Nucleolus Autoantigenic Marker for Systemic Lupus Erthyematosus

Patent No. 6,177,254

Issued: January 23, 2001

Inventors: Jerome Bernard Rattner, 35 Point McKay Court, Calgary, Alberta T3B 5B7 Canada; and Clark Whitehead, 1894 20th Street NW, Rochester, MN 55901

Systemic lupus erythematosus (SLE), commonly known as Lupus, is an autoimmune disease characterized by dysregulation of the immune system resulting in the production of antinuclear antibodies, the generation of circulating immune complexes, and the activation of the complement system. The immune complexes build up in the tissues and joints causing inflammation, and degradation to both joints and tissues. While the word "systemic" correctly suggests that the disease effects the entire body and most organ systems, the disease most often involves inflammation and consequent injury to the joints, skin, kidney, brain, the membranes in body cavities, lung, heart, and gastrointestinal tract. An individual with SLE often experiences unpredictable acute episodes or "outbreaks" and equally unexpected remissions. The pathologic hallmark of the disease is recurrent, widespread, and diverse vascular lesions resembling a rash or changes on the surface of the skin.

Physicians have known Lupus since 1828 when it was first described by the French dermatologist, Biett. Early studies were simply descriptions of the disease, with emphasis on the skin rashes typically present in those afflicted with the disease as well as other easily visible symptoms. Forty-five years later a dermatologist named Kaposi noted that some patients with lupus erythematosus (LE) skin lesions showed signs of affected internal organs. In the 1890s, Sir William Osler, a Canadian physician, observed that SLE could affect internal organs without the occurrence of skin changes. In 1948, Dr. Malcolm Hargraves of the Mayo Clinic isolated and described the particular morphology of the LE cell. This cell was found in the blood of patients with SLE. Hargraves' discovery has enabled physicians to identify many more cases of SLE by using a simple blood test. As a result, the number of SLE cases diagnosed has steadily risen.

SLE is not a rare disorder. Although reported in both the extremely old and the extremely young, the disease is chiefly found in women of childbearing age. Among children the occurrence of SLE is three times more likely in females than in males. In the 60% of SLE patients who experience the onset of this disease between puberty and the fourth decade of life, the female to male ratio is 9:1. Thereafter, the female preponderance again falls to that observed in prepubescent children (i.e. 3:1). In addition, the disorder appears to be three times more common in persons of African and Asian descent than in persons of Caucasian descent.

The prevalence of SLE in the United States is an issue of some debate. Estimates of occurrence range from 250,000 to 2,000,000 persons. Problems with identifying SLE are part of the problem in providing estimates of the numbers of individuals affected. The root of this identification problem is the fact that the clinical features of SLE can be mimicked by many other disorders, such as infectious mononucleosis or lymphoma. In this way the actual number of individuals affected is masked.

The etiology of SLE remains unknown. A genetic predisposition, the systemic proliferation of sex hormones, and an environmental trigger likely result in the disordered immune response that typifies the disease. A role for genetics is suggested by the increased percentage of two histocompatibility antigens in patients with SLE, HLA-DR2 and HLA-DR3. In addition, there is an increased frequency of the extended haplotypes HLA-A1, B8, and DR3 in affected individuals. The role for heredity is further supported by the concordance for this illness among monozygotic twins. The polygenic nature, however, of this genetic predisposition as well as the contribution of environmental factors is suggested by the occurrence rate, which is only moderate and reported to be between 25% and 60%.

The body's immune system normally makes proteins called antibodies to protect he body against viruses, bacteria and other foreign materials. These foreign materials are called antigens. In an autoimmune disorder, such as SLE, the immune system loses its ability to tell the difference between antigens and its own cells and tissues. The immune system then makes antibodies directed against "self." These antibodies, called "auto-antibodies," react with the "self autoantigens" to form immune complexes. The immune complexes build up in the tissues, causing inflammation, injury to tissues, and pain.

Since 1954, the various unusual antibodies found to be associated with SLE have been used to study the disease. The above difficulty in identifying the disease, and those afflicted with it, has led to an effort to use the presence of these antibodies as a tool to diagnose the disease. However, the presence of these antibodies may be the result of factors other than SLE, and to date no one autoantibody has been found to be universal to all individuals with SLE.

Briefly stated, the invention described herein discloses the identification, expression, production, and purification of a novel human protein. Its cDNA and amino acid sequences have been determined and are disclosed. This autoantigenic protein, termed ASE-1, has an approximate molecular mass of 55 kDa and is a component of the nucleolus found in all nucleated human cells. ASE-1 co-localizes with RNA polymerase-I and autoantibodies to ASE-1 are found at a higher frequency in individuals with systemic lupus erthyematosus (SLE). This finding makes ASE-1 a reliable serum marker for SLE, and therefore useful in the detection and characterization of SLE. The cDNA sequences for this nucleolar protein was isolated and then inserted into the pGEX plasmid. The plasmid constructed in this way was then transformed into the E. coli strain JM109 for expression. The E. coli expression vector accurately transcribed and expressed the ASE-1 protein sequence.

Indirect immunofluorescence analysis using polyclonal antibodies generated to cloned and expressed regions of ASE-1 indicate that this protein resides at the fibrillar centers of the nucleolus and the nucleolus organizer regions of mitotic chromosomes during cell division.

To identify the presence of ASE-1 antibodies in an individual patient's serum, a sample is taken and screened against the cloned ASE-1 protein. The presence of the ASE-1 antibody protein complex can be observed through the use of several methods including an ELISA assay, western blot techniques, by binding the autoantigens or specific autoantigenic epitopes to microspheres and identifying reactive sera by flow cytometry, or other known means.

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