Bacterial and Viral Products Affect Differential Pattern Recognition Receptor Activation of Chicken Thrombocytes Evidenced through RNA Sequencing.

It is now well understood that thrombocytes (nucleated platelets) express TLRs and respond to both bacterial and viral products. Release of proinflammatory molecules can be expected following relatively short exposure times to LPS, lipoteichoic acid (LTA), thymidine homopolymer phosphorothioate oligonucleotide [Poly(dT)], and polyinosinic-polycytidylic acid [Poly(I:C)]. This study reports the varied expressions of genes encoded for components of the TLR, nucleotide binding oligomerization domain-like receptor, and retinoic acid-inducible gene RIG-like receptor signaling pathways in response to the TLR ligands listed above. Highly sensitive RNA-sequencing technologies were used to analyze the complete transcriptome of thrombocytes treated with all four microbial products for a period of 1 h. A total of 14,326 gene transcripts were found in chicken thrombocytes across all ligand exposures. After 1 h of stimulation with ligands, 87, 138, 1013, and 22 genes were upregulated for LTA, LPS, Poly(dT), and Poly(I:C), and 12, 142, 249, and 16 genes were downregulated for LTA, LPS, Poly(dT), and Poly(I:C), respectively, with at least a 1-fold change relative to unexposed thrombocytes. Summarizations of biological processes, protein classes, and biochemical pathways reveal the role of chicken thrombocytes in proinflammatory responses linked to key signaling pathways. TLR, nucleotide binding oligomerization domain-like receptor, and retinoic acid-inducible gene RIG-like receptor pathways were mapped based on the transcriptome results with gene expression for common signal and proinflammatory mediators highlighted. The information reported in this study is useful for defining a limited set of proinflammatory molecules to evaluate in cases of either bacterial or viral disease monitoring.

Human oviductal epithelial cells express Toll-like receptor 3 and respond to double-stranded RNA: Fallopian tube-specific mucosal immunity against viral infection.

BACKGROUND: The aim of this study was to evaluate the site-specific immunoregulatory mechanisms against viral infection in human Fallopian tubes. METHODS: We therefore investigated the effects of double-stranded RNA (dsRNA) on the production of interleukin (IL)-6, IL-8 and granulocyte chemotactic protein-2 (GCP-2) by cultured oviductal epithelial cells (OECs) using enzyme-linked immunosorbent assays. Phosphorylation of inhibitor kappaB-alpha (IkappaB-alpha) protein after dsRNA stimulation and the expression of Toll-like receptor (TLR) 3 in these cells were also evaluated by western blot analysis. RESULTS: Polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that antagonizes TLR3, stimulated the secretion of IL-6, IL-8 and GCP-2 by OECs. Poly I:C-induced production of these cytokines by OECs was inhibited by the pretreatment of these cells with anti-TLR3 antibody. The phosphorylation of IkappaB-alpha protein was detected in OECs after stimulation by poly I:C. The expression of TLR3 was also detected in OECs. CONCLUSION: These results suggest that the epithelial cells of the human Fallopian tube have evolved a unique, site-specific mechanism for recognizing viral infection. TLR3-mediated production of proinflammatory cytokines and chemokines in OECs in response to viral dsRNA may be important for antiviral immunity in the human female reproductive tract.

Polydeoxyribonucleotide C photoconjugated with lysine or arginine present unique epitopes for human anti-DNA autoantibodies.

Studies have been carried out to synthesize and characterize the photoconjugates between positively charged amino acids (lysine and arginine) and the polydeoxyribonucleotide C [poly(dC)]. Poly(dC) was covalently crosslinked with lysine or arginine under ultraviolet light. Both lysine and arginine were found covalently photoconjugated to poly(dC), resulting in the formation of photoadduct. Photoaddition of lysine or arginine to poly(dC) rendered them thermodynamically more stable than their native form. A strong recognition of photoadducts was observed with anti-DNA autoantobodies found in the sera of systemic lupus erythematosus (SLE) patients. Poly(dC)-lysine was recognized more strongly than poly(dC)-argine photoadduct. Poly(dC)-lysine photoadduct appears to provide an immunodominant epitope for SLE autoantibody recognition. The result suggests for the possible involvement of these photoadducts as a potential trigger for anti-DNA autoantibody production.

Effect of cytofectins on the immune response of murine macrophages to mammalian DNA.

DNA, depending on base sequence, can induce a wide range of immune responses. While bacterial DNA is stimulatory, mammalian DNA is inactive alone and can, moreover, inhibit the response to bacterial DNA. To determine whether the mode of cell entry affects the immune properties of mammalian DNA, we have investigated the effects of the cytofectin agents Fugene 6 (Roche Diagnostics Corp., Indianapolis, IN), Lipofectin and Lipofectamine (Life Technologies, Grand Island, NY) on the responses of murine macrophages to DNA from calf thymus and human placenta. Whereas calf thymus and human placenta DNA alone failed to stimulate J774 or RAW264.7 cell lines or bone marrow-derived macrophages, these DNAs in complexes with cytofectin agents stimulated macrophages to produce nitric oxide but not interleukin 12. Both single-stranded and double-stranded DNAs were active in the presence of cytofectins. Macrophage activation by the DNA-cytofectin complexes was reduced by chloroquine, suggesting a role of endosomal acidification in activation. As shown by flow cytometry and confocal microscopy, the cytofectins caused an increase in the uptake of DNA into cells. Our findings indicate that macrophages vary in their response to DNA depending on uptake pathway, suggesting that activation by DNA reflects not only sequence but also context or intracellular location.

Modulation of fine specificity of anti-DNA antibody by CDR3L residues.

The light chain of 2C10, an anti-double stranded DNA (dsDNA) autoantibody, is not favorable for DNA binding and it was suggested that the light chain might modulate the specificity of the antibody in DNA binding. We studied several mutant scFvs expressing mutated VL and normal VH of 2C10 to explore the role of the light chain in determining the fine specificity of the antibody, which we define as the preferential binding to a specific sequence of bases or a helical conformation compared to dsDNA from calf thymus. The wild-type Fab and scFv of 2C10 bind to poly(dA-dC).(dG-dT) better than to dsDNA. However, in the absence of the light chain domain, the VH domain bound dsDNA better than poly(dA-dC).(dG-dT), indicating the possible involvement of the light chain in determining the fine specificity in DNA binding. The mutations we studied were located in either CDR1L or CDR3L of the antibody. The CDR1 mutants, D28A, D30A, D31A, and D32A have been previously shown to cause an increase in the affinity of 2C10 scFv to DNA. The fine specificity of 2C10 was not affected by the CDR1 mutants which bound to poly(dA-dC).(dG-dT) better than dsDNA. However, CDR3L mutants, D92A and N93A, which had been shown to be involved in direct interaction with DNA, preferred dsDNA to poly(dA-dC).(dG-dT) in their binding. Our results indicate that the fine specificity of 2C10 in DNA binding is modulated primarily by Asp at 92 and Asn at 93 in CDR3L. The effects of CDR1L mutations indicate that this region affects only the affinity but not the fine specificity of 2C10.

Induction of anti-DNA antibodies by immunization with anti-DNA antibodies: mechanism and characterization.

Two well-characterized IgG monoclonal antibodies, reactive with double-stranded (ds) DNA and nucleosomes, were administered to normal BALB/c mice to examine the reproducibility and the biology of a previously reported model of anti-DNA antibody induction by immunization with anti-DNA antibodies. The monoclonal antibodies were purified either with or without a high-salt wash to remove nucleosomal antigens bound to them during the cell culture. Both monoclonal antibodies, but not normal IgG, induced significant IgG anti-dsDNA antibody production from 1 week to 25 weeks after the last immunization. The antibodies produced in this manner possess different binding preferences to ds synthetic polynucleotides than the antibodies used for the immunization, and they did not react with nucleosomes. The monoclonal antibodies purified with the high-salt wash were more effective in anti-DNA antibody induction than those purified without the high-salt wash. Even when bound to these monoclonal antibodies, neither dsDNA, nucleosomes, or ds synthetic polynucleotides exert significant antigenicity. For example, anti-DNA antibodies produced by mice immunized with an immune complex formed by poly(dA-dT) and one of the monoclonal antibodies that has a high affinity to this polynucleotide did not show an increased affinity to poly(dA-dT). Together, these results suggest that anti-DNA antibody molecules or processed antibody peptides, and not DNA/nucleosomes carried by anti-DNA antibodies, play a role in this model of anti-DNA antibody production.

Monoclonal antibody against DNA adducts with osmium structural probes.

Osmium tetroxide complexes with nitrogen ligands (Os,L) have been widely used as probes of the DNA structure. A monoclonal antibody OsBP7H8 against DNA adducts with Os,L was produced in mice. OsBP7H8 does not bind to proteins or total yeast RNA modified with Os,2,2'-bipyridine (bipy) nor to the unmodified nucleic acids and proteins. The antibody recognizes DNA modified with Os,bipy (DNA-Os,bipy) or with OsO4,1,10-phenanthroline (DNA-Os,phen) but it does not cross-react with oxidized DNA and with DNA adducts of osmium tetroxide complexes with other ligands (such as pyridine, TEMED and bathophenanthroline disulfonic acid). The affinity of OsBP7H8 to DNA-Os,phen is about five-fold higher as compared to DNA-Os,bipy. The antibody can be thus applied either for recognition of single-stranded and distorted regions in DNA (after DNA modification with Os,bipy) or for detection of both single-stranded and double-stranded DNAs (after DNA modification with Os,phen). A new simplified procedure for the dot-blot analysis is proposed, not requiring the purification of DNA-osmium adduct prior to its application to the membrane.

Sequence and structure specific antibodies from phage display libraries.

A large combinatorial phage display library was panned against five nucleic acid antigens, calf thymus DNA, poly[d(GC)], poly[d(AT)], poly(dA) x poly(dT) and poly(rA) x poly(dT). After the third and fourth rounds of panning, many positive clones were selected against poly[d(GC)], poly(dA) x poly(dT) and poly(rA) x poly(dT). The specificity of these antibodies was tested by both direct and competitive solid phase radioimmune assays. All the clones derived from panning with poly[d(GC)] were non-specific and bound to all nucleic acids. The poly(rA) x poly(dT) derived clones were specific for single-stranded nucleic acids, with some sequence preferences, and the poly(dA) x poly(dT) derived clones showed considerable specificity for this antigen. The sequences of these phage-derived antibodies showed no similarities with DNA-binding antibodies from other sources. Even after six rounds of panning no positive clones were detected which bound to poly[d(AT)] and after seven rounds only two were derived from panning with calf thymus DNA. Therefore, sequence- and structure specific antibodies can be recovered from phage display libraries but not all sequences may be represented in the repertoire.

Specificity of monoclonal antibodies produced against phosphorothioate and ribo modified DNAs.

A large number of phosphorothioate DNAs and mixed ribo/deoxyribo duplexes were prepared and their immunogenicity was studied in mice. Only those polymers which were nuclease-resistant were immunogenic and in these cases monoclonal antibodies were prepared. The specificity of the antibodies was measured by direct and competitive Solid Phase Radioimmune Assay (SPRIA) and on this basis four types of antibody could be identified. Type I antibodies are specific for the immunizing polymer and show very limited crossreactivity. For example, Jel 384 binds only to poly(dsA).poly(dT); Jel 453 and 462 bind only to poly(dsG).poly(dC) and poly(dsG).poly(dm5C). Type II antibodies bind to most polymers containing the appropriate modification but will not bind to unmodified DNAs. For example, Jel 343 binds to most thio DNAs regardless of sequence; Jel 346 binds well to most ribose-containing polymers and may be a useful reagent for the detection of the 'A' family of conformations. Type III antibodies bind to most nucleic acids whether modified or not. Their specificities are similar to autoimmune antibodies. Type IV antibodies are single strand-specific such as Jel 383 which binds to poly(dT). There were no examples of antibodies which bound specifically to the immunizing DNA and the unmodified polymer. Thus, modified DNAs cannot be used to prepare sequence-specific reagents. Also, the immunogenicity of modified nucleic acids may limit their usefulness in antisense technologies.

Characterization of a new monoclonal antibody to triplex DNA and immunofluorescent staining of mammalian chromosomes.

A monoclonal antibody, Jel 466, was prepared from mice immunized with poly[d(Tm5C)].poly[d(GA)]. The binding of Jel 466 to nucleic acids was characterized by solid phase radioimmunoassays and competition experiments. There was no binding to single-stranded DNAs or to duplexes which could not form triplexes. In addition, the antibody preferred the triplex form of poly[d(TC)].poly[d(GA)]; it bound weakly to the triplex derived from poly[d(G)].poly[d(C)], but there was no interaction with poly[d(T)].poly[d(A)].poly[d(T)]. This pattern of specificity is very different from that of Jel 318, a triplex-specific antibody that will bind to poly[d(T)].poly[d(A)].poly[d(T)]. The amino acid sequence of Jel 466 also showed very little homology with Jel 318, although both contain many positively charged amino acids. The immunofluorescent staining of mouse and human chromosomes with Jel 466 was studied. In all cases, there was a marked reciprocal relationship between the pattern of Jel 466 on the one hand and that of Hoechst 33258 and Jel 318 on the other. Jel 466 was negative for C-band and G-band but positive for R-band, whereas the opposite was found for Hoechst and Jel 318. Since C and G-bands are AT-rich and R-bands are GC-rich, these staining patterns match the sequence preferences of the two antibodies. Thus the base composition of triplex-forming DNA differs from domain to domain.

Structure of an immunoglobulin Fab fragment specific for poly(dG).poly(dC).

Jel 72 is a murine monoclonal antibody that is highly specific for the right-handed DNA duplex formed by poly(dG).poly(dC). The three-dimensional structure of the antigen-binding fragment (Fab) of Jel 72 has been determined by x-ray crystallographic methods and refined to 2.7-A resolution. The structure has been determined using a combination of molecular replacement and multiple isomorphous replacement techniques. Crystals of Jel 72 contain two Fab fragments/asymmetric unit. The orientation of each of the four separate constant and variable domain pairs was determined from rotation function searches against appropriately oriented model Fab fragments. The rotated Fab domain pairs were placed within the unit cell using a phased translation search procedure with low resolution phases calculated from three derivatives. The two crystallographically independent Fab fragments possess "elbow" angles (relating the variable and constant domain pairs) that differ by 14 degrees (146 degrees versus 132 degrees). The shape and electrostatic surface potential of the combining site suggest a possible model for the recognition of double helical DNA. The model proposes that residues in the third hypervariable region of the heavy chain interact with the DNA bases via the major groove of poly(dG).poly(dC). This model may be applicable to the recognition of double-stranded DNA by autoimmune antibodies that occur in systemic lupus erythematosus.

Structure of an immunoglobulin Fab fragment specific for triple-stranded DNA.

Triple-stranded DNA of the form poly(Pyr).poly-(Pur).poly(Pyr) (where Pyr represents a pyrimidine, and Pur represents a purine) has become the subject of intense research because of its potential use in the control of gene expression and in the development of sequence-specific reagents for cleaving DNA. In a triplex of this type, the second pyrimidine strand is in a parallel orientation to the purine strand and forms Hoogsteen base pairs with it via the major groove. We describe here the three-dimensional crystal structure determination of the antigen-binding fragment (Fab) from the murine monoclonal antibody Jel 318. Jel 318 is specific for triple-stranded DNA, with a preference for the sequence poly[d(T.m5C)].poly[d(G.A)].poly[(d(m5C+.T)]. The structure has been solved by the molecular replacement method and refined by molecular dynamics to an R value of 0.20 at a resolution of 2.8 A. The crystals have cell dimensions that are very similar to those of the previously determined structure of Fab Kol, but the Fab fragments pack within the unit cell in a completely different manner. The protein is in an extended conformation, with an elbow angle of 154 degrees. The shape and electrostatic surface potential of the antibody combining site suggest a possible model for the recognition of triplex DNA in which residues of CDR-H2 (where CDR represents complementarity-determining region) make specific contact with the DNA bases in the minor groove of the triplex.

Hydralazine induces Z-DNA conformation in a polynucleotide and elicits anti(Z-DNA) antibodies in treated patients.

We studied the effect of hydralazine, an antihypertensive drug with lupus-inducing side effects, on the conformation of poly(dG-m5dC).poly(dG-m5dC) and a plasmid with a 23 bp insert of (dG-dC)n.(dG-dC)n sequences. Using an e.l.i.s.a. with a monoclonal anti-(Z-DNA) antibody Z22, we found that hydralazine provoked the Z-DNA conformation in poly(dG-m5dC).poly(dG-m5dC) at 250-500 microM concentration. The supercoiled form of hydralazine-treated plasmid bound to Z22 in a gel-retardation assay. To examine further whether Z-DNA could act as an inciting agent in anti-nuclear antibody production in patients, we analysed 65 sera from 25 hypertensive patients taking hydralazine and found anti-(Z-DNA) antibodies in 82% of these sera. Sera from age-matched normal controls showed no binding to Z-DNA. Data on sera drawn sequentially from four hypertensive patients showed that antibodies were present after the drug treatment. These data demonstrate the presence of a high incidence of anti-(Z-DNA) antibodies in patients treated with hydralazine and suggest that a possible mechanism for the production of autoantibodies in drug-related lupus might involve the induction and stabilization of Z-DNA by drugs.

Human autoantibody binding to multiple conformations of DNA.

Systemic lupus erythematosus and rheumatoid arthritis in humans are characterized by circulating and tissue fixed autoantibodies reactive with self antigens including nucleic acids and other nuclear components. Native calf thymus DNA (B-form), DNA.RNA hybrid (A-form), and left handed DNA (Z-form) were reactive with autoantibodies derived from SLE sera. Inhibition studies suggest that antibodies are recognizing multiple conformations presented by altogether different polymers and A- or Z-DNA might be the immunogenic stimulus for the production of antibodies cross reactive with native DNA.

Spectrophotometrical and immunochemical studies on the conformational changes in poly(dG-dC).poly(dG-dC) after modification by 4-hydroxyaminoquinoline 1-oxide.

Poly(dG-dC).poly(dG-dC) was modified by the reaction with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP. The conformations of 4HAQO-modified poly(dG-dC).poly(dG-dC) and of poly(dG-dC).poly(dG-dC) were studied by circular dichroism spectra under various salt concentration conditions. 4HAQO residues to guanine bases are inefficient in inducing the transition of poly(dG-dC).poly(dG-dC) from B-form to Z-form conformation. We have elicited monoclonal antibodies against 4HAQO-poly(dG-dC).poly(dG-dC). They were characterized using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and binding to supercoiled DNA. These antibodies reacted with 4HAQO-poly(dG-dC).poly(dG-dC) specifically but not with 4HAQO-modified DNA or poly(dG).poly(dC). However, they cross-reacted with N-acetoxy-2-acetylaminofluorene-modified poly(dG-dC).poly(dG-dC) in Z-form conformation. These monoclonal antibodies may recognize a unique conformation in poly(dG-dC).poly(dG-dC) after 4HAQO modification.

The effects of polyamines on the immunogenicity of polynucleotides.

Polyamines--putrescine, spermidine, and spermine--are small organic cations that are present in all living cells. Recent studies revealed that polyamines could provoke a left-handed Z-DNA conformation in poly(dA-dC).poly(dG-dT) and related alternating purine-pyrimidine sequences. In order to examine whether polyamine-induced Z-DNA conformation of poly(dA-dC).poly(dG-dT) is capable of eliciting anti-Z-DNA antibodies, we immunized rabbits with poly(dA-dC).poly(dG-dT) in the presence and absence of spermidine and spermine. Rabbits immunized with the polynucleotide alone produced antibodies reacting toward poly(dA-dC).poly(dG-dT) and heat-denatured calf thymus DNA (ssDNA). In contrast, immunization with poly(dA-dC).poly(dG-dT) complexed with spermidine or spermine produced antibodies reacting with Z-DNA in addition to those binding toward poly(dA-dC).poly(dG-dT) and ssDNA. Antibodies elicited by polynucleotide.polyamine complexes had no reactivity toward polyamines. Solution inhibition studies suggested that anti-poly(dA-dC).poly(dG-dT), anti-ssDNA and anti-Z-DNA antibodies are distinct populations that favor each one of these antigens. Our results suggest that natural polyamines are capable of altering the immunogenicity of polynucleotides by mechanisms involving the stabilization of Z-DNA conformation. This result may have implications in the recent findings of high levels of polyamines and anti-Z-DNA antibodies in the sera of lupus patients and autoimmune mice.

Antibodies against DNA-psoralen crosslink recognize unique conformation.

The DNA-psoralen crosslink induced precipitating antibodies in rabbits with a titer of 1:102,400 by direct binding ELISA. The antiserum showed considerable binding with Z-DNA and calf thymus DNA brominated under high salt concentration which has been shown to attain Z-/analogous conformation. Inhibition experiments substantiated the results of direct binding assay. However, the affinity purified IgG showed high degree of specificity for the immunogen and did not recognize nDNA, Z-DNA and brominated DNA as inhibitor. Poly(dG.dC).poly(dG.dC)-psoralen photoadduct was found to be inhibitory. These results indicate that the antibodies are probably recognizing the unique conformation at the site of psoralen crosslinking. The DNA-psoralen crosslink showed significant binding with SLE sera known to have high levels of anti-native DNA antibodies. Affinity purified SLE-IgG in a competition assay pointed out the autoantibody recognition of altered conformation of DNA-psoralen crosslink.

Quantitative analysis of carbodiimide modified DNA and immunoprobing by adduct specific antibodies.

Antibodies have been raised against N-cyclohexyl-N-(4-methylmorpholinium)ethyl carbodiimide (CMC) modified single-stranded DNA and characterized by competitive and non-competitive immunoassays to be highly specific for CMC base adduct in homopolymers poly(dG), poly(dT) and DNA. The antibodies recognize picogram concentrations of CMC treated DNA with no cross reactivity to at least 1000-fold excess of unmodified DNA or CMC treated poly(dA). The detection limit of antibodies at 1.4 fmol CMC adduct allows quantitation at a CMC/base ratio of 4.6.10(-7). Based upon single modified base-containing synthetic oligomers, a 7-fold higher binding preference is observed for CMC modified thymine than guanine bases. CMC binding to supercoiled DNA is found to depend upon reaction temperature and ionic strength. CMC-modified supercoiled SV40 and ColE1 DNA, exhibit specific antibody binding proportional to the DNA concentration and extent of CMC modification. However, antibody binding observed is independent of the conformation or strandedness of CMC-modified DNA. DNA extensively modified with CMC retains its inherent capacity to specifically and quantitatively hybridize with complementary DNA immobilized to membranes upon direct blotting or Southern transfers from gels. Hybridized CMC-DNA, through antibody binding, provides for the sensitive and non-isotopic detection of the target DNA sequences.

Autoantibodies from patients with systemic lupus erythematosus can affect DNA and RNA synthesis in cultured cells.

Sera reacting positively for anti-DNA antibodies from systemic lupus erythematosus (SLE) patients were tested for their effect on DNA and RNA synthesis in permeabilized cultured cells and isolated nuclei. The immunoglobulin fraction obtained by ammonium sulfate precipitation of serum was shown to exert considerable influence on DNA and RNA synthesis in cultured cells and nuclei. A component of this antibody population is anti-DNA. These antibodies exert different effects on DNA template activity which is a function of their conformational specificity. Intracellular penetration of autoantibodies as noted in SLE may be one of the reasons for clinical manifestations of disease in these patients.

Specific binding to methylated polynucleotides in monoclonal anti-poly(dT) antibodies from autoimmune mice.

Six monoclonal antibodies (mAbs) reactive to synthetic polynucleotide, poly(dT), were established from spontaneous autoimmune MRL/MpJ-lpr/lpr mice and male BXSB mice by spleen cell hybridization method, and were analyzed for cross-reactivity with polydeoxy-5-methylcytidylic acid [poly(dmC)] in comparison with its unmethylated counterpart poly(dC). By direct binding tests, these mAbs, all of which had preponderant binding activity to poly(dT) relative to poly(dU), were divided into two groups: (i) four mAbs showing reactivity to poly(dmC) as well as to natural DNA preparations and (ii) two mAbs with limited reactivity to poly(dT) but no binding to poly(dmC) or natural DNAs. Inhibition binding tests with these synthetic polynucleotides demonstrated that one mAb (TP-A9) in the first group reacted specifically to poly(dmC), as well as to poly(dT). In the second group, one mAb (TP-B5) showed highly specific reactivity to poly(dT) that could not be inhibited by poly(dU), poly(dC), or poly(dmC). However, another mAb (TP-C8) in the second group showed reactivity to poly(dT) that could be inhibited by poly(dU) as well as by poly(dT). Thus, these findings indicate that monoclonal anti-poly(dT) antibodies can cross-react with poly(dmC) with different specificities and suggest that the methylated base may be one of the major antigenic sites of DNA molecules recognized by anti-DNA antibodies spontaneously produced in autoimmune mice.