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K M Pollard

Publications and source records attributed to K M Pollard.

51 records · Page 3Linked to original sources

Monoclonal autoantibody from a (New Zealand black x New Zealand white)F1 mouse and some human scleroderma sera target an Mr 34,000 nucleolar protein of the U3 RNP particle.

A monoclonal IgG2a antinucleolar autoantibody (72B9) was obtained by fusion of spleen cells from a (New Zealand black x New Zealand white)F1 mouse with myeloma cells (P3x63Ag8.653). Antibody 72B9 recognized a highly conserved nucleolar antigen present in both animal and plant cells. The staining pattern produced by antibody 72B9 in different cell substrates was identical with those obtained by scleroderma antibodies reactive with a basic (pI 8.5) nucleolar protein of Mr 34,000, which is associated with the U3 RNP particle. Western blotting further confirmed its reactivity with this scleroderma-related U3 RNP protein.

Animals↗

Structural requirements of DNA used in the Farr assay to detect antibodies directed against double-stranded DNA.

The measurement of antibodies to DNA in SLE requires the use of double-stranded DNA (dsDNA), demonstrably free of single-stranded regions. Such dsDNA preparations can, however, contain other structural components. In this study DNA preparations with defined structure, both secondary (single- and double-stranded and random base-paired) and tertiary (superhelical and open circular), were used in the Farr assay to measure the DNA binding of sera from patients with SLE and related connective-tissue diseases. The presence of true single-stranded DNA regions in denatured DNA, native DNA, and dsDNA containing single-stranded regions increased the DNA binding measured in all sera. DsDNA, whether intact or containing small regions of random base-pairing, was bound by sera from the majority of patients with SLE but not by non-SLE sera. Superhelical dsDNA from bacteriophage PM2 was bound by SLE sera to a greater extent than linear dsDNA was. Inhibition experiments suggested that this difference in binding to DNA according to tertiary, as opposed to secondary, structure is because there are fewer available binding sites on superhelical dsDNA. DNA binding, as measured by the Farr assay, can thus be influenced by both secondary and tertiary DNA structure. Using superhelical DNA, advantage can be taken of the dsDNA form plus tertiary structure to enhance DNA binding of SLE sera beyond the levels achieved using linear dsDNA.

Antibodies, Antinuclear↗

Monoclonal autoantibodies to nuclear antigens from murine graft-versus-host disease.

Systemic lupus erythematosus-like graft-versus-host (GVH) disease was induced in 10-week-old male (C57BL/10 X DBA/2) F1 mice by the intravenous injection of spleen and thymus cells (2:1) from 10-week-old male DBA/2 mice. GVH mice were bled at regular intervals 1 month after injection. Antibody to nuclear antigens (ANA) were detected by immunofluorescence using HEp-2 cells as substrate, and antibody to histones and DNA were detected by enzyme-linked immunosorbent assay (ELISA). The titer and frequency of ANA were found to relate directly to the number of donor cells injected. In order to determine the spectrum of ANA in GVH disease, mice were reinjected with optimum cell numbers (120 X 10(6], and splenocytes from two mice with high titer ANA were fused to mouse myeloma cell line P3/X63Ag8.653. Hybridomas were analyzed for ANA by immunofluorescence and ELISA. Sixty-eight clones were found which secreted ANA. Of these, 59% produced antibody to double-stranded DNA, single-stranded DNA, and/or histones and the remainder gave a variety of nuclear immunofluorescence patterns including speckled, homogeneous, nuclear matrix, and nucleolar. This study indicates that GVH disease provides an excellent source of splenocytes for the production of ANA-producing hybridomas as well as a model for the study of autoimmunity.

Animals↗

IgG subclasses of autoantibodies in systemic lupus erythematosus, Sjogren's syndrome, and drug-induced autoimmunity.

The IgG subclasses displayed by autoantibodies were examined in patients with systemic rheumatic diseases. Solid-phase assays performed with purified antigens were combined with a set of four mouse monoclonal antibodies specific for each human subclass to provide quantitative data for all the major autoantibody specificities. IgG1 accounted for an average of 55% of the total antibody activity to native and denatured DNA, Sm antigen, and histone and constituted significantly more anti-SS-B and anti-nRNP (84% and 92%, respectively). The remaining antibody activity consisted largely of IgG3, and this subclass was particularly prominent with anti-histone and anti-Sm in patients with systemic lupus erythematosus. In contrast, IgG2 constituted 3 to 12% of the anti-native and anti-denatured DNA and less than 5% of the anti-SS-B/La activity in only three patients with Sjogren's syndrome. IgG2 was essentially undetectable in antibodies to Sm and RNP antigens. IgG4 was also uncommon, although this isotype was significantly more prevalent in anti-histone from patients treated with procainamide showed that the isotype distribution of anti-histone and anti-denatured DNA remained remarkably constant. However, during periods of large increases in autoantibody activity, a shift from predominantly IgG3 to predominantly IgG1 occurred, consistent with the interpretation that there might be a sequential activation of heavy chain constant regions as the immune response matures. The disproportionately high levels of IgG1 and IgG3 displayed by all the autoantibody specificities examined may indicate that a common immunogenic feature of autoantigens or a common control mechanism underlies the regulation of autoantibody expression.

Antibodies, Monoclonal↗

Measurement of serum DNA binding in chronic active hepatitis and systemic lupus erythematosus using the Farr assay.

Antibodies to double-stranded DNA (dsDNA), generally regarded as highly specific for systemic lupus erythematosus (SLE), have also been reported in chronic active hepatitis (CAH). Using the Farr assay and E. coli DNA, fractionated by benzoylated-naphthoylated-DEAE cellulose chromatography into dsDNA and dsDNA containing single-stranded regions, we compared the serum DNA binding of CAH and SLE patients. Although CAH sera were found to have dsDNA binding significantly above the normal control group such binding was of low level and we could find no evidence of markedly elevated dsDNA binding in CAH. However 12 of the 20 CAH sera studied did bind preferentially to dsDNA containing single-stranded regions suggesting the presence of anti-single-stranded DNA antibodies. We conclude that the description of elevated anti-dsDNA antibodies, as measured by the Farr assay, in CAH is due to the interaction of anti-single-stranded DNA antibodies or other serum components with single-stranded DNA contaminating dsDNA preparations.

Adult↗

Polynucleotide specificities of murine monoclonal anti-DNA antibodies.

Three IgM monoclonal anti-DNA antibodies were produced by hybridoma techniques from an MRL-lpr/lpr mouse using denatured DNA (dDNA) as the selection antigen. All three antibodies also bound poly(dT), poly(rA), and the single-stranded random copolymer poly(dI,dT), and each antibody displayed a unique preference for a limited array of other ribo- and deoxyribopolynucleotides based on direct binding as well as inhibition studies. Inability to identify a common primary structure in the polynucleotides reactive with each antibody suggested that higher ordered structures may be important. This notion was supported by the finding that oligomers of thymidine of 25-30 nucleotides or less were ineffective in blocking antibody binding to dDNA or poly(dT). However, deliberate destabilization of putative secondary structures by decreasing counterion concentration and increasing temperature had little effect on antibody binding to poly(dT). Since the antigenic polynucleotides in general contain little known secondary structure and considerable flexibility, antibody binding may be accompanied by local conformational changes in the polynucleotide that result in a better fit to the antibody combining site.

Animals↗

Purification of the Sm nuclear autoantigen. Detection and clinical significance of IgM antibody.

Sm antigen from rabbit thymus acetone powder was purified using a combination of ammonium sulphate precipitation, DEAE-Sephacel and hydroxyapatite chromatography. This preparation was devoid of previously identified nuclear antigens including ribonucleoprotein (U1-RNP), proliferating cell nuclear antigen (PCNA), Sjögren's syndrome antigen A (SS-A/Ro), Sjögren's syndrome antigen B (SS-B/La), Sjögren's lupus antigen (SL), scleroderma antigen 70 (Scl-70), DNA and histones. The purified material was used in an enzyme linked immunosorbent assay (ELISA) to detect anti-Sm antibody. All sera with precipitating Sm antibody detected by immunodiffusion gave reactions in ELISA greater than 0.40 OD405 and contained predominantly IgG anti-Sm antibody. Of 112 sera which did not have anti-Sm by immunodiffusion there were five which gave reactions greater than 0.40 OD405. Four of these five sera contained only IgM antibody and the fifth contained both IgM and IgG. Of these five, one came from a 'normal' control who had a positive anti-nuclear antibody (ANA), facial rash and diabetes, two were from patients with systemic lupus erythematosus (SLE) and two were from patients with mixed connective tissue disease (MCTD). These findings demonstrate that there are patients whose anti-Sm response may be restricted to IgM and in some of these patients the clinical presentation may be different from that of classical SLE.

Animals↗

Partial purification of anti-DNA antibodies from systemic lupus erythematosus serum by dye-ligand chromatography.

Purification of anti-DNA antibodies from systemic lupus erythematosus (SLE) serum is usually achieved by DNA-affinity chromatography. However, using DNA-cellulose, the present study has found that this technique results in very low yields of DNA binding activity, much of which is contaminated with DNA simultaneously released during chromatography. In comparison it has been found that the anionic dyes Cibracon blue F3GA and Procion red HE3B, bound to crosslinked agarose, give more than 80% recovery and purification of DNA binding activity from whole SLE serum of 11- and 7-fold respectively. The majority of serum immunoglobulin did not bind to the dyes, but that which did, including anti-DNA antibody, bound by ionic interaction. Dye-ligand chromatography is therefore suggested as a useful technique for high yield recovery of partially purified DNA binding activity which can be subjected to further purification procedures, such as preparative isoelectric focusing.

Antibodies, Antinuclear↗

In vivo anti-nuclear antibodies in epithelial biopsies in SLE and other connective tissue diseases.

In vivo anti-nuclear antibody (ANA) was observed by direct immunofluorescence microscopy in epithelial cell nuclei in forty-four biopsies from thirty-three patients. The tissue containing the ANA was macroscopically normal in twenty-seven patients. The thirty-three patients with in vivo biopsy ANA included twenty-three with SLE, three with mixed connective tissue disease, two each with multi-system Sjögren's syndrome, dermatomyositis, and progressive systemic sclerosis, and one with rheumatoid arthritis. Features of sicca syndrome were noted in seventeen patients. The patterns of the in vivo biopsy ANA in the thirty-three patients were speckled (21), homogeneous (6), nodular (2), and both speckled and homogeneous (4). Complement was not detected in the epithelial cell nuclei. Immunoglobulin(s) and/or complement were deposited along the dermoepidermal junction in thirty-two of the forty-four biopsies, and in dermal blood vessels in twenty-two biopsies. Each patient had serum ANA against rat liver substrate; twenty-seven had high titre ANA (1 in 1000 or greater). Elevated levels of DNA-binding were found in twenty patients (61%), but the level of DNA-binding did not correlate with the intensity of in vitro biopsy ANA staining. Serum antibody to ribonucleoprotein (RNP) was present in eight of the twenty-three patients tested (35%), all eight patients having clinical features of sicca syndrome. Hypocomplementaemia was found in thirteen patients (40%), all of whom had active SLE. In vivo biopsy ANA appears to be a real phenomenon of unknown aetiology, and not an artifact, which is found in some patients with active multisystem autoimmune disease, especially SLE.

Adult↗

Autoantibodies in scleroderma.

In scleroderma a profusion of circulating autoantibodies have now been defined. They include autoantibodies to Scl-70 or DNA topoisomerase 1, and to centromere/kinetochore proteins of 17.80 and 140 kilodaltons. In addition, there are several antigens which are resident primarily in the nucleolus and they are RNA polymerase 1, PM-Scl, fibrillarin and 7-2 ribonucleoprotein. Antibody to Scl-70 has been found primarily in the diffuse form of scleroderma and antibody to the centromere/kinetochore proteins in the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia) subset of scleroderma. Autoantibodies to the nucleolar antigens RNA polymerase 1, PM-Scl, fibrillarin and 7-2 RNP have been detected in at least 10% of all patients with scleroderma. For several reasons which are discussed, it appears that the autoantibody response in scleroderma is antigen-driven and further that the autoantigens involved in this disease are present at some time in the nucleolus. These observations may be providing clues to some of the basic mechanisms initiating autoimmunity.

Autoantibodies↗

Induction of DNA-antibodies by d-penicillamine.

As no critical evaluation of native-DNA antibodies due to d-penicillamine (dP) has been made, we have examined this using highly purified DNA fractions obtained by benzoylated-naphthoylated-DEAE-cellulose chromatography. The nDNA, dsDNA, and ds/ss DNA binding was determined in 10 normals, 24 rheumatoid arthritis (RA) patients never on dP, 17 RA patients on dP and in serial sera from 15 of the latter before, during, and after cessation of dP. The results indicate that dP does induce both dsDNA and ssDNA antibodies which persist for many months after its cessation.

Antibodies, Antinuclear↗

Autoantibody-defined epitopes on nuclear antigens are conserved, conformation-dependent and active site regions.

Antinuclear antibodies in the systemic rheumatic diseases have been powerful reagents for identifying and characterizing nuclear antigens and for elucidating immune mechanisms which drive the autoimmune response. Concepts which have emerged from these studies include the following: the autoimmune response is antigen driven, autoantigens are components of subcellular particles, autoantigens are involved in important biosynthetic functions and epitopes recognized by autoantibodies are active sites or functional domain regions. PCNA (proliferating cell nuclear antigen) is a nuclear protein of 36 kDa and autoantibodies are present in patients with systemic lupus erythematosus. PCNA is a component of the DNA replication complex and is associated with DNA polymerase delta in continuous strand DNA synthesis at the replication fork. Studies have shown that epitopes on PCNA recognized by lupus antibodies are conformation-dependent. Composite peptides synthesized by joining discontinuous linear sequences were used to immunize rabbits and one such composite peptide induced antibodies with properties strikingly similar to human autoantibodies. Immunogens in the systemic autoimmune diseases are likely to be intranuclear or other intracellular particles composed of protein-protein or protein-nucleic acid complexes which are involved in cellular biosynthetic functions.

Antigens, Nuclear↗

Increasing diversity of the U.S. population.

Demographic changes have shaped the nation's past and will continue to shape its future. During the first half of the 1990s, the U.S. population grew, on average, by 2.7 million people each year, reaching 262.8 million in 1995. Population growth is projected to continue for the next 50 years, although at a slower rate. The forecast is for more than 390 million Americans by the year 2050. As the U.S. population grows, it will increasingly become more diverse along many socioeconomic dimensions. This increasing diversity will represent an historic shift in America's racial and ethnic composition with long-range implications for how we view racial issues, how we define racial categories and how the political landscape will be refashioned. By the middle of the 21st century the "minority" population will almost equal the size of the non-Hispanic white population. The minority population grew 14 percent during the first half of the 1990s compared with a 3 percent growth in the non-Hispanic white population. But even within the minority population, growth rates varied. Between 1990 and 1995, the Asian population grew 23 percent, the Hispanic population 20 percent and the African American population increased their numbers by 8 percent. Hispanics are projected to outnumber African Americans within the next 15 years. In part, these demographic changes are shifting because of U.S. immigration policies. Until the early 1960s, immigrants to the United States were primarily of white, European stock. Nowadays, Europeans account for about 20 percent of the immigrants. Three-quarters of legal immigrants in the mid-1990s now come from Latin America, the Caribbean and Asia. The increasing racial and ethnic diversity in the United States will create both challenges and opportunities for U.S. schools and businesses in the future. The magnitude of these numbers and their geographic location will be important factors to consider as we prepare for the 21st century.

Cultural Diversity↗

A portrait of Asians and Pacific Islanders in the United States.

Asians and Pacific Islanders (API) have an increasingly visible presence in the United States. This diverse population--encompassing persons with ancestry from East and Southeast Asia, the Indian subcontinent, and the Pacific islands--has grown at a faster rate than any other major racial or ethnic group. In 1996 Asian Americans numbered approximately 9.7 million (up from 3.8 million in 1980), nearly 4 percent of the U.S. population. The Census Bureau projects that this population group will reach 34.4 million by 2050, representing roughly 9 percent of all Americans. While immigration has fueled much of this growth, Asians' young age structure also will help boost their numbers in the next century. Fifty-six percent of Asian Americans live in three states--California, New York and Hawaii. Asian Americans comprise very small proportions of the populations of most other states. About 77 percent of the 2.8 million API households in 1996 were families, compared with 69 percent of white households. Roughly one in six Asian American households has five or more persons, compared with one in 12 white households. Educationally, Asians tend to be high achievers--42 percent of all API adults have at least a bachelor's degree, compared with 26 percent of while adults. Two-thirds of Asian Americans participated in the civilian labor force in 1996. Among employed Asians, one-third held managerial and professional jobs. Both proportions were roughly the same as for whites. Although the median income for API households was 9 percent higher than for white households in 1995, this difference is largely due to Asian households having more workers contributing to the household income. Despite these apparent measures of success, the poverty rates for Asian American families and individuals are nearly twice as high as those for whites.

Adult↗