HCV poly U/UC sequence-induced inflammation leads to metabolic disorders in vulvar lichen sclerosis.

Vulvar lichen sclerosis (VLS) is a dermatologic disorder that affects women worldwide. Women with VLS have white, atrophic papules on the vulva. They suffer from life-long intense pruritus. Corticosteroids are the first-line of treatments and the most effective medicines for VLS. Although VLS has been speculated as an autoimmune disease for a long time, its pathogenesis and the molecular mechanism is largely unknown. We performed a comprehensive multi-omics analysis of paired samples from VLS patients as well as healthy donors. From the RNA-seq analysis, we found that VLS is correlated to abnormal antivirus response because of the presence of Hepatitis C Virus poly U/UC sequences. Lipidomic and metabolomic analysis revealed that inflammation-induced metabolic disorders of fatty acids and glutathione were likely the reasons for pruritus, atrophy, and pigment loss in the vulva. Thus, the present study provides an initial interpretation of the pathogenesis and molecular mechanism of VLS and suggests that metabolic disorders that affect the vulva may serve as therapeutic targets for VLS.

A Polyuridine Insertion in the 3' Untranslated Region of Classical Swine Fever Virus Activates Immunity and Reduces Viral Virulence in Piglets.

Low-virulence classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Few data are available on the molecular determinants of CSFV virulence. The aim of the present study was to assess a possible role for CSFV virulence of a unique, uninterrupted 36-uridine (poly-U) sequence found in the 3' untranslated region (3' UTR) of the low-virulence CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3' UTR (vPdR-36U) and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of 20 5-day-old piglets were infected with vPdR-36U and vPdR-5U. Ten contact piglets were added to each group. Disease progression, virus replication, and immune responses were monitored for 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality, and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. Ninety percent of the piglets infected with vPdR-36U seroconverted, while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient alpha interferon (IFN-α) responses in serum after 1 week of infection, while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first 2 weeks. Taken together, these data show that the 3' UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission.IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3' untranslated region (3' UTR) plays an important role in flavivirus replication, the present study showed for the first time that a long polyuridine sequence acquired in the 3' UTR by an endemic CSFV isolate can activate immunity, control viral replication, and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants is also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8+ T Cells upon Stimulation with a TLR7 Ligand.

The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.

Uridine composition of the poly-U/UC tract of HCV RNA defines non-self recognition by RIG-I.

Viral infection of mammalian cells triggers the innate immune response through non-self recognition of pathogen associated molecular patterns (PAMPs) in viral nucleic acid. Accurate PAMP discrimination is essential to avoid self recognition that can generate autoimmunity, and therefore should be facilitated by the presence of multiple motifs in a PAMP that mark it as non-self. Hepatitis C virus (HCV) RNA is recognized as non-self by RIG-I through the presence of a 5'-triphosphate (5'-ppp) on the viral RNA in association with a 3' poly-U/UC tract. Here we define the HCV PAMP and the criteria for RIG-I non-self discrimination of HCV by examining the RNA structure-function attributes that impart PAMP function to the poly-U/UC tract. We found that the 34 nucleotide poly-uridine "core" of this sequence tract was essential for RIG-I activation, and that interspersed ribocytosine nucleotides between poly-U sequences in the RNA were required to achieve optimal RIG-I signal induction. 5'-ppp poly-U/UC RNA variants that stimulated strong RIG-I activation efficiently bound purified RIG-I protein in vitro, and RNA interaction with both the repressor domain and helicase domain of RIG-I was required to activate signaling. When appended to 5'-ppp RNA that lacks PAMP activity, the poly-U/UC U-core sequence conferred non-self recognition of the RNA and innate immune signaling by RIG-I. Importantly, HCV poly-U/UC RNA variants that strongly activated RIG-I signaling triggered potent anti-HCV responses in vitro and hepatic innate immune responses in vivo using a mouse model of PAMP signaling. These studies define a multi-motif PAMP signature of non-self recognition by RIG-I that incorporates a 5'-ppp with poly-uridine sequence composition and length. This HCV PAMP motif drives potent RIG-I signaling to induce the innate immune response to infection. Our studies define a basis of non-self discrimination by RIG-I and offer insights into the antiviral therapeutic potential of targeted RIG-I signaling activation.

PolyI:polyC12U adjuvant-combined intranasal vaccine protects mice against highly pathogenic H5N1 influenza virus variants.

The highly pathogenic avian H5N1 influenza virus has the potential to incite a global pandemic. Therefore, there is an urgent need to develop effective vaccines against these viruses. Because it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to develop vaccines that will confer cross-protective immunity against variants of the influenza virus. Recently, we reported that the Toll-like receptor 3 agonist, polyI:polyC(12)U (Ampligen), has been proven to be safe in a Phase III human trial, and is an effective mucosal adjuvant for intranasal H5N1 influenza vaccination. Intranasal administration of an Ampligen adjuvanted pre-pandemic H5N1 vaccine (NIBRG14), which was derived from the A/Vietnam/1194/2004 strain, resulted in the secretion of vaccine-specific IgA and IgG in nasal mucosa and serum, respectively, and protected mice against homologous A/Vietnam/1194/2004 and heterologous A/Hong Kong/483/97 and A/Indonesia/6/2005 viral challenge.

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster.

Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

TLR2-activated human langerhans cells promote Th17 polarization via IL-1beta, TGF-beta and IL-23.

The cytokines IL-6, IL-1beta, TGF-beta, and IL-23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ-resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC-like cells TLR2-dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor gammabeta was detected. To clarify if IL-17(+)cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c(+)/langerin(+) monocyte-derived LC-like cells (MoLC), macrophage-activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL-6, IL-1beta, and IL-23. TGF-beta, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti-TLR2 inhibited secretion of IL-6, IL-1beta, and IL-23 by MoLC, while TGF-beta was unaffected. The amount of IL-17 and the ratio of IL-17 to IFN-gamma expression was higher in MoLC- than in monocyte-derived DC-cocultured Th cells. Anti-IL-1beta, -TGF-beta and -IL-23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN-stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.

Intranasal immunization with H5N1 vaccine plus Poly I:Poly C12U, a Toll-like receptor agonist, protects mice against homologous and heterologous virus challenge.

The avian H5N1 influenza virus has the potential to cause a new pandemic. Since it is difficult to predict which strain of influenza will cause a pandemic, it is advantageous to produce vaccines that confer cross-protective immunity. Mucosal vaccine administration was reported to induce cross-protective immunity by inducing secretion of IgA at the mucosal surface. Adjuvants can also enhance the development of fully protective mucosal immunity. Here we show that a new mucosal adjuvant, poly I:poly C12U (Ampligen), a Toll-like receptor 3 agonist proven to be safe in a Phase III human trial, is an effective adjuvant for H5N1 influenza vaccination. Intranasal administration of a candidate influenza vaccine with Ampligen resulted in secretion of IgA, and protected mice that were subsequently challenged with homologous A/Vietnam/1194/2004 and heterologous A/HK/483/97 and A/Indonesia/6/2005 virus.

Immunological responses after immunisation of mice with microparticles containing antigen and single stranded RNA (polyuridylic acid).

Certain toll-like receptor (TLR) agonists, e.g. CpG DNA, can be used as potent vaccine 'adjuvants'. It is known that some sequences of single stranded (ss) RNA stimulate proinflammatory and antiviral responses following interaction with TLR 7 and 8. We have encapsulated ovalbumin (OVA) in the presence and absence of polyuridylic acid (poly-U) inside polylactide microparticles. In comparison to microparticles containing only OVA, bulk cultures of bone marrow-derived plasmacytoid and myeloid dendritic cells produced more (P<0.05) IL-12 and interferon (IFN)-alpha when stimulated with microparticles containing OVA and poly-U. Subcutaneous injection of comicroencapsulated OVA and poly-U resulted in statistically elevated levels of serum anti-OVA IgG1 (P<0.05 versus naïve mice). Conversely, anti-OVA IgG1 levels in C57 BL6 mice immunised with OVA loaded microparticles (without RNA) were statistically indifferent to naïve animals. Furthermore, injection of coencapsulated OVA and poly-U resulted in (P<0.05) greater numbers of OVA specific IFN-gamma secreting T-cells as compared with mice injected with OVA loaded microparticles. A similar trend was seen in mice immunised with OVA loaded microparticles decorated with CpG or solutions of admixed OVA and CpG (P<0.05). These data demonstrate, for the first time, that appropriately formulated ssRNA can act as a potent adjuvant and modulator of adaptive immunological responses.

Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.

Interferons (IFNs) are critical for protection from viral infection, but the pathways linking virus recognition to IFN induction remain poorly understood. Plasmacytoid dendritic cells produce vast amounts of IFN-alpha in response to the wild-type influenza virus. Here, we show that this requires endosomal recognition of influenza genomic RNA and signaling by means of Toll-like receptor 7 (TLR7) and MyD88. Single-stranded RNA (ssRNA) molecules of nonviral origin also induce TLR7-dependent production of inflammatory cytokines. These results identify ssRNA as a ligand for TLR7 and suggest that cells of the innate immune system sense endosomal ssRNA to detect infection by RNA viruses.

Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).

Human papilloma virus (HPV) found in 99.7% of cervical cancers represents an attractive immunotherapeutic target for novel adjuvant dendritic cell (DC) immunotherapy. DC primed with HPV antigens have been shown to be capable of inducing CTL responses powerful enough to eradicate established murine tumours expressing HPV16 antigen. The use of tumour lysate has been found to be an effective means of priming DC with tumour associated antigens in animal models and in clinical trials leading to significant anti-tumour responses. Autologous DC primed with sonicated HPV expressing tumour lysate have been shown to be capable of inducing HPV specific classes I and II T-cell immunity in a pilot clinical study.Synthetic double stranded polyribonucleotides are effective in vitro activation/maturation agents capable of inducing a stable mature DC phenotype producing high levels of IL12. However, the prototype polymer poly [I]:poly [G] has proved to be clinically toxic. Preliminary in vitro data have demonstrated that a novel clinically non-toxic analogue polymer poly [I]:poly [C(12)U] (Ampligen R) can effectively induce in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. Human monocyte derived DC primed with tumour lysate and matured with synthetic dsRNA may therefore offer an effective way of optimising Th1 specific anti-cancer T-cell responses in cancer patients. This strategy is currently being tested in a clinical trial in patients with cervical cancer.

Characterization of the mouse autoantigen La (SS-B). Identification of conserved RNA-binding motifs, a putative ATP binding site and reactivity of recombinant protein with poly(U) and human autoantibodies.

To facilitate the study of autoimmunity to the nuclear Ag La (SS-B) we have isolated and characterized cDNA encoding the mouse La (SS-B) protein. Mouse La (SS-B) protein has 76.7% identity to both human and bovine La (SS-B) proteins and the previously recognized RNP-binding motifs were noted to be highly conserved across the three species. Examination of the primary sequence of human and bovine La (SS-B) newly identifies the site of a potential ATP-binding motif G/AXXXXGK which is preserved in mouse La (SS-B) despite a unique 16 amino acid insertion present in this region of the mouse protein. Analysis of mouse genomic DNA suggests a single gene encodes mouse La (SS-B) and no restriction fragment polymorphisms were identified in the three mouse strains investigated. Two alternate 5' untranslated regions were identified in mouse La (SS-B) cDNA and La (SS-B) mRNA was identified in a wide range of murine tissues consistent with its ubiquitous expression. Recombinant mouse La (SS-B) protein purified from Escherichia coli was shown to bind to poly(U) agarose and to react with sera containing anti-La (SS-B) antibodies obtained from patients with Sjögren's syndrome. The ability to produce recombinant mouse La (SS-B) should provide a valuable reagent for the study of the cellular autoimmune response as well as immunologic tolerance to La (SS-B) Ag in mice.

A monoclonal antibody to the double-stranded polyribonucleotide complex poly(A) X poly(U).

A monoclonal antibody to the double-stranded polyribonucleotide complex poly(A) . poly(U) was derived from the fusion of spleen cells from immunized DBA/2 mice and the P3 X X63-Ag8 plasma cytoma. Specificity studies using radioimmunoassays showed that the anti-poly(A) . poly(U) does not cross-react with single-stranded polyribonucleotides. RNA X DNA hybrids or DNAs. In addition to RNA duplexes associating adenine and uracil, it recognizes synthetic poly(I) . poly(C) and naturally occurring reovirus RNA. It is thus directed against a conformational epitope with an absolute requirement for two polyribose phosphate chains. However, the antibody does not cross-react with poly(G) . poly(C) and is therefore able to distinguish between RNA double helices.

[Availability of DNP-modified 5'- and 3'-ends of the poly(U) fragment bound to the 30S ribosomal subunit for interaction with antibodies]. / Dostupnost' DNF-modifitsirovannykh 5'- i 3'-kontsov fragmenta poli(U), sviazannogo s 30 S-subchastitsei ribosomy, dlia vzaimodeistviia s antitelami.

Modification of the 5'- or 3'-terminal nucleotide residues of poly(U) by dinitrophenyl haptens was carried out. The accessibility of the modified ends of short poly(U) (20-70 nucleotide residues) bound to the 30S subunit of E. coli ribosomes for the interaction with antibodies that are specific against the dinitrophenyl group, was investigated. Some peculiarities of the structural organization of the "entry" (3'-end) and "exit" (5'-end) sites of the template polynucleotide on the 30S subunit and the possibility of the template polynucleotide folding on the ribosome are discussed.

Elevated levels of antibodies to polyuridylic acid detected and quantitated in systemic scleroderma patients by solid phase radioimmunoassay.

A solid support radioimmunoassay has been developed to detect immunoglobulin specific circulating antibodies to polyuridylic acid (Pol U), single-stranded RNA (ss RNA), and single-stranded DNA (ss DNA) in scleroderma and other connective tissue diseases. The assay system uses flex-vinyl microtiter plates on which bovine methyl albumin, the respective polynucleotide, a 1:80 dilution of patient serum, and tritiated high affinity anti-IgG, -IgA, or -IgM are layered. The individual wells containing the sandwich assay are then counted for the presence of labeled immunoglobulins and the results are reported in microgram/ml. Of the 30 scleroderma patients tested, only patients with diffuse systemic scleroderma had antibody levels reactive to Poly U > 4.0 microgram/ml and to ss RNA < 3.0 microgram/ml. Patients with linear scleroderma or morphea had antibody levels to Poly U < 3.0 microgram/ml and very little antibody to ss DNA or ss RNA in their sera. Partial cross reactivity to Poly U was found only in SLE patients with high levels of Ab to ss DNA. Insignificant levels of Poly U antibody were found in patients with other connective tissue diseases and in normal controls. High levels of serum antibody in patients which reacted with Poly U suggest active diffuse systemic scleroderma.

Adjuvant treatment with polyadenylic-polyuridylic acid (Polya.Polyu) in operable breast cancer.

Adjuvant immunotherapy with polyadenylic-polyuridylic acid (PolyA.PolyU) was tested in a randomised trial on 300 patients with operable breast cancer, all of whom were treated by surgery with or without radiotherapy. They were randomly divided into an experimental group of 155 patients who were treated with 30 mg PolyA.PolyU intravenously per week for 6 weeks and a control group of 145 patients who received normal saline intravenously on the same schedule. The mean follow-up time was more than 50 months in both groups. The overall survival was significantly higher in the treated group (p less than or equal to 0.05), in whom the 5-year "relapse-free" surival was also increased. In node-positive patients, treatment increased the relapse-free survival (p less than or equal to 0.03) and overall survival (p less than or equal to 0.07). No side-effects were noted. Thus, immunotherapy with PolyA.PolyU appears to be a simple, non-toxic, and efficient adjuvant treatment in operable breast cancer.

Immunological recognition of three polynucleotide structures associating adenylic and uridylic acids.

The reactions of antibodies directed against the two double-stranded synthetic polyribonucleotide complexes poly(A).poly(U) or poly(I). poly(C) with three polynucleotide structures associating equimolar amounts of adenylic and uridylic acids were studied by immunodiffusion, quantitative precipitation and readioimmunoassay. The three sequence isomers of the same base composition but different nucleotide distribution were: the double helical complex poly(A).poly(U) containing two homopolymeric strands; the copolynucleotide poly(A-U) composed of a strictly repeating riboadenylic acid and ribouridylic acid sequence in both strands; and the copolymer poly(A,U) where the two strands contain both residues but in a random distribution. The three antigens reacted with anti-poly(A).poly(U) and antipoly(I).poly(C) antisera but not to the same extent. The random copolymer poly(A,U) reacted poorly with sera of both specificity. In contrast the reactivity of the alternating copolnucleotide poly(A-U) was widely different according to the specificity of the sera used. Whereas it was recognized by the anti-poly(A).poly(U) antibodies almost to the same extent than the homologous complex, its activity was much lower with anti-poly(I).poly(C) antisera. In addition, the relative efficiency of poly(A-U) was approximately 50 times lower than that of poly(A).poly(U) when tested with anti-poly(I).poly(C) antibody, thereby indicating recognition of different antigenic determinants in both duplexes. Antibodies directed against defined conformational determinants are therefore able to distinguish between three polynucleotide structures of the same base composition but different sequence.

Isolation of a foot-and-mouth disease polyuridylic acid polymerase and its inhibition by antibody.

A template-dependent polyuridylic acid [poly(U)] polymerase has been isolated from BHK cells infected with foot-and-mouth disease virus (FMDV). Enzyme activity in a 20,000 x g supernatant of a cytoplasmic extract was concentrated by precipitation with 30 to 50% saturated ammonium sulfate. The poly(U) polymerase was freed of membranes by sodium dodecyl sulfate and 1,1,2-trichlorotrifluoroethane extraction, and RNA was removed by precipitation with 2 M LiCl. The solubilized poly(U) polymerase required polyadenylic acid as template complexed to an oligouridylic acid primer and Mg2+ for activity, but was inhibited by Mn2+. Antisera from animals infected with FMDV had previously been shown to inhibit the activity of FMDV RNA replicase complexed to the endogenous RNA template. The same antisera also inhibited the activity of poly(U) polymerase. Antisera depleted of antibody by absorption with the virus infection-associated antigen of FMDV no longer inhibited replicase and polymerase activities. The evidence suggests that FMDV RNA replicase, poly(U) polymerase, and the virus infection-associated antigen share a common protein.

Immunogenicity of rice dwarf virus-ribonucleic acid.

Using rice dwarf virus (RDV)-RNA which was extracted from RDV and further purified by MAK-column chromatography, anti-RNA antibodies were produced in rabbits immunized with RDV-RNA antibodies were prods immunized with RDV-RNA-methylated bovine serum albumin complexes. The antisera, as analzyed by complement fixation, cross-reacted with synthetic double stranded RNAs (poly (A)-poly (U), poly (I)-poly (C)), but not with native or denatured DNA, rRNA, tRNA, 5 S RNA and nucleic acids from rice plants. RDV-RNA treated with heat or dimethylsulfoxide was markedly reduced in reactivity to the antisera. When RDV-RNA was digested with RNase A at low salt concentration, its complement fixation activity was abolished. In double diffusion tests, two different precipitation lines were demonstrated between the antiserum and RDV-RNA. One of the precipitation lines connected with those was formed between the antisera, poly (A)-poly (U) and poly (I)-poly (C). As regards immunoglobulin classes of the antibodies, one of the two rabbits employed had antibody activity only in IgG.

Immunochemical characterization of the anti-RNA antibodies found in scleroderma and systemic lupus erythematosus. II. Reactivity with hsa-coupled, uridine-containing, monophosphoric ribodinucleotides.

Sera from patients with scleroderma have been found to have anti-RNA antibodies which react with human serum albumin (HSA)-coupled uridine and uridine monophosphate (UMP) and are inhibited by uracil, uridine and UMP. Scleroderma sera react uniformly with 5'-polyuridylic acid (poly(U)) and fail to react with polyadenylic, polyuridylic acid poly(A) - poly(U)) which is also indicative of their uracil specificity. Anti-RNA antibodies found in systemic lupus erythematosus (SLE) are immunochemically different from those found in scleroderma in that, instead of being uniformly specific to uracil, they are markedly heterogeneous and may react with uracil, uridine and/or UMP. SLE sera frequently react with poly(A) - poly(U), indicating also their ability to recognize the double helical structure of double-stranded RNA. Thirty-seven scleroderma and thirty-four SLE sera from as many patients with either of these conditions were tested against HSA-coupled, uridine-containing monophosphoric dinucleotides in an attempt to characterize further their anti-RNA antibodies. Scleroderma sera were found to react primarily with dinucleotides in which uridine was the base proximal to the carrier protein and, except for sera that also contained antibodies to adenosine which reacted with UpA, they failed to react with dinucleotides in which uridine was in a terminal position only. Reaction with dinucleotides in which uridine was proximal to the carrier protein could be inhibited by uracil but not by the corresponding terminal base. Some lupus sera were found to react with both dinucleotides that contain the same bases in opposite sequence, e.g. ApU and UpA, while others were found to react with only one of the sequences. They were also found to react more frequently with dinucleotides in which HSA was coupled to a base other than uridine, suggesting that the reaction is primarily due to anti-DNA antibodies. Because immunization with dinucleotides coupled to protein prepared by the same method we have used, yields higher specificity to the base attached to the carrier protein, our findings suggest that, in scleroderma, a single event, akin to that of immunization with a purified antigen, gives rise to the anti-RNA antibodies, whereas in systemic lupus erythematosus there is a considerably wider immunological aberration.