BCG infection in mice is promoted by naïve mesenchymal stromal cells (MSC) and suppressed by poly(A:U)-conditioned MSC.

Mesenchymal stromal cells (MSC) transplantation is an actively studied therapeutic approach used in regenerative medicine and in the field of control of immunoinflammatory response. Conditioning of MSC in culture can form their predominantly pro- or anti-inflammatory phenotypes. We demonstrated that poly(A:U)-conditioning of bone marrow-derived mouse MSC induced predominantly pro-inflammatory phenotype. The effects of administration of naïve MSC (nMSC) or conditioned MSC (cMSC) on the course of mycobacterial infection were studied. BALB/c mice infected i.p. with 5 × 106 M. bovis BCG were successively injected i.v. with 0.75 × 106 of nMSC or cMSC in 11 and 12.5 weeks after infection and sacrificed at the week 14. Histological and bacteriological examination of BCG-infected animals revealed low bacterial loads in liver, lungs and spleen; the bacterial load in spleen was higher than in other organs. Treatment with nMSC induced 3-fold increase of the number of bacteria in spleen granulomas, while cMSC decreased significantly the number of bacteria in BCG-positive granulomas. Analysis of preparations of organ homogenates by luminescent microscopy, MGIT cultures and CFU count on Lowenstein-Jensen medium revealed that nMSC promoted mycobacterial growth whereas cMSC suppressed mycobacterial growth significantly. We concluded that MSC therapy can be effective in mycobacterial infection, but only in a case of appropriate conditioning of the cells.

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.

Immunomodulating actions of nucleotides: enhancement of immunoglobulin production by human cord blood lymphocytes.

We have shown previously that polynucleotides enhance in vitro antibody and Ig production in response to T-dependent antigens in mice and augment Ig production by adult human peripheral blood mononuclear cells. Herein, we report their effects on umbilical cord blood mononuclear cells (CBMNC) obtained from full-term babies. CBMNC produced much less IgM/IgG and an almost negligible amount of IgA in response to various stimuli compared with adult peripheral blood mononuclear cells. The supplementation of yeast RNA augmented spontaneous and T-dependent IgM (p < 0.01) but not IgG production by CBMNC. This action was largely attributable to polynucleotides, which appeared to exert their actions in a dose-dependent manner at the initial stages of culture. Their actions were dependent upon the presence of T cells, but they also enhanced spontaneous IgM production by CBMNC in the absence of T cells. Preincubation of T cells from CBMNC and peripheral blood mononuclear cells with RNA for 3 h before the culture resulted in enhanced IgM production, independent of the stimulants used. Thus, polynucleotides appear to exert actions on immature human T cells as well as other lineage cells in vitro. Their actions may be dependent on the presence or absence of antigens or other stimuli and the nature of the stimuli (T dependent versus T independent). These findings may further support the potential importance of nucleotides contained in human breast milk.

Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies.

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity of human carcinoma cell nuclei for polyinosinic acid demonstrated by monoclonal antibodies.

The anti-nuclear cross reactivity of the monoclonal anti-actin antibodies M 372/809 was studied in some detail. The reactivity against a number of nuclear constituents was examined in the ELISA test and the capacities of these constituents to block the M 372/809 anti-nuclear and anti-actin reactions were evaluated in indirect immunofluorescence tests against tissue sections and monolayer cultures of fibroblasts and Vero cells. The repetitive polynucleotides polyinosinic and polyguanylic acid and their deoxyanalogues, actin and vimentin, were found to have the antigenic epitope. The epitope was covered or otherwise inactivated in the presence of polycytidylic acid. Using the M 372/809 antibodies as a reagent, carcinoma cell nuclei were found commonly to have an affinity for polyinosinic and polyguanylic acid. This was seldom noted with non-neoplastic cells.

Specificity of mouse hybridoma antibodies to DNA. III. Antigenic determinants of nucleic acids recognized by poly(dG) specific monoclonal antibodies.

Immunochemical properties of monoclonal autoantibodies to single stranded DNA (ssDNA), which were derived from systemic lupus erythematosus (SLE) prone mice and were specific for poly(dG), were studied using a hapten inhibition assay with oligo- and mononucleotides. Deoxyguanosine monophosphate (dGMP) completely inhibited the ssDNA binding of poly(dG) specific monoclonal antibodies. The affinity to deoxyguanosine was approximately 70% of dGMP, and to guanosine monophosphate (GMP) and to guanosine was 38% and 24%, respectively. In contrast, other purine monomers such as deoxyadenosine monophosphate (dAMP), adenosine monophosphate (AMP) and adenosine exhibited virtually no inhibitory effect on the ssDNA binding of these monoclonal hybridoma autoantibodies. By the same competitive inhibition enzyme immunoassay using deoxyguanosine oligonucleotides of various chain lengths, the minimal antigenic size of ssDNA encompassed by the combining site of the Fab portion of anti-ssDNA monoclonal autoantibodies was determined to be the tetra- or pentanucleotide.

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.

Supplement-induced cytotoxic cells (SICC) generated from mouse thymus or spleen cells cultured in the presence of interleukin 2 and/or polyinosinic acid.

Mouse thymocytes and spleen cells from unprimed C57BL/6 donors generate broadly reactive cytotoxic cells during 5 days of culture in vitro with polyinosinic acid (5') (poly(I] and/or supernatant from PMA-treated EL4 leukemia cells which contains interleukin 2 (IL-2) activity. We refer here to such cytotoxic cells as "supplement-induced cytotoxic cells" or SICC. Thymocytes are dependent on the supernatant factor(s), whereas spleen cells are usually stimulated by poly(I) alone. Polyinosinic acid acts synergistically with supernatant factor(s) to stimulate generation of SICC by both thymocytes (SICC-T) and spleen cells (SICC-S) when the IL-2 activity of the supernatant is inadequate alone. SICC can be generated by both splenocytes and thymocytes in medium supplemented with fetal calf serum or syngeneic plasma. SICC are active in 4 hr 51Cr-release tests against syngeneic, allogeneic, and xenogeneic tumors but not against lipopolysaccharide-induced lymphoblasts. Embryonic fibroblasts, too, are sensitive to SICC generated by thymocytes. In complement-dependent depletion tests, cytotoxic activity is partially sensitive (SICC-T) or fully sensitive (SICC-S) to anti-Thy-1 and -H-2 but not to anti-Lyt-1, -Lyt-2, or -asialo GM1.

[Changes in cellular transcription on terms of the type of proliferative stimulus in immune-reactive lymphocyte cultures]. / Modificari ale transcriptiei celulare în functie de tipul stimulului proliferativ în culturi de limfocite imunreactive.

Transcriptase activities were investigated in cell extracts of human circulating lymphocyte cultures stimulated for proliferation with polyclonal mitogens like PHA, PPD, and endotoxin from Salm. typhimurium S, and also with two polynucleotides. Enzymic activity of the cell extracts was tested differentiatelly for direct (DNA/RNA) and reverse (RNA/DNA) transcription, using appropriate template-primers: activated DAN and poly (dA). r(pU)10 for direct transcription, and poly (rA.rC. rU), poly (dA) x d(pT)10, and poly (rA). d(pT)12-18 for reverse transcription. All lymphoproliferative stimulators are able to enhance simultaneously the cellular mitosis and the direct transcriptase activity in the cell. There is a good correlation between the two effects. Enzyme affinity to natural DNA is higher than to synthetic poly (dA) r(pU)10 PHA, a mitogen for T lymphocyte, enhances the eukaryote type of reverse transcriptase, while among the mitogens for B lymphocyte the PPD enhances an intermediate (viral-eukaryotic) type of reverse transcriptase, and ENDO is not stimulatory for reverse transcription. Duplex polyribonucleotides have an adjuvant activity: they intensify the action of stimulators but they have not a direct enhancing activity. The different transcriptases involved in the lymphocyte differentiation and proliferation may be targets for clinically useful immunosuppressive drugs differentiated for T and B cells.

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.

[Induction of plant resistance to the tobacco mosaic virus with a double-stranded poly(G)-poly(C) complex]. / Induktsiia ustoichivosti rastenii k virusu tabachnoi mozaiki dvunitevym kompleksom poli(G)-poli(Ts).

Inoculation of double-stranded polyribonucleotide poly(G) . poly(C) complex in a concentration of 50-200 micrograms/mg into tobacco and thornapple leaves was found to produce resistance of the plants to subsequent infection with tobacco mosaic virus (TMV) manifested in decreased number and size of local virus lesions. The induced resistance may spread over the plant and be found in the upper untreated leaves. The level of systemic resistance, however, is much lower than that of the resistance demonstrated in the injected leaves. Actinomycin D (5 micrograms/ml) had no significant effect on the number but stimulated the growth of lesions developing in leaves injected with poly(G) . poly(C) as well as increased their number in the upper leaves of the same plants proximal to the treated ones. Development of tobacco resistance to TMV was accompanied by changes in the activity of terminal oxidases, particularly peroxidase. Possible mechanisms of formation of induced resistance are discussed.

Antibodies to natural double-stranded RNA and to synthetic polyribonucleotides in the sera of patients with systemic lupus erythematosus.

A parallel testing of antibodies to double-stranded ribonucleic acid in 80 sera from patients with systemic lupus erythematosus by the membrane binding method using natural 3H-dsRNA preparation and synthetic 125I-poly I. poly C preparation revealed a good correlation (r = +0.81). In a selected set of patients with SLE we observed a slight tendency to preferential binding of the natural preparation and a lower frequency of anti-dsRNA antibodies when compared to that reported by other investigators. The presence of anti-dsRNA did not correlate with the presence of anti-dsDNA.

Antisera to poly(A)-poly(U)-poly(I) contain antibody subpopulations specific for different aspects of the triple helix.

Rabbit antibodies to the triple-helical polynucleotide poly(A)-poly(U)-poly(I) were fractionated into three major antibody populations, each recognizing a different conformational feature of the triple-helical immunogen. Two distinct populations were purified from precipitates made with poly(A)-poly(U)-poly(U) and poly(A)-poly(I)-poly(I). The former reacted with double-stranded poly(A)-poly(U) or poly(I)-poly(C), and similar populations could be purified with either double-stranded form. The second population recognized the poly(A)-poly(I) region of the triple helix, and the third required all three strands for reactivity. These immunochemical studies suggest that the poly(A) and poly(U) have the same orientation in the triple-helicical poly(A)-poly(U)-poly(I) as in the double-helical poly(A)-poly(U), in which they have Watson-Crick base pairing.