Clin. exp. Immunol. (1976) 24, 379-384.

The occurrence of antibodies against single-stranded DNA in the sera of patients with acute and chronic leukaemia S. IZUI, P. H. LAMBERT, N. CARPENTIER & P. A. MIESCHER WHO Immunology Research and Training Centre, Centre de Transfusion, Hdpital Cantonal, Geneva, Switzerland (Received 17 October 1975)

SUMMARY

One hundred and seventy-five sera from thirty-three patients with acute myeloid leukaemia, forty-two patients with chronic myeloid leukaemia and twelve patients with acute lymphatic leukaemia were examined by a radioimmunological technique for the presence of antibodies against single-stranded and double-stranded DNA. The levels of single-stranded DNA binding activity was significantly higher in all three types of leukaemia compared to those of healthy controls. In contrast, none of these sera exhibited a positive reaction with double-stranded DNA. In some cases the level of serum anti-DNA antibodies increased after the decrease of the leucocyte count. The presence of anti-DNA antibodies in leukaemic patients may have some biological

significance. INTRODUCTION Antibodies to DNA have often been reported in sera from patients with systemic lupus erythematosus (SLE) and related rheumatoid syndromes. Anti-double-stranded DNA (dsDNA) antibodies are mainly observed in sera from patients with SLE, while anti-single-stranded (ssDNA) antibodies are frequently found in other clinical conditions as well as in SLE patients (Hughes, Cohen & Christian, 1971; Koffler et al., 1969; Pincus et al., 1969). Moreover, antibodies to DNA have been demonstrated in sera from patients with infectious diseases such as active hepatitis or trypanosomiasis (Hughes, 1975; Lindsley, Kysela & Steinberg, 1974). Such infectious agents seem to be directly or indirectly implicated in the triggering of the immune response to DNA. In experimental animals, c-type particles have been isolated from New Zealand mice and have been shown to have both oncogenic properties and the capacity to induce anti-nuclear antibodies (Croker et al., 1974). The present study was carried out to investigate the possible occurrence of anti-DNA antibodies in human patients with leukaemia using sensitive radioimmunological techniques.

MATERIALS AND METHODS Sera. One hundred and seventy-five serum samples were used from thirty-three patients with acute myeloid leukaemia (AML), forty-two patients with chronic myeloid leukaemia (CML) and twelve patients with acute lymphatic leukaemia (ALL). Sera were kept frozen at -20°C. Mostof the patients have been treated with classical chemotherapy. In addition, patients with chronic myeloid leukaemia have been included in a vaccination programme with BCG or BCG combined with cultured lymphoblastoid cells from leukaemic patients according to Sokal, Aungst & Grace (1973). DNA. Highly polymerized calf thymus DNA (type V, Sigma, St Louis, Missouri) was used without further purification, Denatured DNA was prepared by heating native DNA (0.5 mg/ml in phosphate-buffered saline, pH 7.0) at 100°C for 10 min. then immediately cooling in an ice bath. Radioimmunological detection of anti-DNA antibodies. The titration of anti-DNA antibodies was carried out according to Wold et al. (1968) by precipitation of globulin-bound radiolabelled DNA with 50% saturated ammonium sulphate. DNA was labelled with 125I according to Commerford (1971). 125I-labelled ssDNA and dsDNA were purified by fractionation on Correspondence: Dr P. H. Lambert, WHO Immunology Research and Training Centre, Centre de Transfusion, H6pital Cantonal, 1211 Geneva 4, Switzerland.

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methylated albumin kieselguhr column (Sueoka & Cheng, 1967). Details of titration have been previously described (Fourni6, Lambert & Miescher, 1974). The results were expressed as a percentage of I25I-labelled DNA precipitated specifically. The specific precipitation of radio-labelled DNA was calculated after correction for the non-specific precipitation obtained in the presence of normal human serum (NHS) pools. The formulae described by Farr (1958) were used for this correction. The purity of the '25I-labelled dsDNA used in this assay was tested using a mouse antibody specific for ssDNA but not dsDNA. This antibody precipitated < 5% of the radioactivity. Double antibody methodfor measuring anti-DNA antibodies. Selected sera were also measured by a double antibody method. Titrations were done as follows: 0 1 ml of heat-inactivated test sera (1: 10) was incubated with 10 ng of "25I-labelled ssDNA in 0 1 ml of borate buffer at 4VC overnight. Then 0 3 ml of heated rabbit anti-human IgG antisera was added and incubated at 4VC overnight. The reaction mixtures were washed twice with borate buffer by centrifugation, 1000 g for 30 min, and the radioactivity was measured on the precipitates.

RESULTS The results of the '25I-labelled ssDNA binding activity of 205 sera from thirty-three AML, forty-two CML, twelve ALL patients and thirty normal blood donors are summarized in Fig. 1. After correction for the non-specific precipitation of 125I-labelled ssDNA obtained in a pool of fifty normal blood donors' sera, the mean percentage of 125I-labelled ssDNA precipitated in sera of thirty normal blood donors was -03+ 1.3%. It was found that in the groups of AML, CML and ALL, the serum 125I-labelled ssDNA binding activity was significantly increased when compared to the normal blood donors (P

The occurrence of antibodies against single-stranded DNA in the sera of patients with acute and chronic leukaemia.

Clin. exp. Immunol. (1976) 24, 379-384. The occurrence of antibodies against single-stranded DNA in the sera of patients with acute and chronic leuka...
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