Possible role of antiidiotypes in the loss of anti-topoisomerase I antibodies: comment on the article by Kuwana et al.
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Antibodies, Anti-Idiotypic↗
Biomedical subjects
H A Stafford
Publications and source records attributed to H A Stafford.
Autoantibodies to the 20-kDa ribosomal proteins: identification, characterization, and new aspects on prevalence in systemic Lupus erythematosus.
Autoantibodies to the 20-kDa ribosomal proteins (L12/S10) are not well studied, especially in juveniles with systemic lupus erythematosus (SLE). Randomly selected sera from American juveniles and adults with SLE were screened for antibodies to either 20-kDa protein and P proteins and then assayed for anti-L12 and anti-S10 by immunoblot assays. In a pilot study of patients with anti-P (Cohort 1), IgG antibodies to either 20-kDa protein and, specifically, to L12 were observed in 72 and 42% of juveniles and adults, respectively. IgG antibodies to S10 were detected less frequently. In Cohort 2 patients who were chosen irrespective of autoantibody status, twice as many juveniles as adults had IgG antibodies to either 20-kDa protein. Prevalences of IgG anti-L12 and IgG anti-S10 antibodies in the juveniles were 28 and 16% and in the adults were 13 and 12%, respectively. Anti-L12 were strongly but not invariably associated with anti-P, and usually arose temporally to these antibodies. Anti-S10 activity was due to anti-Sm antibodies. We conclude that IgG anti-L12 are more prevalent in SLE than previously reported, and are responsible for the majority of activity toward the 20-kDa ribosomal proteins, especially in juveniles.
Adolescent↗
A murine monoclonal anti-idiotype to anti-ribosomal P antibodies: production, characterization, and use in systemic lupus erythematosus.
Overt anti-ribosomal P (anti-P) autoantibodies are restricted to a subset of systemic lupus erythematosus (SLE) patients, and are potentially pathogenic. Covert anti-P are detected in all other individuals. An idiotype (Id) network is nonoperational in those with overt anti-P, whereas it is functional in all others. The aim of this study was to produce a murine monoclonal (mAb) anti-Id to characterize the anti-P Id network in SLE. BALB/c mice were immunized with F(ab')(2) fragments of IgG anti-P from a patient with a broadly cross-reactive Id. One mAb was chosen (mAb41) that reacted preferentially to the immunogen. This IgG1 mAb bound comparably in ELISAs to affinity-purified anti-P from 11 SLE patients with overt anti-P. This binding was partially inhibited with ribosomal P antigen. In contrast, it did not bind to affinity-purified control autoantibodies, nor to normal human IgG. mAb41 inhibited anti-P binding to ribosomal P antigen in immunoassays and on Jurkat cells. No change was detected in patients' anti-P antibodies over time when mAb41 was used in Id-specific ELISAs. We conclude that mAb41 is an anti-Id that recognizes a public idiotope within the antigen-combining site of anti-P antibodies. Thus, it is analogous to its human counterparts, and potentially, would modulate the pathogenicity of anti-P autoantibodies in vivo.
Animals↗
Prevalence of autoantibodies to ribosomal P proteins in juvenile-onset systemic lupus erythematosus compared with the adult disease.
OBJECTIVE: To determine the prevalence of anti-ribosomal P (anti-P) proteins in several groups of patients with juvenile-onset systemic lupus erythematosus (SLE) in comparison with the prevalence in adult SLE. METHODS: Serum samples were pooled together from 3 cohorts of patients with juvenile-onset SLE in 3 different medical centers and from a miscellaneous group of juvenile-onset SLE patients whose samples were sent by regional physicians. Sera were studied for the presence of anti-P using 2 assays: Western blot with ribosomes as antigen, and an enzyme-linked immunosorbent assay with the COOH-terminal 22 amino acids of the ribosomal P protein in a multiantigenic peptide format as antigen. Sera found positive by both tests were considered positive for anti-P antibodies. Findings from similar studies involving a large cohort of patients with adult-onset SLE from Oklahoma City were used for comparison. RESULTS: The prevalence of anti-P antibodies in the pooled sample of juvenile-onset SLE sera was 45 of 108, or 42%, while in the adult cohort from Oklahoma City, 20 of 260, or 7.7%, were positive for anti-P (odds ratio [OR] 9.6, P < 10(-8) by Fisher's exact test). In addition, it was shown that 12 of 13 patients with both anti-P and anti-double-stranded DNA (anti-dsDNA) in the juvenile SLE cohort had nephritis, while only 8 of 22 patients without both antibodies were nephritic (OR 21.0, P < 10(-8)). It was also shown that in 9 illustrative cases, the levels of anti-P and anti-dsDNA antibodies usually varied together and in concordance with the clinical activity as measured by the SLE Disease Activity Index (SLEDAI). Finally, anti-P-positive and anti-P-negative patients had a similar prevalence of anti-dsDNA, anti-Ro/SSA, and anti-La/SSB antibodies, but patients with anti-P had a higher prevalence of anti-U1 RNP and anti-Sm (P = 0.041 and P = 0.0385, respectively, by Fisher's exact test). CONCLUSION: Antibodies to ribosomal P protein are more prevalent in juvenile-onset SLE than in adult-onset SLE. Levels of antibodies to ribosomal P protein vary with the clinical disease activity as measured by the SLEDAI, often in concordance with the levels of anti-dsDNA. The presence of both anti-P and anti-dsDNA antibodies was powerfully associated with nephritis in the cohort of patients for whom comprehensive clinical and serologic data were available.
Adolescent↗
Anti-idiotypic antibodies prevent the serologic detection of antiribosomal P autoantibodies in healthy adults.
A subset of SLE patients has serologically detectable autoantibodies to the ribosomal P proteins (anti-P). We reported the discovery of covert anti-P antibodies and their masking IgG-inhibitory antibodies in the sera of healthy adults. The aim of this study was to determine if these IgG-inhibitory antibodies are anti-idiotypic antibodies (anti-Ids). IgG and IgG-depleted fractions of plasma from two healthy adults were assayed for inhibition of anti-P F(ab')2 binding to the ribosomal P proteins in immunoblot. Anti-P antibody activity was completely inhibited by plasma IgG, whereas there was no inhibition by IgG-depleted plasma. IgG-inhibitory antibodies recognized a cross-reactive epitope among anti-P from different SLE patients. Plasma IgG from one healthy adult was depleted of pepsin agglutinators and generic anti-F(ab')2 antibodies by adsorption with an affinity column prepared with normal IgG F(ab')2. Unretained IgG bound exclusively to anti-P F(ab')2 in ELISA. Using four affinity columns, we isolated IgG anti-Ids to anti-P antibodies from four healthy adults. These purified anti-Ids bound to anti-P F(ab')2 from a healthy adult and SLE patients. They did not bind to F(ab')2 fragments prepared from normal IgG or anti-dsDNA. Ribosomal antigens blocked this anti-Id-Id interaction. Purified anti-Ids inhibited the binding of anti-P F(ab')2 from patients to ribosomal P proteins. SLE patients without overt anti-P antibodies also possessed IgG anti-Ids to anti-P antibodies. We conclude that IgG-inhibitory antibodies are anti-Ids to anti-P antibodies, and are directed to public idiotopes on anti-P antibodies. These anti-Ids may be part of an Id network that regulates anti-P antibody expression, and perhaps pathogenicity.
Adult↗
The presence of masked antiribosomal P autoantibodies in healthy children.
OBJECTIVE: To determine if antiribosomal P (anti-P) autoantibodies are present in healthy children. METHODS: Sera from healthy children were screened for anti-P by conventional enzyme-linked immunosorbent assay and immunoblot techniques. Sera were also treated with immobilized ribosomal P antigens on nitrocellulose strips; affinity-purified fractions were tested for anti-P by high-sensitivity immunoblot. The relative binding affinities were compared for affinity-purified anti-P antibodies from healthy children and adults, and patients with systemic lupus erythematosus. IgG fractions of anti-P-depleted sera from healthy children were assessed for inhibition of autologous anti-P activity. RESULTS: Conventional serologic screening showed no IgG nor IgM anti-P in 88 untreated sera. IgG anti-P were unmasked in all 79 sera treated by the membrane batch affinity technique. IgM anti-P were identified in 27 of the treated sera; the percentage of positive sera decreased with increasing age (chi(2) for linear trend P = 0.00081). Affinity-purified anti-P from children had relative binding affinities similar to those of anti-P from other groups. Sera from healthy children contained inhibitory IgG antibodies to anti-P. CONCLUSION: These results show that anti-P autoantibodies are present in all healthy children. The majority of these autoantibodies are masked by IgG antibodies, suggesting concordant development of a regulatory network.
Adolescent↗
Anti-ribosomal and 'P-peptide'-specific autoantibodies bind to T lymphocytes.
Patients with systemic lupus erythematosus (SLE) frequently have anti-lymphocyte autoantibodies, some of which also bind to surfaces of neurons. Since anti-ribosomal P protein autoantibodies (anti-P) from SLE patients also bind to surfaces of neurons, we hypothesized that anti-P are anti-lymphocyte antibodies. A panel of human T lymphocytes was evaluated for anti-P binding by indirect immunofluorescence. Affinity-purified anti-ribosomal antibodies were used as a source of anti-P. These autoantibodies bound to the surfaces of all transformed T cell lines tested. This binding was not mediated by Fc receptors. It was inhibitable by ribosomes. Anti-P bound to circulating T lymphocytes from healthy adults and children. They also bound to thymocytes and cord blood T cells from normal neonates. Circulating T cells from SLE patients with anti-P bound less anti-P than cells from healthy controls. Two patients were studied on multiple occasions. The capacity of their T cells to bind anti-P correlated inversely with titres of anti-ribosomal antibodies. Anti-ribosomal antibodies, other than anti-P, also appear to bind to T cells. The surface of T cells contains a protein with the size and antigenicity of the ribosomal P protein, P0. We conclude that anti-ribosomal antibodies are a subset of anti-lymphocyte autoantibodies. Their possible role in the pathogenesis of lymphopenia or lymphocyte dysfunction in SLE has to be defined in further studies.
Adolescent↗
Development of the anti-Ro autoantibody response in a patient with systemic lupus erythematosus.
OBJECTIVE: To characterize the initial events in anti-Ro production by a patient with systemic lupus erythematosus, in whom this autoantibody is developing. METHODS: The immune response to the Ro ribonucleoprotein and other autoantigens were studied by enzyme-linked immunosorbent assay for IgG and IgM, by isoelectric focusing, and by inhibition studies to determine apparent avidity. RESULTS: The patient's sera showed an oligoclonal response to Ro that increased in complexity and affinity with time. IgM anti-Ro appeared shortly before IgG anti-Ro, and disappeared as IgG anti-Ro increased in titer and affinity. IgG antiribosomal P autoantibodies also appeared during the patient's course, but in contrast to anti-Ro, were not preceded by IgM antiribosomal P. CONCLUSION: These data are consistent with the Ro autoantigen being presented and processed in a manner similar to heterologous antigen, and with differences in the mechanisms that lead to the production of IgG anti-Ro autoantibodies as opposed to antiribosomal P autoantibodies.
Adult↗
Recombinant ribosomal P2 protein can unmask anti-ribosomal P autoantibodies from healthy adults.
Autoantibodies to ribosomal P proteins (anti-P) are detected almost exclusively in the serum samples from patients with systemic lupus erythematosus when conventional enzyme-linked immunosorbent assay and immunoblotting techniques are used. Anti-P are not detected in serum samples from healthy adults by these techniques. By treating serum from healthy adults with ribosome-coated beads, we unexpectedly unmasked anti-P in virtually all individuals. This unmasking of anti-P occurs by the displacement of an antibody inhibitor from anti-P. We wanted to determine whether anti-P from healthy adults could also be unmasked by treatment of their serum or plasma with isolated ribosomal P proteins. Recombinant human ribosomal P2 protein was produced in bacteria as a TrpE fusion protein, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and isolated as strips of membranes corresponding to the size of the P2 fusion protein. Serum or plasma from six healthy adults and three patients with systemic lupus erythematosus were incubated with these strips overnight rather than for 2 hours, as is done in conventional immunoblots. Acid eluates were obtained from the strips and analyzed for antibody activity by immunoblot. Eluates from healthy adults and patients contained antibodies reactive with recombinant ribosomal P2 protein. They also reacted with the three ribosomal P proteins in purified rabbit ribosomes. Their anti-P activity was completely inhibited by a peptide corresponding to the immunodominant linear epitope of the ribosomal P proteins. The antibodies in the eluate were immunoglobulin G. We conclude that anti-P autoantibodies from healthy adults can be unmasked by affinity purification on denatured, recombinant ribosomal P proteins and that antigen excess is sufficient for inhibitor displacement.
Adult↗
Unmasking of anti-ribosomal P autoantibodies in healthy individuals.
Autoantibodies to the ribosomal P proteins (anti-P) are found almost exclusively in a subset of patients with systemic lupus erythematosus. To determine if anti-P autoantibodies are present in healthy individuals but in a masked state, sera from healthy adults were applied to affinity columns coated with ribosomes, and the affinity-purified fractions were analyzed for anti-P Abs by ELISA and immunoblot. Anti-P Abs were detectable in serum only after affinity chromatography, were predominantly of the IgG isotype, stained HEp-2 cells in the characteristic anti-P pattern, and demonstrated specificity for all three ribosomal P phosphoproteins. Using batch affinity chromatography, we identified anti-P autoantibodies in the affinity-purified serum fractions of 54 of 55 healthy individuals. Anti-P Abs from healthy adults generally bound less ribosomal P Ag per mg/ml IgG than anti-P from patients. Inhibition studies revealed that autologous serum contained an IgG inhibitor of anti-P Ab. We conclude that masked anti-P autoantibodies are present in the healthy population and hypothesize that detection of these autoantibodies in systemic lupus erythematosus represents disruption of these regulatory networks.
Adolescent↗
PIG-A, DAF and proto-oncogene expression in paroxysmal nocturnal haemoglobinuria-associated acute myelogenous leukaemia blasts.
Failed surface expression of the complement decay-accelerating factor (DAF) due to mutation of the PIG-A gene is a hallmark of affected paroxysmal nocturnal haemoglobinuria (PNH) blood elements. Previous findings that acute myelogenous leukaemia (AML) blasts evolving in a PNH patient differed from idiopathic AML blasts in that they exhibited DAF negativity suggested that the leukaemic blasts derived from an affected PNH cell. To investigate whether these cells differ from untransformed PNH cells in PIG-A genetic alterations or in DAF mRNA processing, or are distinguishable from conventional AML blasts in proto-oncogene activation or chromosomal structure, their DNA and RNA were examined using PIG-A, DAF and proto-oncogene probes and their karyotype was analysed. Analyses of the PIG-A genome revealed dual exchanges of A1110-->G and T1130-->A resulting in conversions of T370 to R and I377 to N in the coding region but no deletions or rearrangements. Investigations of DAF mRNA processing showed mRNA species differing in 3' UT regions from those in untransformed cells but similar to those in DAF-positive leukaemia cell lines. Studies of c-myb, c-myc, c-fos and c-fms showed no gross genetic alterations, amplifications or variations in mRNA transcripts deriving from these genes. Karyotypic analysis showed no alterations. The results indicate that in AML blasts evolving in PNH: (1) the PIG-A genome exhibits multiple point mutations but no gross genetic changes; (2) DAF mRNA transcripts exhibit differentiation-dependent variations that do not affect GPI-anchoring; (3) c-myb, c-myc, c-fos and c-fms activation show no differences from idiopathic AML; and (4) no karyotypic abnormalities are associated with AML transformation.
Antigens, CD↗
Flavonoid evolution: an enzymic approach.
Flavonoid evolution in land plants is discussed from an enzymic point of view, based on the present day distribution of the major subgroups of flavonoids in bryophytes, lower and higher vascular plants. The importance of varied functions in the origin of pathways with a series of sequential steps leading to end-products is considered; it is argued that the initial function is that of an internal regulatory agent, rather than as a filter against ultraviolet irradiation. The basic synthases, hydroxylases, and reductases of flavonoid pathways are presumed to have evolved from enzymes of primary metabolism. A speculative scheme is presented of flavonoid evolution within a primitive group of algae derived from a Charophycean rather than a Chlorophycean line, as a land environment was invaded. Flavonoid evolution was preceded by that of the phenylpropanoid and malonyl-coenzyme A pathways, but evolved prior to the lignin pathway.
Journal Article↗
Normal polymorphic variations and transcription of the decay accelerating factor gene in paroxysmal nocturnal hemoglobinuria cells.
In paroxysmal nocturnal hemoglobinuria (PNH), an acquired hemolytic anemia, deficiency of decay accelerating factor (DAF) renders blood cells susceptible to increased deposition of autologous complement activation fragments (C3b) and complemented-mediated injury. To investigate the mechanism of the DAF defect, DNA and mRNA from normal and PNH leukocytes were compared in blot hybridization assays by using DAF cDNA and oligonucleotide probes. Southern analyses of DNA from normal cells revealed a single gene spanning approximately equal to 35 kilobases of DNA. Six HindIII banding patterns were distinguishable among normal individuals. In family studies, the patterns segregated as three homozygous and three heterozygous genotypes deriving from three haplotypes: A, B, and C with frequencies of 0.47, 0.36, and 0.17, respectively. Oligonucleotide mapping localized the polymorphic HindIII sites to two noncoding regions in the vicinity of exons encoding (i) the protein oligosaccharide-rich domain and (ii) the mRNA 3'-untranslated region. Analyses of DNA from DAF-negative leukocytes of eight PNH patients demonstrated restriction fragment profiles identical to those of normal individuals for all enzymes studied. Three patients had the BC (normals = 3/32), three patients had the AA (normals = 6/32), and two patients had the AC (normals = 8/32) HindIII genotype. Of the three PNH patients exhibiting the BC genotype, family studies of two demonstrated the expected inheritance patterns, and RNA gel blot analyses of two showed mRNA transcripts indistinguishable from those in normal cells. The absence of DAF gene or mRNA alterations in affected PNH cells that lack other glycolipid-anchored proteins as well as DAF argues that the lesion underlying PNH cells resides in the glycolipid-anchor pathway.
Blood Proteins↗
Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh.
Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C(18) columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3'-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid.
Journal Article↗
Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydromyricetin to Its Flavan-3,4-Diol (Leucodelphinidin) and to (+)-Gallocatechin by Reductases Extracted from Tissue Cultures of Ginkgo biloba and Pseudotsuga menziesii.
Extracts of callus or cell suspension cultures from petioles of Ginkgo biloba catalyzed the production of (+)-gallocatechin (2,3-trans-3,5,7,3',4',5'-hexahydroxy-flavan) from (+)-dihydromyricetin (5'-hydroxy-dihydroquercetin) along with the expected 3,4-cis-diol intermediate, leucodelphinidin, in a NADPH-dependent double-step reductase reaction at pH 7.4. The latter diol, isolated from the above incubation mixture, produced (+)-gallocatechin in a NADPH-dependent reaction. Extracts from tissue cultures derived from needles of Pseudotsuga menziesii (Douglas fir) also produced significant amounts of the 3,4-diol from dihydromyricetin. (+)-Dihydromyricetin, purified via paper chromatography from leaves of Leptarrhena pyrolifolia, was reduced by NaBH(4) to the presumed 3,4-trans-diol and acid epimerized to the 3,4-cis-diol.
Journal Article↗
Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir.
Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols.
Journal Article↗
Incorporation of [C]Phenylalanine into Flavan-3-ols and Procyanidins in Cell Suspension Cultures of Douglas Fir.
L-[(14)C]Phenylalanine, fed to cell suspension cultures of Douglas fir, (Pseudotsuga menziesii Franco) was incorporated simultaneously, but at different rates, into (+)-catechin, (-)-epicatechin, and procyanidins of increasing molecular weight. Asymmetric labeling of dimers and polymers was demonstrated, with more label appearing in the upper than in the lower or terminal unit. In addition, the total pool of free monomers was 10 to 30 times more highly labeled than was this lower, terminal unit of dimers and higher oligomers. Since the dimer, epicatechin-catechin, contained more label than catechin-catechin, it is concluded that the carbocation with the 2,3-cis stereochemistry of (-)-epicatechin was formed more rapidly than was that of the 2,3-trans type of (+)-catechin.
Journal Article↗
Enzymic and nonenzymic reduction of (+)-dihydroquercetin to its 3,4,-diol.
A NADPH-dependent reductase activity, capable of converting (+)-dihydroquercetin (2,3-trans) to its 3,4-diol (a leucocyanidin), has been demonstrated in crude, soluble protein extracts derived from cell suspension cultures of Douglas fir (Pseudotsuga menziessi). Neither NADH nor ascorbate substituted as the H-donor. Quantitative analyses were based on the production of cyanidin, the formation of an adduct with vanillin, and on absorbance at 280 nanometers. Nonenzymic reduction of (+)-dihydroquercetin with NaBH(4) produced two presumably isomeric flavan-3,4,-diols. One of these was similar to the enzymically produced diol, based on products isolated by chromatography on paper, on thin-layer cellulose and on C(18) reversed-phase columns (high performance liquid chromatography), and on the conversion of the diol to the all-trans dimer of (+)-catechin upon the addition of (+)-catechin.
Journal Article↗