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S Whittingham

Publications and source records attributed to S Whittingham.

At least 37 records · Page 2Linked to original sources
PubMed1989

Autoepitopes reactive with anti-SS-B/La.

Sera from 120 patients with suspected autoimmune rheumatic disease and antinuclear antibodies of anti-SS-B/La specificity were examined by Western blotting for reactivity with the SS-B/La polypeptide of HeLa cells and recombinant SS-B/La derived from a 1.4 kilobase (kb) cDNA encoding approximately 90% of the SS-B/La molecule. All sera reacted with the HeLa cell and the recombinant SS-B/La. One hundred and fourteen (95%) reacted with a set of three Staph. aureus V8 protease-resistant peptides of Mr 30,000, 29,00 and 28,000 from a methionine-rich region of HeLa cell SS-B/La designated the X domain, and 98 (82%) reacted with another set of two protease-resistant peptides of Mr 24,000 and 23,000 from a phosphorylated region of HeLa cell La designated the Y domain. One reacted weakly with the Y domain only. All sera that reacted with X and Y reacted more strongly with X, suggesting that X was the major epitope. Antibodies affinity purified from the X domain reacted strongly with the X peptides but not with the Y peptides and conversely, antibodies affinity purified from the Y domain reacted with the Y peptides but not with the X peptides. Both antibodies reacted with a fusion protein comprising 102 amino acids at the carboxyl terminus of the SS-B/La molecule. This protein contained no methionine, demonstrating that methionines were not involved in the antibody-binding site. Over 80% of patients whose only criteria for selection was the presence of anti-SS-B/La had the clinical, histologic, serologic and phenotypic features of Sjögren's syndrome whilst the remaining 20% had at least two of the features.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1988

Characteristics and epitope mapping of a cloned human autoantigen La.

The La (SS-B) polypeptide is a ribonucleoprotein against which high titer antinuclear antibodies (ANA) react in the human autoimmune disease primary Sjögren's syndrome. To identify the autoepitopes with which the ANA anti-La (anti-SS-B) reacts, we isolated a 1.4-kb cDNA clone for La from a lambda gt10 library made from a human Burkitt's cell line. This clone contained an open reading frame of 1065 bp, encoding a 40.1-kDa polypeptide that corresponded to the carboxyl-terminal end of the La protein. The predicted polypeptide sequence of the recombinant protein was highly charged and unrelated to any previously published sequence. We also compared this clone to a previously published cDNA sequence for La and demonstrated significant differences, particularly that the open reading frame in our cDNA continued for 926 additional bases 3' to a putative termination codon in the previously reported sequence. The recombinant La protein was expressed in Escherichia coli and tested for reactivity with 200 sera containing ANA of various specificities. Only the sera containing anti-La antibodies reacted with the cloned La. By expressing subclones of the La cDNA as fusion proteins with beta-galactosidase, we have localized at least one epitope for the binding of anti-La antibodies to the carboxyl-terminal 103 amino acids of the La protein. No anti-La binding could be demonstrated to the region of the La protein that had previously been predicted to contain an autoepitope for the binding of anti-La (SS-B) antibodies. Studies of cloned autoepitopes could provide important clues to the role ANA play in disease and lead to targeted intervention in the treatment of primary Sjögren's syndrome.

Amino Acid Sequence↗
PubMed1988

Antinuclear antibodies as molecular and diagnostic probes.

Much progress has been made in the past decade in defining the specificities of antinuclear antibodies (ANA) that are present in the blood of patients with multi-system autoimmune rheumatic diseases, and ANA now have an important place in diagnostic immunology. Disease-specific ANA have been defined and the intracellular autoantigens against which they react have been characterized. ANA have also established an important place in cell biology. Their use as probes has enabled molecular biologists to isolate, purify and assay the function of the highly conserved molecules with which they react, revealing new insights into the role these molecules play in gene transcription and translation. Cloning of the genes encoding these molecules in addition to providing information on primary structure has also provided human recombinant proteins for use as pure substrates for the development of simple and highly sensitive diagnostic assays in rheumatology. Studies on the epitopes with binding sites for ANA should provide new knowledge on how the immune response to these molecules evolves, how it is maintained and what role ANA play in the pathogenesis of disease.

Antibodies, Antinuclear↗
PubMed1988

Striational autoantibodies: quantitative detection by enzyme immunoassay in myasthenia gravis, thymoma, and recipients of D-penicillamine or allogeneic bone marrow.

Striational autoantibodies (StrAb) are a useful serologic marker of thymoma in patients with myasthenia gravis (MG). We compared a standard immunofluorescence method with a new enzyme immunoassay (EIA) for detection of StrAb. Retrospective testing of 264 stored sera by the two methods yielded well-correlated results (58 sera were positive by both assays; r = 0.8). For 104 patients with spontaneously acquired MG or thymoma, results were 100% concordant, of which 53% were positive. For 34 recipients of D-penicillamine, StrAb were found in 15% by EIA and in 6% by immunofluorescence. StrAb were detected in two of four bone marrow recipients by EIA and in one by immunofluorescence. Prospective testing of 434 fresh sera (of which 49 were positive by the two methods) yielded discordant results in only 4. Serial EIA quantitation of StrAb in two patients with MG and thymoma proved useful in monitoring immunosuppressant therapy and in a third patient predicted recurrence of the tumor. A high prevalence of StrAb was detected by both assays in elderly patients with spontaneous MG, but StrAb were more readily quantifiable by EIA. The EIA method proved to be highly sensitive and specific for detecting StrAb in patients with thymoma with and without MG, in patients treated with D-penicillamine, and in those with graft-versus-host disease after bone marrow transplantation.

Adolescent↗
PubMed1988

Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase.

Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency (greater than 95%) of autoantibodies to a Mr 70,000 mitochondrial antigen, a component of the M2 antigen complex. We have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase (EC 2.3.1.12) of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.

Acetyltransferases↗
PubMed1988

Autoantibodies in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease in which the predominant autoantibodies are antinuclear antibodies (ANA) reactive with DNA and histones, and antibodies reactive with the non-histone extractable nuclear antigens (ENA), Sm and Ro. Racial differences demonstrable in predisposition to SLE are also evident in the prevalence of autoantibodies, the frequency of anti-Sm and anti-Ro being 2-4 times higher in Asians with SLE than in Caucasians with SLE. Autoantibodies have also played a role in the classification of lupus and the recognition of multisystem autoimmune diseases that fail to meet the classical criteria for the identification of patients with SLE but appear to be variants of lupus. Anti-Ro is a diagnostic marker for subacute cutaneous lupus, anti-(U1)RNP a marker for mixed connective tissue disease, antibodies to phospholipids a marker for the syndrome comprising stroke, fetal wastage, thromboembolism and thrombocytopenia and antibodies to histones a marker for lupus induced by the drugs hydralazine and procainamide. There is still no unaminity on whether these antibodies play an integral role in the disease process or whether they are "epiphenomena". The challenge for research in the 1980s is the understanding of the relationship of these antibodies to the pathogenesis of SLE.

Antibodies, Antinuclear↗
PubMed1987

Serological diagnosis of primary Sjögren's syndrome by means of human recombinant La (SS-B) as nuclear antigen.

Human recombinant La nucleoprotein was purified from cultures of Escherichia coli containing a vector with a 1.4 kilobase cDNA encoding La; the nucleoprotein was used to test for antinuclear antibodies (ANA) to La. Serum samples from 260 patients with autoimmune diseases associated with ANA and 100 healthy subjects were tested by an enzyme-linked immunosorbent assay (ELISA). Samples from 47 (94%) of 50 patients with primary Sjögren's syndrome and 1 (7%) of 14 patients with secondary Sjögren's syndrome reacted with the recombinant La. No reactivity was demonstrated in 196 patients with other ANA-associated autoimmune diseases or in 100 healthy subjects. The study confirms the high correlation between ANA, anti-La, and primary Sjögren's syndrome and shows how gene cloning can provide large quantities of human autoantigens for use in highly specific and sensitive diagnostic assays.

Antibodies, Antinuclear↗
PubMed1987

Epstein-Barr virus as an etiological agent in primary Sjögren's syndrome.

It is proposed that the initiating event in primary Sjögren's syndrome is infection with Epstein-Barr virus (EBV), and that the autoimmune exocrinopathy that progresses to keratoconjunctivitis sicca and xerostomia is a sequel to this. Our hypothesis is based on the findings that the antinuclear antibody, anti-La(SS-B), is a marker of primary Sjögren's syndrome, and that anti-La reacts with a ribonucleoprotein, the La autoantigen, to which bind not only all cellular RNAs transcribed by RNA polymerase III, but also the viral RNAs, EBER 1 and EBER 2, encoded by the Epstein-Barr virus (EBV). It is proposed that during EBV infection, there are multiple copies of the EBERs available to bind to the La ribonucleoprotein and when infection occurs in subjects who have an impaired T cell-mediated response to EBV, and who are genetically predisposed to autoimmunity, there is loss of immunological tolerance to La with production of anti-La (SS-B). Thus the inflammatory process in exocrine glands which culminates in the sicca syndrome is due to the combined effects of chronic EBV infection and autoimmunity. Two patients are briefly described in whom primary Sjögren's syndrome appeared to be a direct consequence of EBV infection. The hypothesis engages the question of the respective roles of virus infection, specific immunodeficiency to virus, immunogenetic constitutive influences and an autoimmune response to a ribonucleoprotein antigen in the genesis of a particular organ-specific inflammatory reaction.

Antibodies, Viral↗
PubMed1986

A highly conserved 72,000 dalton centromeric antigen reactive with autoantibodies from patients with progressive systemic sclerosis.

An autoantibody reactive with a 72,000 dalton centromeric antigen was detected by immunoblotting with the use of a nuclear enriched HeLa cell preparation in 42 of 77 patients with progressive systemic sclerosis (PSS). Reactivity with the 72,000 dalton polypeptide was associated with anti-centromere autoantibodies (ACA) detected by immunofluorescence (IF), and the antigen was highly conserved, being present in both human cells and Leishmania tropica. Thirty-five (83%) of the 42 sera reactive with the 72,000 dalton polypeptide also reacted with a 19,500 dalton polypeptide, and antibodies eluted from both the 72,000 dalton and the 19,500 dalton polypeptides reacted with the centromere when retested by IF on intact HEp2 cells, demonstrating that both polypeptides are antigenic components of the centromere. Only one of the 42 sera had precipitating antibodies to the Scl-70 antigen detected by counterimmunoelectrophoresis, indicating that the 72,000 dalton polypeptide was not related to the previously described Scl-70 antigen. The other 35 of the 77 sera tested were negative for ACA, although all had ANA, with the main patterns of IF being fine speckling of the nucleus (18 sera) and homogeneous or speckled staining of the nucleolus (17 sera). Anti-Scl-70 antibodies were detected in 17 of these 35 patients, 15 (88%) of whom reacted with an 89,000 dalton polypeptide, one with a 140,000 dalton polypeptide, and one with a 74,000 dalton polypeptide. Ten of the 15 sera reacting with the 89,000 dalton polypeptide also reacted with a 74,000 dalton polypeptide, and 2-D gel analysis suggested a relationship between the two molecules. Clinically defined types of scleroderma tended to associate with antibodies to particular molecular antigenic specificities. Thirty-seven (88%) of the 42 patients reactive with the 72,000 dalton polypeptide had sclerodactyly and features of the CREST syndrome, whereas patients reactive with the 89,000 dalton polypeptide and with Scl-70 tended to have more extensive cutaneous and visceral involvement.

Antigen-Antibody Complex↗
PubMed1985

Serum protein polymorphisms in patients with primary Sjögren's syndrome.

The distribution of 4 serum proteins, the protease inhibitor alpha-1-antitrypsin, haptoglobin, transferrin and group-specific component or vitamin D-binding protein, were examined in 26 patients with primary Sjögren's syndrome and 208 healthy donor controls. No significant associations were found between any of the 4 systems and susceptibility to primary Sjögren's syndrome.

Blood Proteins↗
PubMed1985

Primary Sjögren's syndrome after infectious mononucleosis.

A healthy young woman developed primary Sjögren's syndrome after protracted infectious mononucleosis. The diagnosis of primary Sjögren's syndrome was supported by histologic evidence of sialadenitis in labial salivary glands, rheumatoid factor, hypergammaglobulinemia, the HLA-B8 phenotype, and a high titer antibody to the anti-La (SS-B) nucleoprotein that co-precipitated the small ribonucleic acids encoded by Epstein-Barr virus, EBER 1 and EBER 2, as well as "host" RNA. There was strong humoral immunity to the Epstein-Barr nuclear and capsid antigens, but weak T-lymphocyte-mediated cytotoxicity to Epstein-Barr-transformed lymphoblasts, anergy to antigens used to elicit delayed-type hypersensitivity, and a low T-helper/T-suppressor cell ratio. The series of events initiated by infectious mononucleosis was attributed to a genetic defect in the immune response. Association of viral RNA with the La nucleoprotein resulted in a break in immunologic tolerance via a T-cell bypass effect with induction of anti-La (SS-B) by polyclonally activated B lymphocytes leading to autoimmune sialadenitis.

Adult↗
PubMed1985

Reactivity of anti-mitochondrial autoantibodies in primary biliary cirrhosis: definition of two novel mitochondrial polypeptide autoantigens.

Sera that contained autoantibodies to mitochondria (AMA) by immunofluorescence were examined by immunoblotting for reactivity with mitochondrial polypeptides from various mammalian species, yeast, and E. coli. Mitochondrial polypeptides were separated by polyacrylamide gel electrophoresis, were immobilized on nitrocellulose, and were exposed to sera. The sera tested included 18 AMA-positive sera from patients with primary biliary cirrhosis (PBC), two AMA-positive sera from patients without PBC, and 53 AMA-negative sera. All AMA-positive sera reacted with either one or the other, or usually both of two human mitochondrial polypeptides of 70 kilodalton (kD) and 45 kD. The 53 AMA-negative sera were not reactive with the 70 kD polypeptide, but six reacted with the 45 kD polypeptide. The reactivity of the 70 kD and the 45 kD polypeptide was destroyed by brief exposure to trypsin. The counterpart of the 70 kD reactive polypeptide in human mitochondria was a 65 to 70 kD polypeptide in rat and mouse mitochondria, and a 55 kD polypeptide in yeast and in E. coli. The apparent 45 kD polypeptide was similar in all mitochondrial preparations tested, but no counterpart could be identified in E. coli. Beef heart mitochondria were used to show that the reactive polypeptides were present in a semipurified preparation of the F1 portion of mitochondrial H+ ATPase; however, sera did not react with the beta subunit of ATPase, proposed as a candidate mitochondrial autoantigen. The present molecular characterization of two particular antigens should lead to the more precise identification of these antigens, and also to a clearer insight into the pathogenesis of PBC.

Antibody Specificity↗
PubMed1985

Molecular analysis of the RNA and protein components recognized by anti-La(SS-B) autoantibodies.

The aim of this study was to determine whether sera with autoantibodies to the La(SS-B) nuclear antigen react with the same or different sets of cellular or viral ribonucleoproteins (RNPs) and whether patients with anti-La(SS-B) comprised a homogeneous group with respect to phenotypic and serological markers. The 34 anti-La(SS-B) sera studied were detected in the course of screening 2,000 sera referred from patients with suspected or defined multisystem autoimmune disease. Analysis of the molecular components of the small nuclear (sn) RNPs isolated from immune complexes developed in vitro between the IgG fractions of the anti-La(SS-B) sera and cell lines selected for their content of viral and cellular (non-viral) RNA showed that all 34 anti-La(SS-B) sera reacted with the same group of cellular RNAs and with two viral RNAs encoded by Epstein-Barr virus. The La(SS-B) RNPs contained one major 50,000 dalton antigenic polypeptide that resolved into 5-6 heterogeneously charged isospecies on two-dimensional immunoblots. In addition to anti-La(SS-B) reactivity, all 34 sera were shown to contain anti-Ro(SS-A) activity by counterimmunoelectrophoresis (CIEP); however, with three exceptions, the antigenic Ro(SS-A) polypeptide was not detectable by immunoblotting. The homogeneity of this group with anti-La(SS-B) was indicated by the findings that of the 34 cases 31 (88%) had hypergammaglobulinaemia, 33 (97%) had rheumatoid factor and 27 (of 30 tested, 90%) were HLA-B8. Thus all anti-La(SS-B) sera react with the same set of RNAs associated with an antigenic 50,000 dalton nucleoprotein, and the presence of anti-La(SS-B) autoantibodies identified a homogeneous group of patients with the serological and phenotypic features of primary Sjögren's syndrome.

Antibodies, Antinuclear↗
PubMed1984

Effect on immunologic and other indices of adjuvant cytotoxic chemotherapy including melphalan in breast cancer.

Thirty patients with histologically proven node-positive early breast cancer (Stage II) were treated by total mastectomy and axillary clearance and adjuvant chemotherapy regimens including melphalan for 1 year. These patients were studied sequentially, at 3-month intervals, for up to 2 years to assess effects of cytotoxic drugs on immune function, and to determine whether any changes in immune function were related to recurrence. All indices were in the normal range before chemotherapy. The most marked and long-lasting effects of chemotherapy were on numbers of circulating T-cells and B-cells. Mean counts +/- one standard error (X 10(6)/ml) for T-cells before and 12 months after stopping chemotherapy were 1.537 +/- 0.118 and 0.874 +/- 0.120 (P less than 0.01), and for B-cells 0.345 +/- 0.060 and 0.207 +/- 0.030 (P less than 0.01). Functional indices of T-cell and B-cell competence were less compromised than values for cell counts and, in contrast, recovery occurred either during or within 3 months of stopping chemotherapy. This held for both T-cell function measured by delayed-type hypersensitivity (DTH) responsiveness to five recall antigens and mitogenic responsiveness to phytohemagglutinin, and for B-cell function measured by titration of blood group isohemagglutinins. After 4 years the 30 subjects were divided into groups according to whether there was recurrence of cancer (14) or no recurrence (16); the only index predictive of recurrence was depression of DTH to recall antigens. Thus it was found that cytotoxic chemotherapy with melphalan appears to cause long-lasting depression of cell counts but only short-lasting depression of functional indices of immunocompetence, and that levels of immunologic indices during chemotherapy are mostly nonpredictive of recurrence of cancer. The results prompt some caution in the use of adjuvant chemotherapy, at least with melphalan.

Adult↗
PubMed1984

Use of the Bio-Rad silver stain to identify gel purified RNA components of small nuclear ribonucleoprotein antigens.

The ultrasensitive silver staining procedure developed for the detection of proteins in polyacrylamide gels has been used to identify the RNA component of small nuclear ribonucleoprotein antigens recognised by antinuclear antibodies. The technique described is a simple, safe and inexpensive procedure with a sensitivity comparable with that obtained by biosynthetic labelling of RNA with 32P.

Antibodies, Antinuclear↗