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Immunoglobulin preparations from sera of healthy donors contain polyspecific autoantibodies interacting with DNA and other charged antigens. These antibodies belong to the IgG class and can exist in the free or hidden state. The hidden antibody activity can be revealed after ion-exchange chromatography on QAE-Sephadex A-50. Immunoenzyme assay was used to assess the interactions of both free and hidden antibodies with different carbohydrates. The hidden antibodies were only able to interact with different polyanionic carbohydrates and neutral xenoantigen Bdi.

To determine the effects of low dose radiation from 131I and 137Cs resulting from the Chernobyl disaster on the expression of endemic goiter in children, we studied children in regions with and without evidence of radiocontamination but comparable iodine insufficiency. We included 89 children in the study (radiocontaminated) region (SR) and 116 in the control region (CR) because they were either fetuses, infants, or pre-adolescents at the time of exposure. We evaluated thyroid dimensions by clinical examination and ultrasound; thyroid function by hormonal levels, and thyroglobulin measurement; histology of the thyroid by fine needle aspiration; and thyroid autoimmunity. SR subjects had 5 times the frequency of thyroid enlargement as those from CR, greatest in the younger patients. There were no instances of clinically apparent thyroid dysfunction. Antithyroid microsomal and antithyroglobulin antibodies were present in a markedly greater percentage of SR subjects. Fine needle biopsy demonstrated greater cellular proliferation in samples from SR.We have demonstrated findings of autoimmune thyroid disease at markedly increased frequency in a population of children with poor iodine nutriture who were exposed to low level radiation, compared to a more iodine deficient population not so exposed. These results suggest that low level radiation may induce thyroid gland changes in children who have inadequate iodine intake nutriture and reinforce the importance of adequate dietary iodine.

Both free and hidden natural antibodies to DNA or cardiolipin were obtained from immunoglobulins of a normal donor. The free antibodies reacting with DNA or cardiolipin were isolated by means of affinity chromatography. Antibodies occurring in an hidden state were disengaged from the depleted immunoglobulins by ion-exchange chromatography and were then affinity-isolated on DNA or cardiolipin sorbents. We used flow cytometry to study the ability of free and hidden antibodies to bind to rat thymocytes. Simultaneously, plasma membrane integrity was tested by propidium iodide (PI) exclusion. The hidden antibodies reacted with 65.2 +/- 10.9% of the thymocytes and caused a fast plasma membrane disruption. Cells (28.7 +/- 7.1%) were stained with PI after incubation with the hidden antibodies for 1 h. The free antibodies bound to a very small fraction of the thymocytes and did not evoke death as compared to control without antibodies. The possible reason for the observed effects is difference in reactivity of the free and hidden antibodies to phospholipids. While free antibodies reacted preferentially with phosphotidylcholine, hidden antibodies reacted with cardiolipin and phosphotidylserine.

The formation of remote consequences of radiation effects on the thyroid status of the population exposed to low-dose radiation was studied. The authors analyzed epidemiological, clinical, biochemical, hormonal, ultrasonographic, immunological, and morphological characteristics of the prevalence and intensity of endemic goiter in children and adolescents living in two iodine-deficient districts of the Oryol region observed after contamination of their territory with 137-Cs in a dose of 1 to 5 Ci/km2 and in-corporation of 131-I in a dose of 20-30 rad and in a control district free from radiation contamination. The results indicate that under the effect of low-dose combined irradiation endemic goiter in the observed districts acquired atypical features in the six years that passed since the Chernobyl accident in comparison with that in the control region: 1) thyromegalia prevalence is increased, its level being disproportionate with the degree of the existent iodine deficiency; 2) sex-specific differences are completely absent; 3) the maximal strain is observed in children exposed to radioactive iodine in utero 4) morphologically, variants of diffuse goiter with a high risk of node formation predominate; 5) progressive growth of thyroid parenchyma is associated with increased thyroglobulin elimination, this increasing the strain of the humoral component of antithyroid autoimmunity. These data indicate that the modifying effect of low-dose exposure on endemic goiter resulted in the disease pathomorphism associated with oncological risk and increased risk of autoimmune thyroiditis.

Electrochromatography (ECHR) exploits a very high electro-osmotic counterflow developed in porous membranes at discontinuous electrophoresis. This counterflow exceeds considerably the anodic migration of any negatively charged protein and is used as a ‘conveyer belt’ for sequential transfer of immunoreagents to the specific adsorbents (antigens or antibodies) fixed on the nitrocellulose membrane. This approach was applied for simultaneous detection of two antigens (α-fetoprotein and carcino-embryonic antigen) in one sample, for determination of subfractions of α-fetoprotein, different in their epitope specificity, and for detection of L chains with certain idiotype on the background of heterogeneous L fraction. ECHR was used also for the partition of different antibodies to DNA adducts, demonstrating the possibility of applying this method to the study of DNA-binding proteins.

Chemical modification with osmium tetroxide (OsO 4 ) in the presence of 2,2′-bipyridine was employed for selective labeling of certain non-B DNA structures. A murine monoclonal antibody (IgG1) specific for deoxythymidine adduct of the modification was produced. It was demonstrated that the level of single-stranded DNA can be evaluated by enzyme-linked immunosorbent assay using the modification and monoclonal antibody. Site-specific OsO 4 -bipyridine modification followed by dot-blotting was used for probing of some unusual conformations occurring in supercoiled plasmids. The cruciform structures, B-Z junctions, and intramolecular triplex and long (A-T) n inserts were targets for the modification and small quantities of the labeled plasmids can be immunodetected. The method enabled to catch certain structural transitions within the same plasmid in vitro and to detect the left-handed Z DNA labeled inside bacteria. The antibody was also shown to recognize specifically the in situ modified DNA in cell nuclei and polytene chromosomes isolated from Drosophila melanogaster . Preferentially S1-nuclease-sensitive (single-stranded) regions of the nuclear and chromosomal DNA were stained by indirect immunofluorescence after the modification.

A novel simple nonradioactive method for detectionof specific nucleotide sequences has been developed. This method consists of the hybridization of a target DNA with a DNA probe modified with trans-diammine-dichlorplatinum(II) (trans-DDP) followed by detection of DNA/DNA hybrids with affinity-isolated anti-DNA-trans-DDP antibodies and poly-horseradish peroxidase-protein A conjugate. Major advantages of this approach are the low cost and the extreme simplicity of the labeling procedure, which involves only mixing of the reagents. The sensitivity of the proposed technique is sufficient to detect 0.8 pg of DNA in Southern blot hybridization and 25 fg in dot hybridization and permits colony screening.

Polyclonal antibodies that bind selectively to DNA modified by antitumour cisplatin and its analogues were isolated. The reactivity of the antibodies with the epitope was enhanced by thermal denaturation of DNA that had been modified by cisplatin before its denaturation. On the other hand, denaturation of DNA before its modification resulted in considerably less reaction of the antibodies. The conversion of monofunctional cisplatin-DNA adducts to bifunctional lesions increased the capability of the modified DNA to competitively inhibit the antibodies. The double-helical oligonucleotides containing a unique bifunctional adduct formed by cisplatin at the d(GG) site cross-reacted with the antibodies in contrast to the oligonucleotide containing a single monofunctional adduct formed at the d(G) site. In addition, poly(dG-dC) poly(dG-dC) modified by cisplatin did not react with the antibodies. It was concluded that the antibodies recognized monodentate lesions, intrastrand cross-links between two purine nucleosides separated by one or more nucleosides and interstrand cross-links negligibly. The antibodies apparently recognized a chemical nature of the bifunctional adduct formed between two adjacent purines and not an unusual conformational feature of DNA resulting from the formation of this adduct. The antibodies were used to analyse the adducts formed by cisplatin on DNA of cultured cells exposed to this drug. During the subsequent incubation of the already exposed cells in the drug-free medium, a part of the bifunctional adducts of cisplatin was completely removed from DNA or transformed to the adducts not recognized by the antibodies.

The influence of charged polymers on the reaction of immunoglobulins from human lupus sera with cellular proteins was investigated in this study. Through immunoblotting it was shown that polyanions (dextran sulfate, heparin, polyinosinic acid) and polylysine inhibited autoantibody binding to several polypeptides of different molecular mass. Using immunofluorescent staining with affinity isolated monospecific autoantibodies it was demonstrated that the immunoreactivity of two nuclear antigens (p30 and p85) and one cytoplasmic antigen (p40) was sensitive to the presence of charged polymers. The inhibiting effect correlated with the concentration of the polymers. The data obtained suggested the competitive mechanism of inhibition of autoantibody-protein reaction by the charged polymers.

SUMMARYThe effect of low-density lipoprotein (LDL) on the binding of DNA and DNA-anti-DNA immune complexes to cultured human skin fibroblasts was examined. Using radioisotope analysis. ELISA and indirect immunofluorescent staining, a correlation between plasma membrane-bound DNA or DNA-anti-DNA immune complexes and cell-associated LDL was established. It was demonstrated that cytotoxicity and internalization of DNA-anti-DNA immune complexes may be LDL mediated. The data obtained suggest that the binding of the major part of DNA and immune complexes bound to surface of normal skin fibroblasts is due to the formation of a DNA-LDL-LDL receptor linkage. A possible role of LDL-containing immune complexes in the pathogenesis of systemic lupus erythematosus is discussed.

Immunoglobulin preparations from sera of healthy donors contain polyspecific autoantibodies interacting with DNA and other charged antigens. These antibodies belong to the IgG class and can exist in the free or hidden state. The hidden antibody activity can be revealed after ion-exchange chromatography on QAE-Sephadex A-50. Immunoenzyme assay was used to assess the interactions of both free and hidden antibodies with different carbohydrates. The hidden antibodies were only able to interact with different polyanionic carbohydrates and neutral xenoantigen Bdi.

Effects of various histones, poly-L-lysine, spermine, and the synthetic peptide Arg-Lys-Asn-Val-Tyr-Arg (thymohexine) on intact rat thymocytes were studied. Histones and poly-L-lysine displayed cytotoxicity, causing disorders in the membrane permeability of thymocytes and their death. The dose and time dependences of the effects of histones on thymocytes were determined. Preparations of normal human immunoglobulins bound histones but displayed neither cytotoxicity nor interaction with intact thymocytes. The immunoglobulins significantly decreased the number of dead thymocytes in the presence of total histones. However, the number of cells with detectable immunoglobulin molecules was increased in the presence of histone in the incubation medium. It is suggested that cytotoxicity depends on the number of epitopes on the cell membrane available for interaction with positively charged protein molecules.

The presence of antihistone antibodies was determined in different commercial immunoglobulin preparations. The ability of histones to perform the function of cofactor in interaction of immunoglobulin preparations and sera of healthy people with charged molecules such as DNA, cardiolipin and phosphatidylcholine, was determined. The addition of histones of immunoglobulin preparations or serum samples is accompanied by forming the stable complex, components of which interact with the charged molecules. The capacity of histones to play the role of cofactor is less evident in the case when they are preincubated with DNA or cardiolipin. The interaction of natural antibodies with histones can explain their polyspecificity in some cases.

An ability to detect hidden high-avidity DNA-binding protein in human gammaglobulin samples was investigated. Using ion exchange chromatography on QAE-Sephadex A-50 a highly positive Farr assay DNA-binding fraction was reproducibly isolated from several commercial normal human gammaglobulin preparations. The estimated dissociation constant had a value of 1.04 × 10 −11 M thus confirming high avidity protein-DNA complex formation. Anti-DNA antibodies (Ab) ELISA data revealed that immunoglobulin (Ig) G, IgM and IgA participated in the protein-DNA interaction. Inhibitory experiments involving a number of polynucleotides, synthetic and natural polyanions demonstrated that both double-stranded DNA (dsDNA) and heat-denatured DNA but not RNA inhibited the protein- 3 H-DNA binding. Generally, the inhibiting effect was more pronounced when purine base-containing polynucleotides and polydeoxy- rather than polyribonucleotides were used. Synthetic polyanions and normal human sera (NHS) also markedly reduced the binding. The presence of hidden high-avidity DNA-binding antibodies in normal gammaglobulin preparations was suggested.

The influence of systemic lupus erythematosus (SLE) patients' sera on lipid accumulation in the cultured smooth muscle cells (SMC) from unaffected human aortic intima was examined. It was demonstrated that the cholesterol uptake in the SMC cultured in the presence of SLE sera is 1.5- to 6-fold higher than in the cells cultured with normal human sera (NHS) obtained from healthy donors. Incubation of the SMC with circulating immune complexes (CIC) isolated from lupus sera by precipitation with 2.5% polyethylene glycol 6000 (PEG) caused a 3- to 4-fold rise in the intracellular cholesterol level. The atherogenic effect of lupus sera, as well as isolated CIC, strongly correlated (r = 0.98) with the low density lipoprotein (LDL) content in the PEG-precipitated CIC. The cholesterol level in cultured SMC also increased 2- to 3-fold when growth medium was supplemented with LDL, DNA, and anti-DNA autoantibodies (IgG) affinity isolated from lupus sera. Using immunofluorescent staining, it was shown that the addition of a DNA-anti-DNA IgG mixture to the growth medium, together with NHS, stimulated LDL incorporation in the SMC. The results of double-label staining suggest the formation of LDL-DNA-IgG complexes which seem to be entrapped in cells more actively than free LDL. The composition of PEG-precipitated CIC was studied by electrophoresis and immunoblotting. Significant amounts of apolipoprotein B, as well as low molecular weight DNA and immunoglobulins, were found in SLE-derived CIC. The data obtained suggest that the atherogenic effect of human lupus sera in vitro is generally due to the appearance of LDL-containing immune complexes. Different mechanisms possibly involved in the lupus atherogenesis are discussed.

Polyclonal antibodies that bind selectively to adducts formed with DNA by chemotherapeutically inactive trans-diamminedichloroplatinum (II) [trans-[Pt(NH3)2Cl2]) were produced by immunization with calf-thymus double-helical DNA modified by trans-[Pt(NH3)2Cl2] at a ratio of bound platinum/nucleotide (rb) of 0.1. High selectivity was obtained by separation of the antibodies from the antiserum with the aid of affinity chromatography on a Sepharose column. The antibodies were competitively inhibited in an ELISA assay by 25 pM trans-[Pt(NH3)2Cl2] bound to double-helical DNA and 2.5 pM trans-[Pt(NH3)2Cl2] bound to denatured DNA (rb = 0.1). The conversion of monofunctional adducts, formed on DNA at the early stage of its interaction with trans-[Pt(NH3)2Cl2], to bifunctional lesions, decreased the ability of the modified DNA to competitively inhibit these antibodies. They did not cross-react with unmodified, denatured DNA, but they reacted with diethylenetriamine-chloroplatinum(II)-chloride-modified double-helical DNA and with double-helical DNA treated with cis-diamminedichloroplatinum(II) for a short time (10 min). The results of this work best fit a model in which one of the major antigenic determinants of double-helical DNA modified by trans-[Pt(NH3)2Cl2] is the platinum atom coordinated in a monodentate or bidentate manner with non-paired nucleotide residues or perhaps a short segment of single-stranded DNA which occurs around the platination site. Nucleic acids modified by trans-[Pt(NH3)2Cl2] can be used as immuno-probes in hybridization experiments.

A murine monoclonal antibody (IgG) has been generated that binds to DNA modified with osmium tetroxide in the presence of 2,2′-bipyridine and does not interact with unmodified DNA. Reactivity of the antibody was tested by gel retardation assay, ELISA, dot-binding assay and immunoblotting. The results obtained suggest that the antibody does not crossreact with modified or unmodified RNA or proteins. The high specificity of the binding reaction is due to the specific recognition of modified deoxythymidine residue by the monoclonal antibody. A possible way of using the antibody produced is discussed.

The effect of polyanions on the reactivity of human autoantibodies with cellular proteins was studied. The results of immunoblotting revealed that dextran sulfate (DS), heparin, single-stranded (ss) DNA, and polyinosinic acid (poly I) inhibit interaction between immunoglobulins (Ig) from human autoimmune sera and many polypeptides with various molecular mass. These proteins were suggested to belong to a new subclass of autoantigens, the immunoreactivity of which is sensitive to the presence of polyanions. For some of these antigens, molecular mass, intracellular localization and frequency of appearance of positive sera were determined.

Syngeneic thymocytes are shown to restore the capacity of bone marrow treated with rabbit antimouse brain (RAMB) serum to form the spleen colonies. The same effect may be exerted by the V fraction of thymosin or endotoxin, but not by allogeneic thymocytes. It is assumed that RAMB serum acts not on the stem cells but rather on another cell population (accessory cells?) contributing to the differentiation.

Prolonged repeated analyses of sera from 98 patients with systemic lupus erythematosus (SLE) and 76 patients with similar diffuse diseases of connective tissue by a radioisotope membrane binding method for the assessment of antibodies against double-stranded ribonucleic acid (anti-ds-RNA) revealed that these antibodies are most frequently encountered in patients with the evolutive form of SLE (53%), but also in approximately one fifth of patients with non- evolutive SLE and in patients with similar diffuse diseases of connective tissue. Anti-ds-RNA are not linked with stages of clinical activity; their occurrence is on the whole independent on the occurence of antibodies against ds-DNA and nuclear ribonucleoprotein. There is a very close association with the incidence of antibodies against denatured DNA and a substantially small association with antibodies against acid nuclear antigen (Sm). The occurrence of anti-ds-RNA is a favourable prognostic sign, as in the majority of patients who had such antibodies at any time in their serum, later the disease can be suppressed by treatment, while patients who never had anti-ds-RNA in serum remain for a long time in the active stage of the disease.

Aim. To ascertain the role of high antibodies (Ab) titers to microsomal antigen (MA) of the thyroid in clinical manifestations of cytopenic syndromes. Material and methods. Clinical data are presented on 144 patients with depressed hemopoiesis having different levels of thyroid Aab. Results. When the titer was significantly elevated (41% patients with immune cytopenias), hematological malignancies run with recurrences, remission is rare. Treatment of the thyroid pathology improves hematological indices in some cases though does not reduce titers of Ab thyroid MA. Conclusion. Detection of autoimmune pathology of the thyroid is necessary not only for specification of the concurrent process diagnosis but also for initiation of adequate combined treatment.

Aim. To study the level of thyroid antibodies (TAB) in blood diseases. Material and methods. TAB levels dynamics was studied in 413 patients with hematological diseases. Results. Increased incidence of cases with high Ab titer to thyroid microsomal antigen was found. High and moderate tilers were revealed in 33% of all examinees, including 41%, 22%, 32% and 29% among patients with immune cytopenia, chronic lymphoid leukemia, generalized mature-cell lymphoma and myeloproliferative diseases, respectively. These high and moderate titers were rather stable. In 15% patients Ab were elevated insignificantly, this rise being unstable. Conclusion. Stable high titers of antibodies to thyroid antigens point to increased incidence rates of concomitant autoimmune hyroiditis in patients with blood diseases.