A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.

https://www.pnas.org/content/pnas/76/9/4350.full.pdf

Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.

In 1982, the Diagnostic and Therapeutic Criteria Committee of the American College of Rheumatology (ACR)published revised criteria for the classification of systemiclupus erythematosus (SLE) (1). During the ensuing decade several investigators, including Drs. Graham Hughes and Donato Alarcon-Segovia, among others, have described the presence and clinical associations or antiphospholipid antibodies in patients with SLE, as well as the occurrence of theprimary antiphospholipid syndrome (2-5). In 1992, Piette and colleagues suggested that the ACR revised criteria be reevaluated in light of the above discoveries (6).

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780400928

Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus.

For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or para-articular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780290816

Development of criteria for the classification and reporting of osteoarthritis: Classification of osteoarthritis of the knee

https://www.onlinelibrary.wiley.com/doi/epdf/10.1111/j.1538-7836.2006.01753.x

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).

The New York and the Rome diagnostic criteria for ankylosing spondylitis (AS) and the clinical history screening test for AS were evaluated in relatives of AS patients and in population control subjects. The New York criterion of pain in the (dorso) lumbar spine lacks specificity, and the chest expansion criterion is too insensitive. The Rome criterion of low back pain for more than 3 months is very useful. Our study showed the clinical history screening test for AS to be moderately sensitive, but it might be better in clinical practice. As a modification of the New York criteria, substitution of the Rome pain criterion for the New York pain criterion is proposed.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.1780270401

Evaluation of diagnostic criteria for ankylosing spondylitis. A proposal for modification of the New York criteria.

The heavy metal mercury elicits a genetically restricted, anti-nucleolar autoantibody response that targets fibrillarin, a 34-kDa protein component of many small nucleolar ribonucleoprotein particles The mechanisms by which a toxin such as mercury elicits an autoantibody response that predominantly targets a single intracellular protein autoantigen remain uncertain, but may be prefaced by mercury gaining access to the intracellular environment Mercury-induced cell death was associated with loss of fibrillarin antigenicity and modification of the molecular properties of fibrillarin as revealed by aberrant migration under nonreducing conditions in SDS-PAGE Addition of mercury to isolated nuclei also resulted in aberrant migration of fibrillarin, but not other nuclear autoantigens The sensitivity of the HgCl2-induced modification of fibrillarin to 2-ME, iodoacetamide, and hydrogen peroxide suggested interaction of mercury with the two cysteines in the fibrillarin sequence This was confirmed by mutation of the cysteines to alanines, which abolished the aberrant migration of fibrillarin in the presence of HgCl2 The modification of the molecular structure of fibrillarin by mercury reduced immunoprecipitation by anti-fibrillarin autoantibodies, pointing to unmodified fibrillarin as the B cell Ag and implicating mercury-modified fibrillarin as the source of T cell antigenicity These observations demonstrate for the first time that an environmental toxin can alter the physicochemical properties of an autoantigen and may help to explain the antigenic specificity of mercury-induced murine autoimmunity

The autoimmunity-inducing xenobiotic mercury interacts with the autoantigen fibrillarin and modifies its molecular and antigenic properties.

Precipitating anti-PM-Scl antibodies are present in sera from patients with polymyositis, scleroderma, and polymyositis/scleroderma overlap syndromes. By indirect immunofluorescence microscopy, anti-PM-Scl antibodies stained the nucleolus in cells of different tissues and species, suggesting that the antigen is highly conserved. By electron microscopy, anti-PM-Scl antibodies reacted primarily with the granular component of the nucleolus. Drugs that inhibit rRNA synthesis had a marked effect on the expression of PM-Scl antigen. In actinomycin D-treated cells, immunofluorescence staining by anti-PM-Scl was significantly reduced with residual staining restricted to the granular regions of nucleoli. Treatment with 5,6-dichloro-beta-D-ribofuranosylbenzimidazole (DRB) also selectively reduced nucleolar staining. On a molecular level, anti-PM-Scl antibodies precipitated 11 polypeptides with molecular weights (Mr) ranging from 110,000 to 20,000. The Mr 80,000 and 20,000 polypeptides were phosphorylated. Evidence suggests that the PM-Scl antigen complex may be related to a preribosomal particle.

/pdf/immunolocalization-and-partial-characterization-of-a-y7a9tqntu3.pdf

Immunolocalization and partial characterization of a nucleolar autoantigen (PM-Scl) associated with polymyositis/scleroderma overlap syndromes.

Four distinct nucleolar proteins or RNA-protein complexes have recently been identified as targets of human autoimmune antibodies. These autoantigens areRNA polymer-ase I, PM-Scl (a particle possibly related to preribosomes), 7-2 RNP andfibrillarin (a U3-RNP associated protein). The four different nucleolar autoantigens could be assigned to distinct nucleolar subcompartments by light and electron microscopic immunocytochemistry. RNA polymerase I was located in the fibrillar centers, PM-Scl antigen and 7-2 RNP in the granular component and fibrillarin in the dense fibrillar component. Experimental evidence suggests that these naturally occurring antibodies could be helpful tools in further studying nucleolus structure and functions as well as molecular mechanisms involved in ribosome biogenesis. From a clinical viewpoint, we believe that it is important to identify the nature of reactive autoantigens in systemic autoimmune diseases in order to answer questions concerning the mechanisms which render conserved ubiquitous cellular proteins immunogenic. Revealing such mechanisms in return could give clues with regard to the etiology of certain systemic rheumatic diseases. Ribosome biogenesis, a highly dynamic process with its many well-defined intermediate biological steps related to specific nuclear structures could be amenable for such studies.

/pdf/human-autoantibodies-probes-for-nucleolus-structure-and-3g09wyqou8.pdf

Human autoantibodies: probes for nucleolus structure and function.

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus.

Using indirect immunofluorescence (IF) with HEp-2 cells as a substrate serially bled SJL mice were found to gradually develop a high titre of anti-nucleolar antibodies (ANuA) after 3-5 weeks of s.c. injections of 1.6 mg HgCl2/kg body weight every third day. The ANuA showed a clumpy nucleolar pattern of localization and were composed of all IgG subclasses, but contained, in comparison with the antinuclear antibodies (ANA) in MRL-lpr/lpr mice, significantly lower titres of IgG2a and only traces of IgG3. Immunoblotting analysis using purified mouse liver nucleoli revealed that the sera with ANuA identified the same 34-kD nucleolar protein which was targeted by a human scleroderma serum containing autoantibodies monospecific for fibrillarin. In addition, a fraction of the mercury-treated SJL mice developed serum antibodies reacting with 10-15 and 60-70 kD nucleolar proteins in immunoblotting. The presence of serum autoantibodies reacting with the 10-15 kD proteins correlated with significantly increased titres of anti-histone antibodies of the IgG class in ELISA. Some mercury-treated SJL mice also developed a significantly increased titre of anti-histone antibodies of the IgM class. B10.S mice treated with mercuric chloride consistently developed ANuA, which also targeted a 34-kD nucleolar protein. Since anti-fibrillarin antibodies are specific markers of scleroderma, the present animal model may be valuable for studies of the immunological aberrations which are likely to induce this autoimmune response.

Eng M. Tan

Papers

The autoimmunity-inducing xenobiotic mercury interacts with the autoantigen fibrillarin and modifies its molecular and antigenic properties.

Immunolocalization and partial characterization of a nucleolar autoantigen (PM-Scl) associated with polymyositis/scleroderma overlap syndromes.

Human autoantibodies: probes for nucleolus structure and function.

Immunolocalization of 7-2-ribonucleoprotein in the granular component of the nucleolus.

Anti-fibrillarin autoantibodies in mercury-treated mice.