Coexistence of systemic lupus erythematosus, tuberous sclerosis and aggressive natural killer-cell leukaemia: coincidence or correlated?

Among patients with systemic lupus erythematosus (SLE) there is an increased risk of haematological malignancies, especially non-Hodgkin lymphoma. However, the association of SLE with aggressive CD3 negative natural killer (NK)-cell leukaemia has not been reported so far. We present a case of a 39-year-old woman with SLE, aggressive NK-cell leukaemia and tuberous sclerosis complex. The prior probability of developing the combination of these three rare diseases by coincidence is extremely low (<10(-13)). Possible underlying immunological, genetic and toxic/environmental pathways are discussed.

Receptor for advanced glycation end products (RAGE) polymorphisms are associated with systemic lupus erythematosus and disease severity in lupus nephritis.

OBJECTIVE: Interaction of advanced glycation end products (AGEs) with their receptors (RAGE) plays an important role in inflammation in auto-immune diseases. Several functional polymorphisms of RAGE have been described. In this study we analysed the role of RAGE polymorphisms in disease susceptibility for systemic lupus erythematosus (SLE). In addition, we investigated whether these polymorphisms in SLE are associated with serum levels of soluble RAGE (sRAGE), renal involvement (lupus nephritis (LN)) and its outcome. METHODS: For this cross-sectional study DNA samples of 97 SLE patients, 114 LN patients and 429 healthy controls (HC) were genotyped for four RAGE polymorphisms: -429 T/C, -374 T/A, 2184 A/G and Gly82Ser. Differences in genotype frequencies and allele frequencies were tested between patients and HCs. In SLE patients, sRAGE was measured by enzyme-linked immunosorbent assay (ELISA). In addition, association of genotypes with sRAGE and disease severity in LN was analysed. RESULTS: The C allele of -429 T/C, the T allele of -374 T/A and the G allele of 2184 A/G were significantly more prevalent in SLE and LN compared with HC. In LN, the C allele of RAGE -429 T/C, the A allele of -374 T/A and the G allele of RAGE 2184 A/G polymorphism were significantly associated with more proteinuria and worse renal function during the first two years of treatment. No association of genotype with sRAGE was found. CONCLUSION: RAGE polymorphisms are associated with susceptibility to SLE and LN. In addition, some of these polymorphisms are likely to be associated with disease severity and initial response to treatment in LN.

Synchronized turbo apoptosis induced by cold-shock.

In our research on the role of apoptosis in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE), we aim to evaluate the effects of early and late apoptotic cells and blebs on antigen presenting cells. This requires the in vitro generation of sufficiently large and homogeneous populations of early and late apoptotic cells. Here, we present a quick method encountered by serendipity that results in highly reproducible synchronized homogeneous apoptotic cell populations. In brief, granulocytic 32Dcl3 cells are incubated on ice for 2 h and subsequently rewarmed at 37°C. After 30-90 min at 37°C more than 80-90% of the cells become early apoptotic (Annexin V positive/propidium iodide negative). After 24 h of rewarming at 37°C 98% of the cells were late apoptotic (secondary necrotic; Annexin V positive/propidium iodide positive). Cells already formed apoptotic blebs at their cell surface after approximately 20 min at 37°C. Inter-nucleosomal chromatin cleavage and caspase activation were other characteristics of this cold-shock-induced process of apoptosis. Consequently, apoptosis could be inhibited by a caspase inhibitor. Finally, SLE-derived anti-chromatin autoantibodies showed a high affinity for apoptotic blebs generated by cold-shock. Overall, cold-shock induced apoptosis is achieved without the addition of toxic compounds or antibodies, and quickly leads to synchronized homogeneous apoptotic cell populations, which can be applied for various research questions addressing apoptosis.

The role of reactive oxygen species in apoptosis of the diabetic kidney.

Increased levels of reactive oxygen species (ROS) by hyperglycemia can induce apoptosis of renal cells and diabetic nephropathy. The redox balance in the renal cell seems, therefore, of the utmost importance. ROS-mediated apoptosis may be further aggravated by an inadequate cytoprotective response against ROS. When there are insufficient cytoprotective and ROS scavenging molecules, ROS lead to considerable cellular damage and to a point of no return in apoptosis. Induction of cytoprotective proteins may prevent or attenuate apoptosis, renal cell injury, and finally diabetic nephropathy. Here, we discuss some mechanisms of apoptosis and several strategies that have been probed to ameliorate, or to prevent apoptosis in the diabetic kidney.

Incomplete nuclear transformation of human spermatozoa in oligo-astheno-teratospermia: characterization by indirect immunofluorescence of chromatin and thiol status.

BACKGROUND Sperm heterogeneity in the human, as observed in oligo-astheno-teratozoospermia (OAT), is associated with hypospermatogenesis. METHODS The chromatin of sperm from OAT and normospermic males was characterized with antibodies specific for nucleosomes, the histone H3.1/H3.2 isoform, histone TH2B, apoptosis-associated H4 acetylation (KM-2) and protamines. Subsequently, sperm samples were stained with the thiol-specific fluorochrome monobromobimane (mBBr) before and after reduction with dithiotreitol (DTT) as most thiol groups reside in the cysteine-rich protamines. We also used fluorescence-activated cell sorter (FACS) for sperm histograms and sorting for high or low free and total thiol levels. These fractions were further analysed for DNA damage with the TdT-UTP nick end-labelling (TUNEL) assay. RESULTS OAT sperm nuclei stained higher for nucleosomes and KM2-epitopes, and lower for TH2B. For free, and total, thiol groups, OAT sperm were characterized by biphasic distributions, reflecting incomplete thiol oxidation as well as overoxidation, and possibly reduced protamine contents. The TUNEL assay on sperm subfractions, for both control and OAT sperm, revealed that a lower level of free thiol groups is associated with a higher TUNEL incidence, and this relationship was also found for total thiol levels. Hence, both overoxidation and a low total number of thiol groups predestine for sperm apoptosis. CONCLUSIONS Chromatin characteristics reflecting an incomplete nucleosome to protamine remodelling were found in subfertile males. Sperm apoptosis is related to both incomplete remodelling and protamine overoxidation.

Differential binding of chemokines CXCL1, CXCL2 and CCL2 to mouse glomerular endothelial cells reveals specificity for distinct heparan sulfate domains.

INTRODUCTION: Proliferative glomerulonephritis manifests in a range of renal diseases and is characterized by the influx of inflammatory cells into the glomerulus. Heparan sulfate (HS) is an important (co-)receptor for binding of chemokines, cytokines and leukocytes to the endothelial glycocalyx, a thick glycan layer that covers the inside of blood vessels. During glomerulonephritis, HS in the glomerular endothelial glycocalyx plays a central role in chemokine presentation and oligomerization, and in binding of selectins and integrins expressed by leukocytes. We hypothesize that distinct endothelial HS domains determine the binding of different chemokines. In this study we evaluated the interaction of three pro-inflammatory chemokines (CXCL1, CXCL2 and CCL2) with mouse glomerular endothelial cells (mGEnC-1) in ELISA in competition with different HS preparations and anti-HS single chain variable fragment (scFv) antibodies specific for distinct HS domains. RESULTS: HS appeared to be the primary ligand mediating chemokine binding to the glomerular endothelial glycocalyx in vitro. We found differential affinities of CXCL1, CXCL2 and CCL2 for HS in isolated mGEnC-1 glycocalyx, heparan sulfate from bovine kidney or low molecular weight heparin in competition ELISAs using mGEnC-1 as a substrate, indicating that chemokine binding is affected by the domain structure of the different HS preparations. Blocking of specific HS domains with anti-HS scFv antibodies revealed a domain-specific interaction of the tested chemokines to HS on mGEnC-1. Furthermore, chemokines did not compete for the same binding sites on mGEnC-1. CONCLUSION: CXCL1, CXCL2 and CCL2 binding to the glomerular endothelial glycocalyx appears differentially mediated by specific HS domains. Our findings may therefore contribute to the development of HS-based treatments for renal and possibly other inflammatory diseases specifically targeting chemokine-endothelial cell interactions.

Cross-reactivity of human and murine anti-DNA antibodies with heparan sulfate. The major glycosaminoglycan in glomerular basement membranes.

In 30 of 33 human systemic lupus erythematosus (SLE) sera and in 10 sera from MRL/l mice with spontaneous SLE, antibodies against heparan sulfate were detected. The anti-heparan sulfate titers showed a significant correlation with the anti-DNA antibody titers. By inhibition studies it was demonstrated that heparan sulfate could inhibit the binding of anti-DNA antibodies to DNA, whereas DNA could block the binding to heparan sulfate. That this reaction is due to crossreactivity of anti-DNA antibodies was further substantiated by the finding that two monoclonal anti-DNA antibodies also bound to heparan sulfate. Antibodies eluted from human and mouse kidneys with diffuse SLE glomerulonephritis showed a similar binding to DNA and heparan sulfate when these eluted antibodies were tested in vitro. Heparan sulfate is the major glycosaminoglycan constituent of the glomerular basement membrane. Our findings suggest that heparan sulfate might serve as a target antigen in vivo for cross-reactive anti-DNA antibodies.

Anti-nucleosome antibodies complexed to nucleosomal antigens show anti-DNA reactivity and bind to rat glomerular basement membrane in vivo.

Histones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely. We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis.

Selection and characterization of a unique phage display-derived antibody against dermatan sulfate.

Dermatan sulfate (DS) is a member of the glycosaminoglycan (GAG) family and is primarily located in the extracellular matrix. Using a modified phage display procedure, we selected 2 different antibodies against DS of which one antibody, LKN1, was specific for DS. LKN1 was especially reactive with 4/2,4-di-O-sulfated DS, and did not react with other classes of GAGs including chondroitin sulfate and heparan sulfate. Immunohistochemical analysis of kidney, skin and tendon showed a typical fibrillar staining pattern, co-localizing with type I collagen. Staining was abolished by specific enzymatic digestion of DS. Immunoelectron microscopy confirmed the association of the DS epitope with collagen fibrils. The location of DS did not follow the main banding period of collagen, which is in line with the current concept that the core protein rather than the DS moiety of DS-proteoglycans specifically binds to collagen fibrils. This unique anti-DS antibody and the availability of its coding DNA may be instrumental in studies of the structure and function of DS.

Heparan sulfate proteoglycan from human tubular basement membrane. Comparison with this component from the glomerular basement membrane.

Heparan sulfate proteoglycan (HSPG) was extracted from human tubular basement membrane (TBM) with guanidine and purified by ion-exchange chromatography and gel filtration. The glycoconjugate was sensitive to heparitinase and resistant to chondroitinase ABC, had an apparent molecular mass of 200-400 kDa and consisted of 70% protein and 30% glycosaminoglycan. The amino acid composition was characterized by its high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulfonic acid yielded core proteins of 160 and 110 kDa. The heparan sulfate (HS) chains obtained after alkaline NaBH4 treatment had a molecular mass of about 18 kDa. Results of heparitinase digestion and HNO2 treatment suggest a clustering of sulfate groups in the distal portion of the HS side chains. These chemical data are comparable to those obtained previously on glomerular basement membrane (GBM) HSPG (Van den Heuvel et al. (1989) Biochem. J. 264, 457-465). Peptide patterns obtained after trypsin, clostripain or V8 protease digestion of TBM and GBM HSPG preparations showed a large similarity. Polyclonal antisera and a panel of monoclonal antibodies raised against both HSPG preparations and directed against the core protein showed complete cross-reactivity in ELISA and on Western blots. They stained all basement membranes in an intense linear fashion in indirect immunofluorescence studies on human kidneys. Based on these biochemical and immunological data we conclude that HSPGs from human GBM and TBM are identical, or at least very closely related, proteins.

Immunohistochemical quantification of heparan sulfate proteoglycan and collagen IV in skeletal muscle capillary basement membranes of patients with diabetic nephropathy.

In IDDM patients, an increased permeability of the glomerular capillaries has been associated with a general loss of negatively charged heparan sulfate proteoglycans (HSPGs) within basement membranes (BMs). In the present study, we used immunohistochemical staining to quantify heparan sulfate (HS), HSPG core protein, and collagen IV in capillary basement membranes of skeletal muscle biopsies taken from 9 healthy control subjects (C) and 20 IDDM patients: 7 with normal albumin excretion rate (<30 mg/24 h) (D0), 5 with incipient nephropathy (albumin excretion rate 30-300 mg/24 h) (D1), and 8 with clinical nephropathy (albumin excretion rate >300 mg/24 h) (D2). In the capillaries, staining was measured by a scanning and integrating microspectrophotometer. A significant difference in the absorbance of HS was found among the four subgroups (means +/- SD): 0.477 +/- 0.082 (C), 0.627 +/- 0.031 (D0), 0.542 +/- 0.098 (D1), and 0.371 +/- 0.118 (D2) (P = 0.006). Similarly, an overall significant difference in the absorbance of collagen IV was demonstrated (means +/- SD): 0.836 +/- 0.111 (C), 0.838 +/- 0.300 (D0), 0.970 +/- 0.173 (D1), and 0.512 +/- 0.248 (D2) (P = 0.02). No statistical difference in the absorbance of core protein was demonstrated among the groups. Within the diabetic groups, HS was inversely correlated to albuminuria (r = -0.76, P = 0.003) and albuminuria corrected for creatinine clearance (r = -0.69, P = 0.008). Because, in IDDM patients with albuminuria, alterations of the content of HS and collagen IV within the capillary BM have been demonstrated immunohistochemically, not only in the glomerular filtration barrier, but also in the skeletal muscle capillary BM, we suggest that these changes reflect universal quantitative or qualitative alterations within the capillary filtration barrier.

Regulation of glomerular epithelial cell production of fibronectin and transforming growth factor-beta by high glucose, not by angiotensin II.

Accumulation of matrix proteins is a prominent feature of diabetic nephropathy. Glomerular visceral epithelial cells (GVECs) are important contributors to extracellular matrix (ECM) production in the glomerulus. Factors involved with increased accumulation of ECM proteins are high glucose, angiotensin II (ANG II), and transforming growth factor (TGF)-beta. Therefore, we investigated the effects of high glucose and ANG II on fibronectin and TGF-beta production by human GVECs in vitro. We found that ANG II had no effect on the production of fibronectin and TGF-beta by GVECs. Using reverse transcriptase-polymerase chain reaction analysis, no ANG II receptor could be detected on these cells. However, high glucose induced a twofold increase in fibronectin (P < 0.01) and a three- to sixfold increase in TGF-beta (P < 0.001) production. Similar results were obtained by analyzing the mRNA levels of fibronectin (increased 2.7-fold) and TGF-beta (increased 3.5-fold). Addition of increasing concentrations of rTGF-beta to control cells resulted in increased fibronectin production. Neutralizing antibodies against TGF-beta significantly reversed the increase in fibronectin protein and mRNA caused by high glucose back to control levels. We conclude that high glucose concentrations stimulate the synthesis of fibronectin and that this effect is mediated by induction of TGF-beta. These results suggest that in diabetic nephropathy, high glucose levels play a role in changing the matrix composition of the glomerular basement membrane through induction of TGF-beta. Our results indicate that a contribution to this process by an effect of ANG II on GVECs seems unlikely.

Uptake of apoptotic leukocytes by synovial lining macrophages inhibits immune complex-mediated arthritis.

Previously we have shown that synovial lining macrophages (SLMs) determine the onset of experimental immune complex-mediated arthritis (ICA). During joint inflammation, many leukocytes undergo apoptosis, and removal of leukocytes by SLMs may regulate resolution of inflammation. In this study we investigated binding and uptake of apoptotic leukocytes by SLMs and its impact on the onset of murine experimental arthritis. We used an in vitro model to evaluate phagocytosis of apoptotic cells on chemotaxis. Phagocytosis of apoptotic thymocytes resulted in a significant decrease (58%) of chemotactic activity for polymorphonuclear neutrophils (PMNs). If apoptotic cells were injected directly into a normal murine knee joint, SLMs resulted in a prominent uptake of cells. After ICA induction, electron micrographs showed that apoptotic leukocytes were evidently present in SLMs on days 1 and 2. Injection of apoptotic leukocytes into the knee joint 1 h before induction of ICA significantly inhibited PMN infiltration into the knee joint at 24 h (61% decrease). This study indicates that uptake of apoptotic leukocytes by SLM reduces chemotactic activity and inhibits the onset of experimental arthritis. These findings indicate an important mechanism in the resolution of joint inflammation.

Prevalence of "syndrome X" features in parents of type 1 diabetic patients with or without nephropathy.

OBJECTIVE: To compare the prevalence of "syndrome X"-related parameters in parents of type 1 diabetic patients with diabetic nephropathy (DNP) to those in parents of diabetic patients without DNP. RESEARCH DESIGN AND METHODS: In this cross-sectional study, we included 50 parents of type 1 diabetic patients with DNP and 50 parents of diabetic patients without DNP. All parents were investigated in a fasting state for serum lipids including nonesterified fatty acids (NEFAs), glucose, HbA1c, plasma uric acid, fasting insulin levels, and albuminuria. Blood pressure was recorded in the supine position using an automatic device; ankle/brachial index was measured with Doppler ultrasound. Presence of cardiovascular disease was determined by a standardized questionnaire and electrocardiogram registration. Subjects without known diabetes underwent a 2-h oral glucose tolerance test. Anthropometric parameters such as BMI, waist-to-hip ratio, and percentage of body fat were measured. In addition to univariate analysis, a syndrome X score (SXS) was formulated, comprising a number of syndrome X-related biochemical, physiological, and/or anthropometric parameters. RESULTS: Univariate analysis revealed no significant differences in syndrome X parameters between parents of type 1 diabetic patients with or without DNP. Also, the composite SXS was similar in both groups. CONCLUSIONS: In this study, no differences were found in the prevalence of syndrome X features between parents of type 1 diabetic patients with DNP and parents of patients without DNP.

A sensitive haemolytic assay of mouse complement.

Under appropriate conditions the haemolytic activity of mouse complement can be measured in a 51Cr release assay. Modifications that increase the sensitivity are the following: (a) mouse 7 S anti-SRBC antibodies as amboceptor; (b) low amount of target cells; (c) ionic strength of 0.13 M NaCl in the test medium; (d) incubation temperature of 30 degrees C; (e) incubation time of 90 min; (f) total reaction volume of 1 ml.

An assay for the quantitative measurement of in vitro phagocytosis of early apoptotic thymocytes by murine resident peritoneal macrophages.

Research into the mechanisms by which apoptotic cells are phagocytosed has grown considerably over recent years, together with a growing appreciation of the importance of clearance of redundant cells for tissue homeostasis. However, studies addressing the efficacy of phagocytosis have been rare. The few studies reported to date were either attempts to determine apoptotic cell clearance from the circulation or were focused on clearance in inflammation. We now describe an in vitro assay which permits the quantitative measurement of phagocytosis of apoptotic cells by murine resident peritoneal macrophages. The apoptotic cells used in the assay were murine thymocytes incubated with dexamethasone for only 3 h. Most apoptotic thymocytes were annexin V positive and propidium iodide negative and therefore still in the earlier stages of apoptosis. The assay was completed 7 h after the isolation of both macrophages and thymocytes, while macrophage culture time was only 4 h. Because of this short-term culture it is likely that the resident peritoneal macrophages largely maintained their in vivo phenotype. Using BALB/c macrophages and thymocytes, the maximal in vitro phagocytosis exceeded five thymocytes per macrophage in 1 h and two of these thymocytes were taken up within 10 min. Therefore, in vitro phagocytosis by resident peritoneal macrophages was rapid and of high capacity, as it is postulated to be in vivo. Under selected conditions, the mean uptake was 4.45+/-0.70 (mean +/- SD, n = 31) thymocytes per macrophage in 1 h. The inter-assay coefficient of variation, also representing the biological variability, was found to be 15.7%. The average intra-assay coefficient of variation was 13.6%. This assay permits comparisons of phagocytic efficacy between different strains of mice in vitro. In addition, a method of preparation is described which allows long-term storage of experimental results. Finally, our data suggests that internalization, but not binding of apoptotic cells to short-term cultured resident peritoneal macrophages, is critically dependent on the presence of serum. This allows separate analysis of binding and internalization of apoptotic cells with the assay, without the necessity to use agents blocking internalization.

A comparative study of total body irradiation as a method of inducing granulocyte depletion in mice.

Since conventional methods of inducing depletion of polymorphonuclear granulocytes (PMNs) in mice, such as treatment with cytostatic drugs and anti-PMN sera, proved to be insufficient to induce a stable PMN depletion for several days, and were accompanied by considerable toxic side effects, we induced neutrophil depletion in mice by total body irradiation (TBI) in a single dose of 6.0 Gy (600 rads.) at a dose rate of 0.20 Gy/min. This treatment reduced the number of PMNs in the peripheral circulation to values below 150/microliter from day 3-10 after irradiation. The number of lymphocytes fell simultaneously. Platelet counts remained above 60% of normal values during the first 7 days after irradiation. Complement levels were not significantly affected by TBI. The results show that TBI of 6.0 Gy induces pronounced and stable PMN depletion in mice for at least 7 days. Furthermore, under an aseptic regimen the mice can be kept in good condition and losses are less than 5%.

A new ELISA for the detection of anti-heparan sulfate reactivity, using photobiotinylated antigen.

Autoantibodies reacting with a great variety of autoantigens are characteristic for the autoimmune disease systemic lupus erythematosus (SLE). Although reactivity with heparan sulfate (HS) in sera of patients with SLE is found in association with the occurrence of nephritis, the aetiological significance of this association is not clear. The assay which is generally used to measure anti-HS reactivity is subject to false-positive results, as a consequence of the binding of negatively charged moieties within immune complexes to the precoat employed (protamine sulfate). Therefore, we have developed a new ELISA in which photobiotinylated HS is efficiently and reproducibly bound to streptavidin-coated wells. We compared the new ELISA with the classical anti-HS ELISA by testing culture supernatants of 20 murine monoclonal antibodies (mAb) to DNA (containing free anti-DNA and anti-DNA/nucleosome immune complexes) and preparations of these mAb (containing only free anti-DNA), purified under dissociating conditions. In the classical anti-HS ELISA, 14 out of 20 of the culture supernatants reacted positively with HS; after purification no reactivity remained. The discrepancy must be due to anti-DNA/nucleosome immune complexes present in the culture supernatants. In the new ELISA only four out of 20 culture supernatants and one of the purified preparations reacted with HS. This latter reactivity is probably not specific, since this mAb also reacted with streptavidin alone. To find out whether there is a correlation between the occurrence of nephritis and anti-HS reactivity, measured in this new anti-HS ELISA, we tested sera of patients with a renal- or non-renal exacerbation of SLE in the newly developed anti-HS ELISA. We observed a correlation between anti-HS reactivity and nephritis.

Clinical evaluation of a modified ELISA, using photobiotinylated DNA, for the detection of anti-DNA antibodies.

The measurement of anti-dsDNA antibodies is important for the diagnosis and the follow-up of patients with systemic lupus erythematosus (SLE). For routine detection of anti-dsDNA, the Farr assay and the immunofluorescence technique (IFT) on Crithidia luciliae proved to be very useful. The anti-dsDNA ELISA is not used for routine purposes in our institute since it is flawed by false-positive results due to binding of negatively charged (immune) complexes to the employed precoat (protamine sulphate). Recently, a new anti-dsDNA ELISA has been described in which photobiotinylated dsDNA is coated to streptavidin coated plates. To investigate whether this modified ELISA is more specific than the classical anti-dsDNA ELISA, we tested sera of patients with SLE (n = 51), myasthenia gravis (MG, n = 25), rheumatoid arthritis (RA, n = 25) and Sjögren's syndrome (SS, n = 23) and sera of healthy blood bank donors (BBD, n = 25). In both assays the sera of the SLE patients gave significantly higher values than the sera of healthy blood bank donors. In the classical ELISA, 84% of the sera from patients with RA and 28% of sera of patients with MG were found positive. For the modified assay the figures were 8% and 24%, respectively. This modified ELISA was further studied and clinically evaluated by comparing it with the classical anti-DNA ELISA and two other anti-DNA assays (Farr assay and IFT), using 500 sera sent to our institute for routine anti-DNA determination and sera of an additional 75 healthy blood bank donors. Quantitatively, both ELISAs showed the same high degree of correlation with the IFT. The modified ELISA gave a better correlation with the Farr assay than the classical anti-DNA ELISA. From our data we conclude that the ELISA using photobiotinylated DNA is a more reliable assay than the classical anti-DNA ELISA.