Direct effect of dsRNA mimetics on cancer cells induces endogenous IFN-ß production capable of improving dendritic cell function.

Viral double-stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN-ß autocrine loop, dsRNA-elicited IFN-ß production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN-ß at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte-derived DCs. IFN-ß produced by poly I:C-activated human cancer cells increased the capacity of monocyte-derived DCs to stimulate IFN-γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3(-/-) mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1(-/-) mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN-ß production and contribute to the antitumoral response.

Regulation of cytokine gene expression by adjuvants in vivo.

Antibody isotype affects biological activity of the antibodies and therefore should be considered in prevention of disease by vaccination. In previous reports, we demonstrated that adjuvants affect the antibody isotype switching process and favour the production of certain isotypes. The present study extends these findings and shows fundamental differences in the cytokine induction pattern according to the adjuvant used. Cytokine mRNA levels were determined by in situ RNA-RNA hybridization performed on splenocytes isolated from mice injected with different adjuvants. The results revealed that Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), Al(OH)3 and QuilA administration results in a type-2 (humoral) response, increasing IL-4, IL-5 and IL-13 gene expression, while poly I:C exhibits a type-1 (cell-mediated) response, increasing the production of interferon-gamma (IFN-gamma), IL-2 and IL-6 mRNA. Finally, BeSO4 and poly A:U augment IL-5 and IL-6 mRNA production, while lipopolysaccharide (LPS) and LiCl augment IL-6 and tumour necrosis factor-alpha (TNF-alpha) mRNA production. Also, the adjuvants appear capable of overcoming the inherent IL-2/IFN-gamma and IL-4 dichotomy of C57B1/6 and BALB/c mice, respectively, in response to cellular antigens such as Leishmania and herpes simplex virus (HSV). The overall data suggest that adjuvants direct the isotype switching process via induction of certain cytokines, a finding that can be useful in selection of the most efficient isotype of protective antibodies for disease prevention by vaccination.

Antibody-nucleic acid interactions. Antibodies to psoralen-modified RNA as probes of RNA structure.

Antisera elicited by immunization of rabbits with 4'-aminomethyl-trioxsalen (AMT)-modified poly(A,U) complexed with methylated bovine serum albumin was characterized in competition radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). AMT-poly(A,U) was over 10,000-fold more reactive than unmodified poly(A,U) or AMT alone. The antiserum cross-reacted to varying extents with AMT-modified-RNA's and -DNA's. The presence of AMT-uridine usually assured strong reactivity. The amino group of AMT contributed to antibody binding to a small degree. Binding was not significantly affected by high ionic strength, suggesting that binding does not involve ion pair formation. Murine encephalomyocarditis virus replicative intermediates, as well as cellular RNA and DNA were modified by psoralen in intact cells, suggesting that EMCV RNA and cellular RNA's in intact cells possess detectable stretches of base pairs. The antibodies described here will be useful in studying the secondary and tertiary structure of RNA's in vitro and in intact cells.

Formation of triple-helical nucleic acids studied by using antibodies specific for poly(A).poly(U).poly(U).

The formation of the triple helix of poly(A).poly(U).poly(U) was studied by using antibodies specific to poly(A).poly(U).poly(U). the 10-11 base chain length for oligo(A) and the 20-30 base chain length for oligo(U) may be the minimum sizes required to maintain a stable triple helix. Double-stranded poly(A).poly(U) which was the core of triple-stranded poly(A).poly(U).poly(U) could bind poly(U) and produce an analogue of poly(A).poly(U).poly(U) reactive with the antibodies even if the poly(A) or poly(U) was brominated or acetylated to the extent of 35-55%. However, brominated or acetylated poly(U) did not produce a stable triple helix with double-stranded poly(A).poly(U).

Polynucleotide antibodies in connective tissue disease: viral markers or disease mediators?

Antibodies to polynucleotides are seen primarily in systemic lupus erythematosus (SLE), but also occur in a variety of other connective tissue diseases. We looked at the prevalence of antinucleotide antibodies (double- and single-stranded RNA and DNA [dRNA, sRNA, dDNA, and sDNA]) in the sera of patients with SLE (70), rheumatoid arthritis (RA) (31), juvenile rheumatoid arthritis (JRA) (68), osteoarthritis (12), and of 22 patients with a preceding viral illness. In comparison with sera from a control population, elevated mean antibody levels to sRNA were found in the sera of all the patients with connective tissue disease, as well as in the sera of patients with preceding RNA, but not DNA, viral infections. Elevated mean levels of antibodies to dRNA were seen in all groups with the exception of RA. Elevated mean antibody titers to sDNA were not seen in patients with JRA nor were they present in the sera of patients with preceding RNA viral infections. Elevated mean anti-dDNA titers were seen only in sera from patients with SLE. High correlation coefficients between the levels of antibodies to sRNA and dRNA in sera from SLE and RA, and between sDNA and dDNA in sera from SLE suggest cross-reactivity of the antibodies in these diseases. Immunization of an elderly population with influenza (RNA) viral vaccine induced antibodies to sRNA only. These studies further document the prevalence of antipolynucleotide antibodies in the sera of patients with connective tissue diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of the mixed leukocyte reaction by serum from polynucleotide-injected mice.

Sera from mice which have been injected iv with either poly(A) X poly(U) or poly(I) X poly(C) 1 1/2 hr prior to bleeding were found to suppress the mixed lymphocyte reaction. This effect was reduced considerably by 18 hr. Characterization of the suppressive sera revealed it (a) was stable to heating at 56 degrees C for 1 hr and freezing at -20 degrees C for 1 month; (b) had a molecular weight greater than 30,000; (c) could be induced in sera from athymic nude mice; and (d) was present to a lower degree in sera from aging 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.

The mouse immune response to the double stranded polyribonucleotide complex poly(G) . poly(C).

Ten inbred strains of mice were immunized with the double stranded polyribonucleotide complex polyguanylic . polycytidylic acid [poly(G) . poly(C)]. While some immunogenic properties of this duplex were comparable to those of other nucleic acids antigens, differences were also noted. High (SJL/J, BALB/c), low (DBA/2, AKR) and intermediate responders were observed; these differences were not abolished by adsorption of the duplex to MBSA. This pattern of immune response is distinct both from that observed with two other synthetic polyribonucleotide double helices [poly(A) . poly(U) and poly(I) . poly(C)] and with single stranded DNA. The anti-poly(G) . poly(C) activity was localized in the 7S region, whether the sera came from high or low responders, from mice immunized with or without a carrier, after one or several injections. In contrast with anti-poly(A) . poly(U) sera which do not react with poly(G) . poly(C), anti-poly(G) . poly(C) exhibited poly(A) . poly(U) binding activity; no clear relationship between the two activities, however, could be demonstrated. Thus a series of immunological properties differentiates poly(G) . poly(C) not only from the natural polydeoxyribonucleotide single stranded DNA, but also, and more unexpectedly, from two other double stranded polyribonucleotide complexes. These observations suggest that the mechanism controlling the antibody response to poly(G) . poly(C) differs from that regulating poly(A) . poly(U) and/or poly(I) . poly(C), and are to be connected with the fact that the anti-poly(G) . poly(C) antibodies occurring in the sera of patients with systemic lupus erythematosus did not correlate with the antibody activities directed toward the other duplexes.

Immunization of cattle with a variant-specific surface antigen of Trypanosoma brucei: influence of different adjuvants.

Nine different adjuvants were examined for their ability to potentiate the humoral and cell-mediated immune responses of cattle to a soluble glycoprotein antigen prepared from Trypanosoma brucei. Serological responses as measured by the Farr assay were best augmented by the oil-based adjuvants and saponin. Cell-mediated immunity, as assessed by specific lymphocyte transformation in vitro, was enhanced by all oil-based adjuvants at different intervals after immunization. Results from a challenge infection of immunized cattle with the homologous clone of T. brucei and from neutralization tests indicated that protection against infection was better correlated with specific antibodies than with cell-mediated responses. From these considerations, and the absence of tissue reactions at the site of inoculation, saponin was considered more practical than the oil-based or bacterial adjuvants for the elicitation in cattle of antibodies to purified soluble antigens.

The separation of three antibody populations from anti-poly(A).poly(U) antibodies elicited in mice or rabbits and antigenic features of poly(A).poly(U)).

Anti-poly(A).poly(U) antibodies in ascitic fluid of DDY mice immunized with poly(A).poly(U)-methylated bovine serum albumin complexes were fractionated into three major antibody populations, Ab-1, Ab-2 and Ab-3, by precipitating with poly(I).poly(C), poly(A).poly(U), and poly(A).2 poly(U), respectively. Antibody population one, Ab-2, reacted with various double-stranded RNAs [poly(I).poly(C), poly(A).poly(U), and rice dwarf virus ribonucleic acid (RDV-RNA)] and poly(A).2 poly(U). Ab-2 reacted with poly(A).poly(U) and poly(A).2 poly(U). Although both Ab-1 and Ab-2 reacted with poly(A).poly(U), the two populations were distinguishable by their different reactivities against chemically modified antigens and oligonucleotides. In contrast to Ab-2, acetylation at the furanose 2'-position of poly(U) resulted in a dramatic decrease in the complement fixation reactivity of Ab-2. Also, Ab-2 was capable of binding with complexes of hexa- to heptaadenylates and poly(U), whereas Ab-1 required oligoadenylates of longer chain lengths (9-10 chain length) for binding. Therefore, it appears that poly(A).poly(U) possesses unique antigenic determinants which are recognizable only by Ab-2, in addition to those determinants which are common to a variety of double-stranded RNAs.

Immunologic effects of hydralazine in hypertensive patients.

Twenty-seven hypertensive patients (23 blacks, 4 whites) treated with hydralazine had frequent serologic evidence of autoimmunity. However, only 1 patient developed a lupus syndrome. Acetylator phenotype influenced the autoimmune response; slow acetylators had a higher incidence and titers of autoantibodies. The lupus patient not only had high titers of autoantibodies but they were predominantly IgG in contrast to the predominant IgM antibodies found in other slow acetylators. Hydralazine treatment did not alter cell-mediated immune responses and hydralazine antibodies were not detected. However, half the patients tested who received hydralazine had positive lymphoproliferative responses to the drug.

Evidence for the presence of lipopolysaccharide in a ribonuclease-sensitive ribosomal vaccine of Pseudomonas aeruginosa.

To obtain information about the nature of the immunogens in the ribosomal vaccine (fraction II) of Pseudomonas aeruginosa, we studied the specificity of rabbit antibodies to fraction II. Crossed immunoelectrophoresis demonstrated the presence of antibodies which precipitated with ribosomal antigens, but not with lipopolysaccharide (LPS). By means of an enzyme-linked immunosorbent assay, antibodies to LPS were detected in antibodies to fraction II. Antibodies to fraction II could protect mice against a lethal challenge with P. aeruginosa. Absorption experiments demonstrated that the protective ability of antibodies to fraction II was due to antibodies to cell envelope antigens, whereas antibodies to ribosomal antigens did not contribute to the protection. Antibodies to LPS could be detected in mice 1 week after a single vaccination with fraction II. It was concluded that the protective activity of fraction II was due, at least in part, to the presence of LPS in the ribosomal vaccine. Treatment of fraction II with ribonuclease decreased the protective activity of the ribosomal vaccine. Addition of synthetic polyadenylic acid-polyuridylic acid restored the protective activity of ribonuclease-treated fraction II, indicating that RNA in the ribosomal vaccine might act as an adjuvant or a carrier in the presentation of the of the contaminating cell envelope antigens. The protective activity and the toxicity of fraction II were compared with the protective activity and the toxicity of fraction I, which contained cell envelope components, including LPS, and of purified LPS. The results indicated that protection by the ribosomal vaccine was associated with a slightly higher toxicity than was protection by fraction I, whereas purified LPS was the most toxic vaccine.

Antibodies to polyadenylic acid in patients with myasthenia gravis.

Sera from 100 patients with myasthenia gravis and 45 patients with non-myasthenia gravis neuromuscular diseases were studied for antibodies to poly rA, poly rA-rU, native and denatured DNA. All patients with myasthenia gravis had significant anti-acetylcholine receptor antibodies with a mean titre of 1.2 X 10(-7)M. Forty-eight per cent of the myasthenia gravis patients had anti-poly rA antibody levels which were greater than 3 standard deviations from the mean of 65 control patients by Millipore filter radioimmunoassay. The antibody was specific for poly rA and present in a much higher frequency than antibodies to the other nucleic acids tested. Sucrose-gradient ultracentrifugation demonstrated that the antibody was limited to the IgM class alone. Mechanisms relating these findings to a more generalized immunological dysfunction are discussed.

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.

Electron microscopy of the reactions of anti-poly A. poly U and anti-poly I. poly C antibodies with synthetic polynucleotide complexes and natural nucleic acids.

The reactions between purified anti-poly A. poly U and-poly I. poly C. antibodies (IgG and IgM), and synthetic and natural polynucleotides were visualized at the molecular level. This was achieved by the use of fine tungsten bidirectional shadowing of molecules adsorbed onto thin carbon films, combined with dark field electron microscopic observation. A progression was observed from monogamous multivalency (binding of a single multifunctional antigen molecule with several combining sites of the same antibody molecule simultaneously) (Crothers and Metzger, 1972, Immunochemistry, 9, 341-357), to aggregation. Different types of figures were observed, among which loops formed by the coiling of the antigen around a single IgM molecule were very frequently seen. The tendency of IgG antibodies to bind cooperatively to certain antigens was also noted. In contrast, cross-links were seldom encountered. The cross-reactivity of different polynucleotides was also assessed by a quantitative analysis. The length of antigen associated to an antibody molecule (either IgG or IgM) was also measured.

Studies on putative adult worm-derived vaccines and adjuvants for protection against Schistosoma mansoni infection in mice.

Intraperitoneal transfer of viable adult worms of Schistosoma mansoni did not confer protection against a challenge infection to recipient mice. Antigens of schistosome origin were evaluated for their ability, with and without concomitantly administered nonspecific adjuvants, to stimulate protective immunity against S. mansoni. Freshly perfused ground worms or a putative membrane antigen extracted with 0.5 M KC1 from adult worms, when injected together with Corynebacterium parvum (or in a single experiment with poly [A : U]), resulted in a significant reduction in worm burden of a challenge infection with S. mansoni as compared with that of untreated controls. The membrane antigen was maintained carefully at low temperatures in buffers capable of retarding enzymatic degradation while it was being prepared.