Expression of inactive stage anti-dsDNA idiotypes on anti-ssDNA antibodies in a lupus patient during active stage of lupus cerebritis.

The possibility that idiotypes (Ids) defined on anti-double stranded DNA (dsDNA) antibodies during active and inactive stages of lupus (1/84 Id and 4/90 Id, respectively) were expressed on anti-DNA antibodies during a subsequent active period (9/90) of the disease was investigated in a lupus patient with lupus cerebritis. Using rabbit (R)-anti-Ids specific to 1/84 Id and 4/90 Id in inhibition assays, the 4/90 Id was shown to be expressed on the framework regions of anti-single stranded DNA (ssDNA) but poorly on co-existing anti-dsDNA antibodies of active (9/90) stage. The 1/84 Id was poorly expressed on both types of 9/90 anti-DNA antibodies. While the 9/90 anti-ssDNA significantly bound to immobilized ssDNA and several single-stranded polynucleotides, only ssDNA inhibited the binding of the anti-ssDNA to ssDNA, suggesting its monospecificity toward ssDNA. Western blot analysis following isoelectric focusing showed that a spectrotype pattern of 4/90 Id-positive 9/90 anti-ssDNA IgG was similar to that of the 4/90 anti-dsDNA, suggesting that they are of related clonal origin. The present study suggests the idiotypic heterogeneity of anti-DNA antibodies and the shift of antigen specificity within an idiotypically related anti-DNA population during exacerbation of the disease.     

Multi-score estimation of catecholamine fluorescence for clinical purposes

Experience accumulated at multi-score semiquantitation of catecholamine fluorescence of glyoxylic acid-treated stretch preparations of human clinical specimens is presented. The methodology and criteria of quantitation are described in detail. For an example, comparison between 3 different methods for analyzing neural-bound noradrenaline in human myocardial tissue in various heart diseases (obtained during the course of cardiac surgery) is presented: Biochemical determination of tissue noradrenaline content multi-score estimation of catecholamine fluorescence of glyoxylic acid-treated stretch preparations microfluorimetric analysis of the same stretch preparations. The results show that the multi-score estimation method gives a reliable concept of the relative amounts of noradrenaline stored in the intrinsic adrenergic nerve net (corresponding closely to the individual and group differences observed at biochemical noradrenaline determination). In addition, possible regional differences, alterations in the structural integrity of the inbuilt intrinsic nerve net, and other structural changes (e.g. pathological catecholamine accumulations) are easily recognized, whereas biochemical estimation cannot give information on structural aspects, which may have important clinical repercussions. Microfluorimetry does not seem suitable for studies on human myocardial specimens for several reasons which are discussed. The method of multi-score estimation of catecholamine fluorescence described and discussed is recommended for other similar and related studies on human clinical materials.     

Adsorption of anti-dsDNA antibodies by immobilized polyanionic compounds.

Antibodies to dsDNA are characteristically present in serum from patients with systemic lupus erythematosus (SLE), and have been shown to have the capacity to react with various molecules bearing repeating negative charges. After a number of polymeric or monomeric molecules with differently charged groups and hydrophobic molecules had been coupled covalently as ligands on cellulose gel, the adsorption capacities of the ligands for anti-dsDNA antibodies were evaluated. It was found that gels coupled with polyanionic dextran sulphate (DXS) and polyacrylic acid (PA) and monoanionic sulphanilic acid (SA) absorbed anti-dsDNA antibodies effectively. DXS gel also adsorbed antibodies to ssDNA and heparan sulphate, antigens with repeating negatively charged moieties, while no ligand was able to adsorb anti-nRNP antibodies. The finding that DXS gels adsorbed anti-dsDNA antibody in proportion to their charge density, and that the interaction between anti-dsDNA and DXS gel is broken readily by an increase in ionic strength, indicated that the binding is ionic in nature. Moreover, virtually all F(ab')2 anti-dsDNA became adsorbed onto the DXS gels, suggesting that the binding occurred via specific antigen-binding sites on the antibody molecule. Binding of these polyanion-binding autoantibodies with anionic sites in the glomerular basement membrane may therefore cause the tissue damage observed in SLE.     

Microtechnique for simultaneous determination of immobilizing and cytotoxic sperm antibodies. Methodological and clinical studies.

A microtechnique for simultaneous detection of immobilizing and cyctotoxic sperm antibodies was elaborated according to the principles of Terasaki and McClelland. The technique was found reproducible, easy to perform and thus suitable for large scale examinations. The test includes determination of titres for immobilizing and cytotoxic effects and provides at the same time an opportunity to observe both complement-dependent and independent activities. Furthermore, agglutination patterns can easily be recorded. The requirement of only small volumes of test samples makes the test suitable also in testing secretions from the genital tract. In these cases, inclusion of a control for anticomplementary activity of the sample is advisable. In serum from 288 men from infertile couples simultaneous occurence of immobilizing and cytotoxic antibodies was observed in eight cases; all of them also had sperm-agglutinins in serum. In two of these cases, immobilizing and, in one case, cytotoxic activity was revealed in seminal plasma using undiluted samples. Patients with negative findings in serum also revealed negative findings in seminal plasma. Immobilizing and cytotoxic antibodies were not disclosed in serum from 247 women from infertile couples.     

Cationic and high affinity serum IgG anti-dsDNA antibodies in active lupus nephritis.

To investigate differences between cationic anti-dsDNA antibodies during active and inactive nephritis, low- and high-affinity IgG anti-dsDNA antibodies were prepared from sera of a lupus patient and compared for their binding affinity, spectrotype, and idiotype expression. The ratio of high-affinity to low-affinity anti-DNA antibodies and the relative avidity of the high-affinity anti-DNA antibodies decreased when active nephritis became inactive. Isoelectric focusing showed that cationic anti-dsDNA populations were present predominantly in the high-affinity fraction during active nephritis and in the low-affinity fraction during inactive nephritis. Idiotypic analysis by ELISA and Western blotting showed that the high-affinity cationic anti-DNA antibodies during active nephritis were idiotypically different from their low-affinity counterparts during inactive nephritis. The differences in binding affinity and idiotypy of the cationic anti-dsDNA antibodies suggest that certain serum IgG anti-dsDNA antibodies with both cationic charge and high affinity may be associated with active nephritis.     

Linking complement and anti-dsDNA antibodies in the pathogenesis of systemic lupus erythematosus

Systemic lupus erythematosus is a severe autoimmune disease that affects multiple organ systems resulting in diverse symptoms and outcomes. It is characterized by antibody production to a variety of self-antigens, but it is specifically associated with those against anti-dsDNA. Anti-dsDNA antibodies are present before the onset of clinical disease and are associated with severe manifestations of lupus such as glomerulonephritis. Their levels fluctuate with changes in disease activity and, in combination with the levels of complement proteins C3 and C4, are strong indicators of disease flare and treatment response in patients with lupus. The decreased complement levels that are noted during flares of lupus activity are believed to be secondary to increased autoantibody production and immune complex formation that results in tissue damage; however, recent data suggest that complement activation can also drive development of these pathogenic autoantibodies. This review will explore the various roles of complement in the development and pathogenesis of anti-dsDNA antibodies.     

A peptide DNA surrogate that binds and inhibits anti-dsDNA antibodies

Autoantibodies to dsDNA are an important diagnostic marker and pathogenic factor for systemic lupus erythematosus (SLE). Although the anti-dsDNA antibodies present in SLE are indicative of an antigen-driven response, the antigen has not been conclusively identified. The specific SLE anti-dsDNA antibodies were obtained by affinity purification using a dsDNA-coupled Sepharose column. Using the anti-dsDNA antibodies to screen a phage peptide display library, we demonstrated that purified polyclonal anti-dsDNA antibodies and a monoclonal anti-dsDNA antibody specifically bind a 15 mer peptide ASPVTARVLWKASHV. This chemically synthesized peptide could be recognized by anti-dsDNA antibodies in ELISA and Dot blot. This 15 mer peptide can inhibit anti-dsDNA antibodies binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay while a control peptide did not inhibit anti-dsDNA antibodies. This study demonstrates the potential usefulness of the peptide DNA surrogate in diagnostic tests of SLE and in the investigation of the origin of anti-dsDNA antibodies. It may also be used in studies of the DNA-anti-DNA antibody interaction.     

Generation of anti-ssDNA antibodies by persistent immunization of mice with sheep erythrocytes

Immunization of (DBA/2) mice with sheep erythrocytes (1 X 10(8) red cells, once weekly for 2 months) elicited anti-sheep erythrocyte antibodies, a part of which combined with ssDNA. By contrast, immunization with rat (Fisher) erythrocytes (1 X 10(8) red cells, once weekly for 2 weeks) did not elicit antibodies cross-reactive with ssDNA. The antibody response (IgM and IgG) to sheep erythrocytes rose sharply and subsequently tapered off (usually within the first 2 weeks). The level of IgG antibodies cross-reactive with ssDNA increased and, after ca. 1 month, decreased. No increase in anti-trinitrophenyl antibodies was detected. These results suggest the existence of a homeostatic mechanism. The anti-ssDNA antibodies bound to sheep erythrocytes, ssDNA and, marginally, to trinitrophenyl-gelatin; they did not bind to poly-D-glutamic acid, rat erythrocytes or mouse erythrocytes. Treatment of sheep red blood cells with neuraminidase, proteinase K, trypsin, or DNase did not alter the erythrocytes' capacity to bind the anti-ssDNA antibodies; solubilization of the erythrocytes with Triton X-100 abolished the binding. Neither a methanol:chloroform (1:1) extract (which contains the erythrocyte phospholipids) nor the residue (left after the extraction) bound anti-ssDNA antibodies. The determinant mediating the binding could be conformational.     

Evaluation of labelled monoclonal antibodies by simultaneous estimation of the association constant, the immunoreactive fraction, and the number of effective binding sites on the specific target.

The reaction between a labelled monoclonal antibody (MoAb) and its specific target is characterised by three parameters: the association constant (Ka) of the labelled MoAb, the number (N) of effective binding sites on the specific target, and the immunoreactive fraction (F) of the labelled MoAb preparation. Immunological binding parameters are usually estimated graphically, by fitting the experimental data to linear equations derived from the first order law of mass action (FLMA) at equilibrium. However, only two parameters can be estimated simultaneously in a two-dimensional plot. Consequently, graphical estimation of Ka, F and N must be performed stepwise, using at least two different plots. The three parameters are interdependent, and therefore a stepwise estimation procedure might give suboptimal results. In order to investigate whether this is a problem of practical significance in the evaluation of labelled MoAbs, a computerised iterative nonlinear least squares (INLSQ) method was applied to estimate the three parameters simultaneously. The binding parameters in reactions between different 125I-labelled MoAbs and different types of targets were significantly changed when a graphical procedure was replaced by the computerised INLSQ method, and the goodness of fit to FLMA was improved. Hence, the nonlinear least squares method is the preferred procedure. Values were affected when only a subset of the data was included in the estimation procedure, indicating some heterogeneity even in these presumably homogeneous MoAb reactions. The radiolabelling procedure was presumed to be the main reason for this heterogeneity.     

Lack of high avidity IgM anti-ssDNA antibodies in cells from autoimmune-prone and autoimmune mice

Non-autoimmune prone CBA mice were compared with autoimmune prone NZB, NZW, and (NZB x NZW)F1 mice for the ability of their splenic cells to produce anti-ssDNA-forming cells spontaneously in vitro, measured in the plaque forming cell assay. The number of antibody forming cells was measured and the relative avidity of antibody produced determined using a plaque inhibition assay. Splenic lymphocytes from young animals of a non-autoimmune strain (CBA/J) were shown to be capable of generating anti-ssDNA IgM antibody-forming cells in culture which displayed a higher avidity for antigen than that from autoimmune-prone or frankly autoimmune mice. Since an increased switching from IgM to IgG autoantibody production and defects in Fc-mediated signalling by IgG antibody have been identified in autoimmunity, we suggest that the metabolic block, normally in force in non-autoimmune-prone animals, accounts for this elevated avidity of IgM autoantibody.     

Use of dried blood spots for the detection and confirmation of HTLV-I specific antibodies for epidemiological purposes.

AIMS--To modify and evaluate a gelatin particle agglutination test that could provide a sensitive, specific and inexpensive method for the detection of HTLV-I antibody in dried blood spot samples (DBS) collected on filter paper. METHODS--A set of 26 reference samples confirmed as HTLV-I antibody positive were assembled from patients with tropical spastic paraparesis or adult T cell leukaemia and blood donors. Serum samples and simulated antibody positive dried blood spot eluates were tested using the Serodia assay together with two confirmatory tests: HTLV BLOT 2.3, a western blot, and Select-HTLV, an enzyme immunoassay (EIA). Both confirmatory tests use synthetic peptides to differentiate between antibodies to HTLV-I and -II. The modified Serodia assay was then used to test anonymously 10,135 DBS collected from neonates from London. Samples reactive in the modified Serodia test producing a positive result were titrated to an end point and confirmed as before. RESULTS--All 26 eluates made from simulated DBS derived from positive reference samples were identified as positive by the modified Serodia HTLV-I test and were confirmed as anti-HTLV-I positive by EIA. Two eluates derived from relatively low titre reference samples gave indeterminate results on western blotting. Screening of the 10,135 neonatal DBS resulted in six repeat reactives, five of which were confirmed. The remaining reactive sample gave an indeterminate result on western blotting and there was insufficient eluate for testing by EIA. The overall seroprevalence of HTLV-I in this population was 0.05% (five of 10,135). CONCLUSION--The modified Serodia HTLV-I assay provides a sensitive, specific and inexpensive (10 pence/test) method for screening large numbers of DBS. The format of the assay makes it ideally suited for simultaneous screening of antibodies to HIV-1, HIV-2 and HTLV-I using semi-automated equipment.     

Equipment for the quantification of motor performance for clinical purposes

An apparatus is described for the quantitative assessment of important parameters that characterise motor performance in normal subjects and in patients with different types of motor disorders. The apparatus has a handle that can be moved along a straight horizontal track either by the subject (to study voluntary movements) or by a torque motor (to study reflex activity). During voluntary movements the mass and friction of the mechanical part of the equipment are eliminated by feedback of the force exerted at the handle by the subject. The computer program that controls the apparatus gives a choice of four different tests that characterise different aspects of the motor system: the reflex organisation, the regulation of viscoelastic properties mediated in part by reflex activity, the control of fast goal-directed movements, and the performance in a tracking task. The results of a pilot study to the tracking behaviour of clumsy children show that the group of clumsy children differs from a group of normal children in the latency of the tracking response, in the ability to track high-frequency components and in the fact that clumsy children introduce relatively more frequency components in the response that are not present in the tracking signal.     

The origin and pathogenic consequences of anti-dsDNA antibodies in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a multisystem autoimmune rheumatic disease characterized by the presence of autoantibodies, many of which are directed against antigens of nuclear origin. In the clinical setting, these antibodies are important in the management of patients with SLE, providing information about disease activity, subtype and prognosis. The clearance of apoptotic debris is defective in patients with SLE. This causes the inappropriate persistence of nuclear antigens and thus a breakdown in peripheral tolerance with ensuing autoantibody generation. Patients have a variety of self-directed antibodies, and it is increasingly clear that some, but not all, of these antibodies are directly pathogenic. This review discusses the current hypotheses regarding the features by which antibody pathogenicity may be determined and therefore predicted, as well as the mechanisms by which these autoantibodies cause tissue damage in SLE and in lupus nephritis in particular. Identifying these pathogenic antibodies may well hold the key to the development of new, targeted therapies.     

联合分析血小板特异性抗体和瞬逝生物传感器技术检测血小板抗体的方法比较

目的 应用联合分析血小板特异性抗体(specificp platelet antibodies,SASPA)和瞬逝生物传感器技术(evanescent biosensor technology,EVA)分别检测血小板特异性同种抗体和自身抗体,对两者进行方法学比较.方法 收集94例存在HPA-1a、HPA-5b、HLA-I和自身抗体的患者血浆标本,制备血小板膜糖蛋白-血小板特异性抗体复合物,分别用SASPA和EVA方法对复合物进行检测,对SASPA检测所得平均荧光强度值(MFI)和EVA检测所得斜率值(Slope)作统计学分析和比较.结果 SASPA方法对HPA-1a、HPA-5b、HLA-I和自身抗体检测的敏感度分别为74.1％、86.9％、86.9％和40.9％,EVA方法的敏感度分别为88.9％、78.3％、60.9％和72.7％.人类血小板抗原(HPA)-1a抗体检测中,MFI与Slope表现为强相关性,相关性系数为r=O.95(P ＜0.05).结论 SASPA和EVA技术对传统的单克隆抗体特异性血小板抗原固定实验(MAIPA)作了继承和改良,SASPA方法可以用极少量的血小板同时检测多种血小板特异性抗体,提高了检测效率;EVA方法将血小板裂解后的实验时间缩短为10 min,极大地简化了操作过程,减少了检测时间.SASPA和EVA检测方法的建立对临床血小板免疫相关疾病的诊断和血小板输血领域的发展具有深远意义.     

Opsonization by anti-dsDNA antibodies of apoptotic cells in systemic lupus erythematosus

In order to asses the role of the soluble mediators of serum from patients with SLE in the apoptotic cell clearance, we measured the in vitro phagocytosis of apoptotic Jurkat cells by normal healthy donor macrophages in the presence of SLE patients' sera. A significant increase of the phagocytic index (NHD = 1.0 +/- 0.3; SLE = 1.9 +/- 0.6; p < 0.01) was to be observed in the presence of serum from patients with SLE. The increased phagocytic index correlated to the anti-dsDNA antibodies titers. We conclude that anti-dsDNA antibodies present in sera of patients with SLE favor the apoptotic cell phagocytosis by opsonization of the target cells. This may represent a deviation of the clearance process towards inflammation and a new pathologic feature of these autoantibodies in SLE.