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L Aarden

Publications and source records attributed to L Aarden.

48 records · Page 3Linked to original sources
PubMed1985

Complement-fixing antibodies to dsDNA detected by the immunofluorescence technique on Crithidia luciliae. A critical appraisal.

Studies using an adapted immunofluorescence technique (IFT) on Crithidia luciliae to determine the complement fixing ability of antibodies to dsDNA in relation to disease manifestations, i.e., nephritis, have yielded conflicting results. To establish the relevance of these determinations, we studied sera containing antibodies to dsDNA from 64 patients with systemic lupus erythematosus (SLE), and found that anti-dsDNA of 52% of these sera had the ability to fix complement. SLE patients with nephritis demonstrated a much higher incidence of complement fixing anti-dsDNA (83%) than patients without nephritis (17%, p less than 0.01). On the other hand, patients with nephritis also had higher titers of anti-dsDNA (mean 1:400) than patients without nephritis (mean titer 1:75; p less than 0.01). A clearcut correlation between anti-dsDNA titer and complement fixing anti-dsDNA titer (p less than 0.01) was observed which obviously disturbs the correlation between nephritis and complement fixing anti-dsDNA. Comparing matched sera from patients with nephritis and patients without nephritis with the same antidsDNA titer, we found no difference in complement fixing anti-dsDNA. In the IFT used to measure complement fixing anti-dsDNA, incubation of the Crithidia slides with patients' serum was followed by an incubation with fresh normal serum which served as a source of complement. We observed that this incubation with fresh normal serum resulted in elution of anti-dsDNA antibodies from kinetoplast DNA. This elution was caused by IgG present in normal serum.

Antibodies↗
PubMed1982

Influence of pH on the detection of low- and high-avidity anti-dsDNA.

In 2 radioimmunoassays in use to detect antibodies to dsDNA, the Farr assay and the PEG assay, we observed inhibitory effects of normal human serum (NHS) on the DNA binding by SLE sera. This was found to be due by the fact that, during incubation at 37 degrees C, CO2, introduced in the incubation mixture by the serum, evaporates from the mixture. This results in increase in pH to values well above pH 8.0, which in turn leads to a decreased DNA binding by antibody. When SLE sera are tested at low dilution, this phenomenon may lead to false negative results. Proper pH control, by the use of buffers with a greater buffering capacity than PBS, completely prevented the observed inhibitory effects. However, under these conditions NHS bound significant amounts of DNA in both assays. The non-specific DNA binding by NHS was found to be heat-stable, but could be eliminated either by aerosil treatment of the sera or by addition of dextran sulphate to the incubation mixture. Lipoproteins and, to a lesser extent, the complement component C1q appear responsible for this non-specific binding. To avoid false negative results with SLE sera as well as non-specific binding by NHS, we propose the use of stronger buffers in combination with added dextran sulphate to the incubation mixture in both the Farr assay and the PEG assay.

Antibody Affinity↗
PubMed1982

Specificity in systemic lupus erythematosus of antibodies to double-stranded DNA measured with the polyethylene glycol precipitation assay.

Recently, a new radioimmunoassay--the polyethylene glycol (PEG) assay--was introduced to measure antibodies to double-stranded (ds) DNA. In this method, polyethylene glycol precipitation of formed 3H-DNA/antiDNA complexes is used instead of the ammonium sulfate precipitation used in the Farr assay. In contrast to the Farr assay, with which only high-avidity antibodies to dsDNA are detected, the PEG assay also reportedly measures anti-dsDNA of relatively low avidity. We studied whether this gain in antibody measurement results in loss of specificity for systemic lupus erythematosus. When the PEG assay was applied to a selected panel of 440 sera from patients with various well-defined autoimmune diseases and to a group of 197 normal human control sera, matched sex and age to the patients, the method was found to be fairly specific for systemic lupus erythematosus, although the sera from some patients with myasthenia gravis and some with autoimmune liver disease were also found positive. Screening of 352 additional serum specimens, sent to our laboratory for diagnostic reasons, revealed that, with the PEG assay, an extra population of relatively low-avidity antibodies to dsDNA--missed by the Farr assay--was detected. Upon clinical evaluation, we found that the patients in whom such antibodies were detected generally fulfilled a number of the preliminary criteria of the American Rheumatism Association for systemic lupus erythematosus, but that this diagnosis often was not made. We claim that the presence of low-avidity antiDNA characterizes a milder form of the disease in which patients often show only a single clinical feature of the disease. We conclude that results of the PEG assay add valuable diagnostic and clinical information to results obtained by the Farr assay.

Antibody Specificity↗
PubMed1982

Measurement of low avidity anti-dsDNA by the Crithidia luciliae test and the PEG assay.

With the immunofluorescence technique (IFT) using Crithidia luciliae as a substrate, 14,417 sera sent to our laboratory for routine anti-dsDNA determination, were screened for the presence of antibodies to dsDNA. The 1,260 sera that were found IFT positive were then assayed with the Farr radioimmunoassay, in which 3H-labelled PM2-DNA is used as antigen. Only 470 sera (37%) were found to be Farr positive. This discrepancy is, at least partially, caused by the fact that the Farr assay does not detect anti-DNA of low avidity, whereas the Crithidia-IFT does. Sixty-eight percent of the IFT-positive/Farr negative sera were found positive with the PEG assay, a radioimmunoassay that also employs double stranded PM2-DNA as antigen, and that also detects anti-dsDNA of low avidity. The IFT performed on IFT positive/Farr negative sera was found to be rather irreproducible. It was shown that this was due to local increases of the salt concentration resulting from the way the assay was performed. The problem could be overcome by careful control of the assay conditions, i.e. never letting Crithidia slides dry up after washing with PBS. In the PEG assay, these sera sometimes showed a DNA binding that decreased with time. It could be shown that this is caused by a parallel increase in pH during the incubation as a result of CO2 evaporation from the serum.

Antibodies, Antinuclear↗
PubMed1982

Avidity of antibodies to dsDNA: comparison of IFT on Crithidia luciliae, Farr assay, and PEG assay.

The Farr assay is thought to detect only antibodies to DNA of relative high avidity. This is due to the high salt concentration of the employed ammonium sulfate precipitation, which dissociates DNA-anti-DNA complexes of low avidity. A recently introduced method to detect anti-DNA, the PEG assay, circumvents these dissociating reaction conditions by using polyethylene glycol instead of ammonium sulfate to precipitate the complexes; we therefore thought to measure antibodies to DNA of low avidity as well. We tested this assumption in several ways. It was found that the PEG assay detects a population of antibodies to DNA that are missed by the Farr assay. Complexes made with these antibodies were salt labile and could readily be dissociated by means of excess DNA, whereas Farr-positive antibodies formed stable complexes with DNA. Avidity studies using the method described by Celada et al. indicated that the anti-DNA detected by the PEG assay but missed by the Farr assay was of relatively low avidity. An inverse correlation between avidity and slope of the binding curves in the PEG assay was observed. These results confirm the notion that the PEG assay detects antibodies to DNA of low avidity. The fact that the Farr assay does not measure these antibodies confers possible diagnostic importance upon the PEG assay.

Antibody Affinity↗
PubMed1980

The use of polyethylene glycol precipitation to detect low-avidity anti-DNA antibodies in systemic lupus erythematosus.

With a recently introduced method for measurement of low-avidity anti-DNA, the polyethylene glycol (PEG) precipitation assay (Riley et al., 1979), high levels of DNA binding by normal human serum (NHS) were found when circular PM2-DNA was used as antigen. THe nature of this DNA binding was studied. The removal of low-density lipoproteins (LDL) from the serum, e.g., by Aerosil absorption, eliminated DNA binding by NHS. Purified LDL bound DNA to the same extent as NHS. Non-specific binding of NHS or LDL to DNA was prevented by adding dextran sulphate to the incubation mixture. Analysis on sucrose gradients showed that only large DNA-anti-DNA complexes were precipitated by 3.5% PEG. The PEG assay with dextran sulphate is a sensitive assay for low-avidity anti-DNA antibodies. It adds important information to results obtained with the Farr assay, which mainly detects antibodies of high avidity.

Antibodies, Antinuclear↗
PubMed1980

Limiting dilution analysis of the suppressive effect mediated by alloantigen-primed cells.

T cells primed in mixed lymphocyte culture exert both positive and negative allogeneic effects on B cells expressing the appropriate alloantigens. The positive and negative effects can be separated by limiting dilution analysis: positive effects, measured by production of anti-sheep erythrocyte antibody, are revealed when low numbers of primed T cells are added to cultures of B cells and sheep erythrocytes, while suppression of the response occurs at higher T-cell inputs. In the present report, these negative allogeneic effects have been analysed in detail. Suppression was qualitatively and quantitatively similar when helper T cell activity was provided from any of several sources. Helper T cells in the alloantigen-primed population gave rise to active T-cell replacing factors even under conditions in which all microcultures were suppressed and suppressor cells were present at a high multiplicity in every well. The degree of suppression was influenced by the multiplicity of B cells in culture; as the number of B cells increased, more suppressor cells were required to inactivate a microculture. Taken together, these data indicate that the targets of the suppressor cells are B cells and not helper T cells or T-cell replacing factors. Although suppressor cells can prevent the activation of B cells by the more frequent helper cells in the primed T-cell population, detailed analysis of the stoichiometry of the suppression demonstrated that a single suppressor cell is capable of inactivating only a limited number of B cells, suggesting that a 'ratio-dominance' model of suppression is operative in this system.

Animals↗
PubMed1977

Detection and characterization of DNA-anti-DNA complexes in a patient with systemic lupus erythematosus.

DNase digestion of SLE serum, with consequent release of bound DNA antibody has been proposed as a method for the direct demonstration of circulating DNA-anti-DNA complexes. In the present studies on the serum of a girl with active SLE nephritis, circulating DNA-anti-DNA complexes were demonstrated at the precise time of relapse of SLE nephritis. Ultracentrifugation showed that these complexes were of low molecular weight.

Antibodies, Antinuclear↗
PubMedDate not supplied

Both naive and memory T cells can provide help for human IgE production, but with different cytokine requirements.

Most in vitro systems for the induction of IgE production by human B cells require both IL-4 and the presence of T cells. Little is known about the mechanism of T cell help or the ability of different T cell subsets to provide this helper activity. In the present study we demonstrate that, in the presence of exogenous IL-4, anti-CD3 stimulated naive T cells (CD4+CD45RA+) are potent helper cells for human IgE production. In their presence, as little as 750 autologous B cells can produce up to 100 ng/ml IgE. This response was found over a broad range of anti-CD3 concentrations. IgE helper activity by naive T cells was inhibited by IL-2. Under all conditions tested, naive T cells were unable to provide help for IgM production. This is in contrast to activated memory T cells (CD4+CD45RO+), which are very efficient helper cells for IgM or IgE production, provided that IL-2 or IL-2 plus IL-4 are present respectively.

Antibodies, Monoclonal↗
PubMedDate not supplied

Differential induction of interleukin-6 production in monocytes, endothelial cells and smooth muscle cells.

Studying the production of IL-6 (interleukin-6) by monocytes, endothelial cells and smooth muscle cells we observed that cytokine inducers like IL-1, TNF alpha (tumor necrosis factor alpha), LPS (lipopolysaccharide), SAC (Staphylococcus Aureus Cowan 1) and PMA could be divided roughly into two categories. Bacterial products such as LPS or SAC have a potent IL-6 inducing effect on monocytes and minor or no effect on endothelial- and smooth muscle cells. The other category comprising IL-1, TNF alpha and PMA induces IL-6 production in endothelial- and smooth muscle cells. Only IL-1 induces IL-6 production in monocytes as well as in endothelial cells and smooth muscle cells. In addition to IL-6, also IL-1 and TNF alpha are produced by monocytes however with different kinetics. None of the stimuli had any inhibitory effect on IL-6 production with the exception of PMA. Whereas PMA induced IL-6 production in endothelial cells and it potentiated the induction of IL-6 by IL-1 in these cells, it inhibited LPS-stimulated IL-6 production in monocytes. In line with the effects of PMA, staurosporin induced IL-6 production in monocytes and it inhibited IL-1 driven IL-6 production by endothelial cells.

Antigens, Bacterial↗
PubMedDate not supplied

Characterisation of monoclonal antibodies to human IL-4: application in an IL-4 ELISA and differential inhibition of IL-4 bioactivity on B cells and T cells.

Five murine monoclonal antibodies, raised against E. coli derived human IL-4, were established. All mAb were also reactive with natural IL-4. Competition ELISA experiments revealed that mAb 1,2 and 4 recognized a related epitope on IL-4. mAb 5 and mAb 6 recognized another epitope. Two non-competing mAb were used to develop a sandwich IL-4 ELISA. mAb 5 was used for coating and biotinylated mAb I was used as the second antibody. Intra- and interassay-coefficients were 3.3 and 10.1% respectively. The ELISA is specific for IL-4, rapid and sensitive (the detection limit is 2 pg/ml). The capacities of the antibodies to inhibit IL-4 activity were tested in B cell and T cell assays. All antibodies inhibited IL-4 dependent IgE production by human B lymphocytes. A similar inhibition of IL-4 driven T cell proliferation by the antibodies was observed, with the exception that mAb 4 did not affect the activity of IL-4 on T cells. These results led to the suggestion that B cells make use of another (additional) IL-4 receptor chain.

Animals↗