Eng M. Tan

Mixed connective tissue disease-an apparently distinct rheumatic disease syndrome associated with a specific antibody to an extractable nuclear antigen (ENA)

Abstract We describe the clinical and serologic findings in twenty-five patients with an apparently distinct rheumatic disease syndrome which we have termed mixed connective tissue disease. All these patients had hemagglutinating antibody to an extractable nuclear antigen (ENA) which consists mainly of protein and ribonucleic acid (RNA). A marked sensitivity of the hemagglutination antigen to ribonuclease indicated that the specificity of the antibody to ENA circulating in these patients was different from that of antibody to ENA which occurred in about 50 per cent of the patients with systemic lupus erythematosus. Serum from patients with mixed connective tissue disease also contained high titers of speckled pattern fluorescent antinuclear antibody which showed the same response of tissue antigens to enzyme digestion as found with hemagglutinating antibody. There was no detectable Sm antibody. Antibody to native deoxyribonucleic acid (DNA) was infrequent and of low titer, and serum complement levels were normal or elevated. The clinical characteristics of the patients with mixed connective tissue disease included a combination of features similar to those of systemic lupus erythematosus, scleroderma and polymyositis. Most of these abnormalities were responsive to corticosteroid therapy. Thus, the detection of antibody to ENA with a well defined specificity allows recognition of an apparently distinct mixed connective tissue disease syndrome which is characterized by an excellent response to corticosteroid therapy and a favorable prognosis.

Antinuclear Antibodies: Diagnostic Markers for Autoimmune Diseases and Probes for Cell Biology

Publisher Summary Early studies pointed to the directions for subsequent investigations and there has been continuing identification of other circulating autoantibodies, characterization of their respective cellular antigens, and demonstration of the relationship between autoantibodies and clinical syndromes. The antibodies identified in these studies have been used extensively by investigators in molecular and cell biology as powerful probes for understanding the precursor messenger RNA (mRNA) splicing. Autoantibodies designated SS-A or Ro and SS-B or La were identified in patients with Sjogrens syndrome (SS) and SLE, and have been shown to target intracellular proteins that may be involved with regulation of RNA polymerase III function. Investigators in the field had long perceived that these spontaneously occurring auto-antibodies in human disease would turn out to be useful reagents in cell biology. It was also appreciated that it was necessary to characterize the molecular nature of the auto-antigens so that in addition to the immunochemical properties, their structure and function might be determined. One of the purposes of this chapter is to show that the new molecular biology of cellular antigens and auto-antibodies could now be providing insights into comprehending some features of autoimmunity. In addition, there appears to be a need for the synthesis of the wealth of information that has accumulated and an evaluation of what it might signify.

Autoantibodies to Nuclear Antigens (ANA): Their Immunobiology and Medicine

Publisher Summary Autoantibodies to nuclear antigens (ANAs) have assumed an important place in the diagnostic armamentarium of the clinician because of distinct profiles of ANAs in different diseases. Profiles of ANAs have, therefore, been extremely useful in differential diagnosis, where the disease does not have classical or full-blown manifestations.. Immune complexes formed in the circulation or in situ mediate tissue injury by the activation of complement and other inflammatory mediators. Not only do these antibodies precipitate their respective antigens but also other proteins or nuclear RNAs that might be associated with them in special ways. The reasons for these special associations of protein antigens with specific sets of nuclear RNAs is unknown, but the possibility that there might be functional relationships in these complexed particles is not unreasonable. The key question that pervades the minds of many investigators in this field is the reason for the appearance of ANAs in certain individuals. It is highly improbable that the phenomenon is a random immune response to nuclear breakdown products, because the types of ANAs in different diseases are strikingly different. Some known environmental agents are drugs, such as hydralazine and procainamide, that together with lower levels of hepatic acetyltransferase enzyme predispose the host to the development of ANAs. Another agent may be the Epstein–Barr virus that is a ubiquitous environmental agent.

Autoantibodies in the diagnosis of systemicrheumatic diseases

Abstract Distinct profiles of autoantibodies directed to intracellular antigens can be detected in the systemic connective tissue diseases. They aid in establishing the correct diagnosis and are included in many sets of diagnostic criteria, such as the ones developed for systemic lupus erythematosus (anti-Smith antigen and anti-double-strand DNA antibodies), mixed connective tissue disease (anti-U1-nuclear ribonucleoprotein antibodies), and Sjogrens syndrome (SS) (anti-SS A/Ro and anti-SS-B/La antibodies). They are useful prognostic markers in some situations and facilitate clinical and treatment follow-up. Autoantibodies have also been used as probes to gain insights into cell biology, helping to isolate and purify intracellular proteins involved in key cellular functions. We give detailed information on two of the most useful techniques for the detection of autoantibodies in the clinical and research laboratory settings, indirect immunofluorescence and immunoblotting. We also discuss several of the antigen -autoantibody systems found in systemic lupus erythematosus (Smith antigen, U1-nuclear ribonucleoprotein, SS-A/Ro, SS-B/La, proliferating cell nuclear antigen ribosomal ribonucleoprotein, double-strand DNA, histones, antiphospholipids, Ku, Ki/SL), systemic sclerosis (centromere, topo I, RNA polymerases, fibrillarin, polymyositis-Scl, Th/To), polymyositis/dermatomyositis (transferRNA synthetases, signal recognition particle, and others), and SS (SS-A/Ro, SS-B/La, nucleolar organizing region-90, p80-coilin), addressing their clinical significance, common detection methods, immunogenetic associations, and the molecular and cellular biology of the cognate antigens.

Expression of proliferating cell nuclear antigen (PCNA)/cyclin during the cell cycle

The expression of proliferating cell nuclear antigen (PCNA), also called cyclin, was quantified in the cell lines SP2/0 and MOLT-4 and in mouse splenocytes induced to proliferate in vitro with mitogens. Autoantibody from a patient with systemic lupus erythematosus was used to label PCNA in cell suspensions after the cells had been fixed and permeabilized. In the same cells DNA was stained by propidium iodide. The cells were then analysed by flow cytometry for PCNA and DNA content. The PCNA profiles in proliferating spleen cells and the cell lines were similar. Most G0-G1 cells did not express significant amount of PCNA. A dramatic increase in PCNA immunofluorescence was observed in late G1 cells, and further increases were observed in S-phase cells. G2-M cells showed a reduced level of PCNA immunofluorescence relative to S-phase cells but were still elevated relative to G0-G1 cells. Proliferating cells arrested at the G1-S boundary by exposure to cytosine arabinoside showed an increased PCNA immunofluorescence as compared to unstimulated cells.

Antibodies to cellular antigens in Sjögren's syndrome.

An extract of human lymphocytes from continous cell culture was used as the antigen source to detect antibodies in sera of patients with Sjögrens syndrome (SS). Using double diffusion in agarose, 85 per cent of a selected group of patients had precepating antibodies. Three precipitating antigen-antibody systems were detected and were shown to be different from those described previously in othe systemic rheumatic diseases. The SS precipitating antibodies were temporarily classified as precipitins, A, B, and C. SS patinets with sicca syndrome but without clinical rheumatoid arthritis had precipitin systems A and/or B, and SS patients with associated rheumatoid arthritis had precipitin system C. Serum reactants were demonstrated by immuno-electrophoresis to migrate in the gamma globulin region. The precipitating activity of the serum factors was not destroyed by treatment with 2-mercaptoethanol and was not removed by absorption of rheumatoid factor from the sera. The reactivity of the lympuocyte antigens was destroyed by treatment with trypsin but not by deoxyribonuclease or ribonuclease.

Major antigen of liver kidney microsomal autoantibodies in idiopathic autoimmune hepatitis is cytochrome P450db1.

Type 1, liver kidney microsomal autoantibodies (LKM-1) are associated with a subgroup of idiopathic autoimmune type, chronic active hepatitis (CAH). The antigenic specificity of LKM-1 autoantibodies from 13 patients was investigated by immunoblot analysis of human liver microsomal proteins. Polypeptides of 50, 55, and 64 kD were detected with these antisera. A high titer LKM-1 serum was selected to screen a human liver lambda gt11 cDNA expression library, resulting in the isolation of several complementary (c)DNA clones. Autoantibodies affinity purified from proteins expressed by two of the immunopositive cDNA clones, HLD8.2 and HLD13.2, specifically react with a 50-kD protein of human liver microsomes and display immunofluorescence staining of the proximal renal tubular epithelia characteristic of LKM-1 sera. Determination of the sequence of HLD8.2 revealed that it encodes a recently described cytochrome P450db1. A bacterial fusion protein constructed from HLD8.2 proved to be a specific and sensitive diagnostic reagent. All sera from patients with LKM-1 positive liver disease react with this fusion protein. No reaction was seen, however, for sera from patients with other types of autoimmune liver diseases, viral hepatitis, systemic immunological disorders, or healthy controls.

Deoxyribonucleic Acid Antibody: A Method to Detect Its Primary Interaction with Deoxyribonucleic Acid

Antibody to DNA in human serums can be detected by the ammonium sulfate method. This sensitive and specific technique, which measures the primary interaction between DNA and antibody to DNA, is based on the observation that free DNA is soluble in 50-percent saturated ammonium sulfate whereas antibody-bound DNA is insoluble.

The Natural History of Interstitial Cystitis: A Survey of 374 Patients

A survey directed at determining the natural history of interstitial cystitis was conducted at our clinic. Information on demographics, risk factors, symptoms, pain and psychosocial factors was elicited from 374 patients who satisfied the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases criteria for interstitial cystitis and had all been diagnosed as having interstitial cystitis by a urologist. With regard to demographics, patients were predominantly female (89.8%) and white (94.1%), with a mean age of 53.8 +/- 0.7 years (standard error) and age at the first symptoms of 42.5 +/- 0.8 years. Information on 25 potential risk factors included 44.4% of the women reporting hysterectomy, 38.2% of the patients having strong sensitivities or allergic reactions to medication and only 2.7% being diabetic. With regard to interstitial cystitis symptoms, frequency and urgency were reported by 91.7% and 89.3% of the patients, respectively, while pelvic pain, pelvic pressure and bladder spasms were reported by more than 60% of respondents and burning by 56%. Location and degree of pain were also reported. Urination relieved or lessened interstitial cystitis pain for 73.6% of the patients and medication was effective for 46.8%. Other behaviors (for example hot baths, heating pads, lying down or sitting) were less effective. Conversely, stress, constrictive clothing and intercourse increased interstitial cystitis pain in more than 50% of the patients. In addition, acidic, alcoholic or carbonated beverages, and coffee or tea increased interstitial cystitis pain in more than 50% of the patients. More than 60% of the patients were unable to enjoy usual activities or were excessively fatigued and 53.7% reported depression. Travel, employment, leisure activities and sleeping were adversely affected in more than 80% of the patients. Pain location and degree differed significantly between patients with and without ulcers in the bladder. In addition, there was an apparent plateau in the frequency and urgency among patients after approximately 5 years with symptoms.

Molecular definition and sequence motifs of the 52-kD component of human SS-A/Ro autoantigen.

Serum SS-A/Ro autoantibodies are commonly found in patients with Sjogrens syndrome, systemic lupus erythematosus, neonatal lupus, and subacute cutaneous lupus. Two proteins of 60 and 52 kD have been described as targets for these autoantibodies. To define the 52-kD component unambiguously, cDNA clones were isolated from human HepG2 and MOLT-4 cell cDNA libraries. The identity of cDNA was established by (a) the specificity of the antibody affinity purified from the recombinant protein, (b) the reactivity of the purified recombinant protein with prototype SS-A/Ro sera in immunoblot and ELISA, and (c) two-dimensional gel comigration of MOLT-4 cell 52-kD protein and the recombinant protein. A 1.9-kb cDNA encoded the complete 52-kD protein containing 475 amino acids (Mr 54,082). Putative zinc-finger domains and a leucine zipper motif were identified in the amino-terminal half of the 52-kD protein, implicating its possible association with DNA/RNA. Sequence homology detected between the 52-kD protein and human ret transforming protein, and mouse T cell gene expression down-regulatory protein rpt-1, may provide leads to the functional role of the 52-kD protein in addition to the possibility that these proteins might constitute members of a subfamily of finger proteins.