibodies are directed against P450 1A2 (LM). Autoantibodies directed against cytochrome P450 1A2 also are found in patients suffering from hepatitis as a disease component of APS-1.Abbreviations AIH autoimmune hepatitis - APS1 autoimmune polyendocrine syndrome type 1 - APS-1 autoimmune polyglandular syndrome type 1 - LKM microsomal autoantibodies in liver and kidney - HSV-1 herpes simplex virus type 1 - UGT UDP-glucuronosyltransferases     

Identification of monoclonal insulin autoantibodies in insulin autoimmune syndrome associated with HLA-DRB1*0401

We have characterized HLA and insulin autoantibodies in a Japanese female patient with insulin autoimmune syndrome. Serological HLA typing demonstrated the patient had HLA-DR4, and DNA typing showed she had HLA-DRB1*0401 which has not been reported in patients with insulin autoimmune syndrome in Japan. A single binding affinity of insulin autoantibodies was demonstrated by Scatchard analysis and immunoglobulin class of insulin autoantibodies was exclusively IgG-kappa. HLA-DRB1*0406 is strikingly associated with patients with insulin autoimmune syndrome who have polyclonal insulin autoantibodies. The present report demonstrated the first Japanese patient with insulin autoimmune syndrome carrying HLA-DRB1*0401 who was revealed to have monoclonal insulin autoantibodies. The present results indicate that HLA molecules are the major determinants of polyclonal insulin autoantibodies and monoclonal insulin autoantibodies in insulin autoimmune syndrome.     

Epstein–Barr virus and rheumatoid arthritis: is there a link?

Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic, destructive, debilitating arthritis. Its etiology is unknown; it is presumed that environmental factors trigger development in the genetically predisposed. Epstein–Barr virus, a nearly ubiquitous virus in the human population, has generated great interest as a potential trigger. This virus stimulates polyclonal lymphocyte expansion and persists within B lymphocytes for the host's life, inhibited from reactivating by the immune response. In latent and replicating forms, it has immunomodulating actions that could play a role in the development of this autoimmune disease. The evidence linking Epstein–Barr virus and rheumatoid arthritis is reviewed.     

Autoantibodies to fibrillin-1 activate normal human fibroblasts in culture through the TGF-beta pathway to recapitulate the "scleroderma phenotype"

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-beta1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-beta1 with anti-TGF-beta1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-beta1 from fibrillin-1-containing microfibrils in the ECM.     

A possible active segment on the inactive human X chromosome

An idic(Xp-) in which the two X chromosomes are attached short arm to short arm, and which thus has two b regions (the Q-dark segment next to the centromere on Xp) between the inactivation centers, assumed to be situated on the Q-dark region next to the centromere on Xq, showed 63.8% bipartite Barr bodies as compared with 22.2% formed by idic(Xq-). In addition, the mean distance of the two parts of the Barr bodies in the fibroblasts of a patient with idic(Xp-) is significantly greater than in the cases with one or no b region. Contrary to the other patients with abnormal X chromosomes, the buccal cells of a woman idic(Xp-) showed a number of bipartite Barr bodies. — To explain these observations we have put forward the hypothesis that the b region on the Xp always remains active and thus, when the rest of the chromosome forms a Barr body, this segment is extended, allowing the two parts of the X chromatin to get farther apart and at the same time increasing the percentage of bipartite bodies.     

Two different rheumatoid factor-producing cell populations distinguished by the mouse erythrocyte receptor and responsiveness to polyclonal B cell activators

The subsets of human peripheral blood B lymphocytes from which Epstein Barr virus- (EBV) and pokeweed mitogen- (PWM) induced IgM anti-IgG autoantibody-producing B cells arise have been compared. EBV-induced IgM anti-IgG autoantibodies preferentially derive from a subset of B cells that forms rosettes with mouse erythrocytes. In contrast, PWM-induced IgM anti-IgG antibodies preferentially arise from a B cell subset lacking the mouse erythrocyte receptor.     

Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera,and Their Number Is Influenced By Age,Gender, and Disease

The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases.     

Immunocytochemical evidence for methylation of the inactive X chromosome in human fetal oogonia

The state of DNA methylation of the X chromosomes of human interphase oogonia from a 46,XX and a 46,XX/47,XXX fetus at 17 weeks of gestation was tested immunocytochemically with an antibody to 5-methylcytosine (5MeC). Of 1637 oogonial nuclei from the 46,XX fetal ovary, 313 (19.1%) contained Barr bodies, of which 93.6% were positive for 5MeC. Of 1780 oogonia from the 46,XX/47,XXX fetus 327 (18.4%) contained Barr bodies; 175 oogonia had one Barr body and 152 had two. Of the single Barr bodies 145 (82.8%) had positive 5MeC reaction product. Of the 152 oogonia from the XXX line, 97 (63.8%) had positive 5MeC on both Barr bodies, 35 (23%) had one positive and one negative, and 20 (13.1%) had no product on either Barr body. This immunocytochemical evidence supports the hypothesis that the DNA of the inactive X-chromosome of the human 17-week gestation oogonium is methylated.     

Human autoantibodies to spermatogenic antigens and Sertoli cells

Immunofluorescence staining using human autoantibodies is a simple and reliable method for investigation of meiotic and post-meiotic cells. Patients suffering from autoimmune diseases often produce circulating autoantibodies to antigens of germ cells and Sertoli cells. Four hundred human autoimmune sera were screened by indirect immunofluorescence on mouse seminiferous tubule cells. Autoantibodies of several specificities were found: one group reacted with organelles of meiotic prophase spermatocytes or spermatozoa. Included in this group were autoantibodies to synaptonemal complexes, sex vesicle, acrosome, and sperm tail. A second group of autoantibodies was found to stain different spermatogenic cell types uniformly, such as round spermatids or Sertoli cells.     

Cardiac myosin-induced myocarditis. Heart autoantibodies are not involved in the induction of the disease

We recently demonstrated that cardiac myosin is capable of inducing autoimmune myocarditis in genetically predisposed mice. This disease parallels coxsackievirus B3-induced autoimmune myocarditis in many respects and is associated with high-titer autoantibodies specific for cardiac myosin. The following lines of evidence suggest that these autoantibodies are not involved in the induction of autoimmune myocarditis: 1) immunoperoxidase staining of heart sections from cardiac myosin-immunized A/J and A.SW mice revealed IgG depositions only along damaged muscle fibres in infiltrated areas, but not in intact tissue; 2) myosin autoantibodies did not bind to the surface of viable cardiac myocytes isolated from mice, but only reacted with myocytes permeabilized with detergent; 3) mice treated with a single high dose of cyclophosphamide, which reduces the humoral immune response, still developed severe myocarditis, despite the fact that their autoantibody titers were reduced to the level of adjuvant-injected controls; and 4) passive transfer of high-titer myosin autoantibodies failed to induce myocarditis, although the titers in the recipients were comparable to those found in mice with cardiac myosin-induced disease. Together, the results suggest that high-titer myosin autoantibodies are secondary rather than primary to the disease.     

Organ-specific autoimmune disease induced in mice by elimination of T cell subsets. V. Neonatal administration of cyclosporin A causes autoimmune disease

Cyclosporin A (CsA), a potent immunosuppressive drug, caused organ-specific autoimmune disease, such as gastritis with anti-parietal cell autoantibodies or oophoritis with anti-oocyte autoantibodies, in BALB/c mice when the drug was administered daily for 1 wk to newborns. Administration to adult mice did not. CsA abrogated the production of L3T4+ T cells and Lyt-2+ T cells in the thymus. Consequently, these T cells were substantially depleted from the peripheral lymphoid organs, especially when the drug was administered from the day of birth. Autoimmune disease was prevented when CsA-treated newborn mice were inoculated with splenic T cells from normal syngeneic mice. However, removal of the thymus immediately after neonatal CsA treatment produced autoimmune disease with a higher incidence and in a wider spectrum of organs, i.e., thyroiditis, sialoadenitis of the salivary gland, gastritis, insulitis of the endocrine pancreas, adrenalitis, oophoritis, or orchitis. Each autoimmune disease was accompanied by the development of circulating autoantibodies specific for the corresponding organ Ag. Immunopathology of these autoimmune diseases was quite similar to that of human organ-specific autoimmune diseases.     

Mucosal production of antigastric autoantibodies in Helicobacter pylori gastritis

Background. Apart form bacterial virulence factors of Helicobacter pylori , certain host factors influence the pathogenesis of H. pylori gastritis. In particular, antigastric autoantibodies that are detectable in the sera of a substantial proportion of H. pylori were shown to correlate with the development of gastric atrophy. The aim of this study was to analyze the possible antigastric autoimmune response in H. pylori gastritis at the site where the action is, i.e., in the gastric mucosa.
Material and Methods. Gastric biopsy specimens from antrum and corpus mucosa of 24 H. pylori –infected and of 33 noninfected patients were cultured for 3 days, and tissue culture supernatants were analyzed for the amount of locally produced IgA and IgG. Antigastric autoantibodies were screened in the sera and in the supernatants by means of immunohistochemistry.
Results. The infected patients had significantly higher concentrations of locally produced IgA, whereas the IgG concentrations were virtually the same in infected and noninfected patients. IgG or IgA antigastric autoantibodies, or both, were detectable only in the sera (38%) and supernatants (17%) of infected patients. Interestingly, the patient with the strongest local autoimmune response showed body-predominant H. pylori gastritis, with destruction of gastric glands and atrophy of the body mucosa.
Conclusions. These results demonstrate that antigastric autoimmune reactions are detectable at the site of the disease and might be relevant for the pathogenesis of gastric mucosa atrophy in H. pylori gastritis.     

Barr body frequency in esophageal cancer in Indian women

In a retrospective study, Barr body frequency was analyzed in 50 consecutive cases of esophageal cancer in females. The majority (95%) of the tumors had less than 20% Barr body frequency (of which 80% showed less than 10%), while normal controls showed 30% to 45% Barr body frequency. The mechanism(s) involved in lowering the frequency is not known at present.     

Immune dysregulation and self-reactivity in schizophrenia: do some cases of schizophrenia have an autoimmune basis?

Schizophrenia affects 1% of the world's population, but its cause remains obscure. Numerous theories have been proposed regarding the cause of schizophrenia, ranging from developmental or neurodegenerative processes or neurotransmitter abnormalities to infectious or autoimmune processes. In this review, findings suggestive of immune dysregulation and reactivity to self in patients with schizophrenia are examined with reference to criteria for defining whether or not a human disease is autoimmune in origin. Associations with other autoimmune diseases and particular MHC haplotypes, increased serum levels of autoantibodies, and in vivo and in vitro replication of some of the functional and ultrastructural abnormalities of schizophrenia by transfer of autoantibodies from the sera of patients with schizophrenia suggest that, in some patients at least, autoimmune mechanisms could play a role in the development of disease. Recent findings regarding specific autoimmune responses directed against neurotransmitter receptors in the brain in patients with schizophrenia will also be reviewed.     

IgG4 autoantibodies induce dermal-epidermal separation

Bullous pemphigoid (BP) is a sub-epidermal autoimmune blistering disease associated with autoantibodies to the dermal-epidermal junction (DEJ). Patients' autoantibodies induce dermal-epidermal separation when co-incubated with cryosections of human skin and leucocytes from healthy volunteers. IgG autoantibodies trigger complement and/or leucocyte activation resulting in specific pathology in several autoimmune conditions. In these diseases, IgG1 and IgG3 isotypes, but not the IgG4 subclass, are thought to trigger inflammatory pathways resulting in tissue damage. The capacity of IgG4 autoantibodies to mediate tissue damage has not yet been demonstrated. In this study, we isolated IgG1 and IgG4 autoantibodies from bullous pemhigoid patients'serum and analysed their blister-inducing potential in our cryosection assay. As expected, complement-fixing IgG1 autoantibodies induced sub-epidermal splits in this experimental model. Purified IgG4 did not fix complement, but, interestingly, like IgG1, activated leucocytes and induced dermal-epidermal separation. The potential of IgG4 autoantibodies to induce Fc-dependent dermal-epidermal separation was significantly lower compared to IgG1. Our results demonstrate that IgG4 autoantibodies are able to activate leucocytes and point to a hitherto less recognized function of IgG4. Moreover, for the first time, we clearly demonstrate that BP IgG4 autoantibodies have the capacity to induce leucocyte-dependent tissue damage.     

Autoantibodies to peroxiredoxin I and IV in patients with systemic autoimmune diseases

Anti‐oxidative enzymes protect living bodies from various oxidative stresses. In the systemic autoimmune diseases, autoantibodies to oxidized molecules and to anti‐oxidative enzymes have been reported. To promote understanding of the relationships between autoimmunity and oxidative stress, we here investigate whether autoimmunity to the anti‐oxidative peroxiredoxin (Prxs) enzymes exists in patients with systemic autoimmune diseases. Specifically, we detected autoantibodies to recombinant Prx I and Prx IV respectively by ELISA and western blotting. Next, clinical parameters were compared between the anti‐Prx I or IV‐positive and ‐negative patients. We found that 33% of the 92 patients with autoimmune diseases tested possessed autoantibodies to Prx I (57% in systemic lupus erythematosus (SLE), 19% in rheumatoid arthritis (RA), 5% in Behçet disease, and 46% in primary vasculitis syndrome). In contrast, autoantibodies to Prx IV were detected in only 17% of the same patients. No significant correlation was found between occurrence of the two autoantibodies. Clinically, possession of anti‐Prx I autoantibodies correlated with lower serum levels of CH50, C3, and C4. Taken together, our data demonstrate the existence of autoantibodies to Prxs for the first time. The autoantibodies to Prx I may be involved in the pathophysiology of systemic autoimmune diseases such as SLE and vasculitis.     

Humanization of autoantigen

Transmissibility of characteristic lesions to experimental animals may help us understand the pathomechanism of human autoimmune disease. Here we show that human autoimmune disease can be reproduced using genetically engineered model mice. Bullous pemphigoid (BP) is the most common serious autoimmune blistering skin disease, with a considerable body of indirect evidence indicating that the underlying autoantigen is collagen XVII (COL17). Passive transfer of human BP autoantibodies into mice does not induce skin lesions, probably because of differences between humans and mice in the amino acid sequence of the COL17 pathogenic epitope. We injected human BP autoantibody into Col17-knockout mice rescued by the human ortholog. This resulted in BP-like skin lesions and a human disease phenotype. Humanization of autoantigens is a new approach to the study of human autoimmune diseases.     

Autoantibodies against a 43 KDa muscle protein in inclusion body myositis

Background

Inclusion body myositis (IBM) is a poorly understood and refractory autoimmune muscle disease. Though widely believed to have no significant humoral autoimmunity, we sought to identify novel autoantibodies with high specificity for this disease.

Methodology/Principal Findings

Plasma autoantibodies from 65 people, including 25 with IBM, were analyzed by immunoblots against normal human muscle. Thirteen of 25 (52%) IBM patient samples recognized an approximately 43 kDa muscle protein. No other disease (N = 25) or healthy volunteer (N = 15) samples recognized this protein.

Conclusions

Circulating antibodies against a 43-kDa muscle autoantigen may lead to the discovery of a novel biomarker for IBM. Its high specificity for IBM among patients with autoimmune myopathies furthermore suggests a relationship to disease pathogenesis.