The CCR4-NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression.

A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus.

Immunostimulatory RNA oligonucleotides induce an effective antitumoral NK cell response through the TLR7.

RNA oligonucleotides containing immune-activating sequences promote the development of cytotoxic T cell and B cell responses to Ag. In this study, we show for the first time that immunostimulatory RNA oligonucleotides induce a NK cell response that prevents growth of NK-sensitive tumors. Treatment of mice with immunostimulatory RNA oligonucleotides activates NK cells in a sequence-dependent manner, leading to enhanced IFN-gamma production and increased cytotoxicity. Use of gene-deficient mice showed that NK activation is entirely TLR7-dependent. We further demonstrate that NK activation is indirectly induced through IL-12 and type I IFN production by dendritic cells. Reconstitution of TLR7-deficient mice with wild-type dendritic cells restores NK activation upon treatment with immunostimulatory RNA oligonucleotides. Thus, by activating both NK cells and CTLs, RNA oligonucleotides stimulate two major cellular effectors of antitumor immunity. This dual activation may enhance the efficacy of immunotherapeutic strategies against cancer by preventing the development of tumor immune escape variants.

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.

Interactions of single and combined human immunodeficiency virus type 1 (HIV-1) DNA vaccines.

DNA immunization permits evaluation of possible antagonistic or synergistic effects between the encoded components. The protein expression capacity in vitro was related to the immunogenicity in vivo of plasmids encoding the HIV-1 regulatory genes tat rev, and nef. Neither Tat nor Rev expression was influenced by co-expression in vitro of all three proteins, while Nef expression was slightly inhibited. With the combination of genes, the T-cellular responses of mice against Rev and Nef were inhibited compared with those when single gene immunization was used. No interference was detected for the Tat T-cell response. Thus, co-immunization with certain genes may result in inhibition of specific immune responses.

Reactive oxygen species modified thymine and poly(dT) present unique epitope for human anti-DNA autoantibodies.

Hydroxyl radical, one of the most potent of all reactive oxygen species has been implicated in many human degenerative diseases and is known to modify adenine and thymine in cellular DNA. In the present studies, adenine, thymine and their synthetic homopolymers poly(dA), poly(dT) were ROS-modified and subsequently used as inhibitors of native DNA binding to human anti-DNA autoantibodies. Besides nDNA, modified thymine and poly(dT) were effective inhibitors of DNA-anti-DNA antibody interaction. The relative affinity of ROS-modified poly(dT) was better than that of native DNA. Visual detection of modified thymine and poly(dT) binding to affinity purified anti-DNA IgG by an indirect band shift assay support competition inhibition data. The enhanced recognition of ROS-DNA by anti-DNA autoantibodies, as reported earlier, could be due to the ROS-induced modification of thymine.

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.

Human immunoglobulin M paraproteins cross-reactive with Neisseria meningitidis group B polysaccharide and fetal brain.

Three hundred fifty-nine serum samples from patients with immunoglobulin M (IgM) or IgG monoclonal gammopathies were tested for binding to the capsular polysaccharide (PS) of Neisseria meningitidis group B (MenB PS, poly-alpha[2-->8]-N-acetylneuraminic acid). Of 159 IgM paraproteins, 7 (4.4%) were positive, compared with 0 of 200 IgG paraproteins (P < 0.05). Since MenB PS reactivity was limited to the IgM paraproteins, the 159 IgM paraproteins were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with seven other bacterial PSs. None reacted with meningococcal A or C, Haemophilus influenzae type b, or Streptococcus pneumoniae type 3, 6, 14, or 23 PS. The specificity of the MenB PS-reactive antibodies was confirmed by demonstration of binding to N. meningitidis group B cells but not to a capsular PS-deficient mutant and by specific inhibition of binding to solid-phase MenB PS by soluble MenB PS in an ELISA. Five of five antibodies tested protected infant rats from bacteremia caused by Escherichia coli K1, an organism with a PS capsule that also is composed of poly-alpha[2-->8]-N-acetylneuraminic acid. Each of the seven MenB PS-reactive paraproteins had autoantibody activity as defined by binding to homogenates of calf brain in a radioimmunoassay. For six of the seven antibodies, binding to calf brain was inhibited by the addition of soluble MenB PS. Thus, approximately 4% of human IgM paraproteins have autoantibody activity to poly-alpha[2-->8]-N-acetylneuraminic acid, an antigen expressed in fetal brain and cross-reactive with the MenB capsular PS. The reason for this skewing of the IgM paraprotein repertoire toward reactivity with poly-alpha[2-->8]-N-acetylneuraminic acid antigenic determinants is unknown.

Production of a monoclonal antibody reactive with poly(A) and its application in the isolation of poly(A)+ RNA.

Hybridoma cells producing antibody reactive with poly(A) were isolated following fusion of spleen cells from a BALB/c mouse immunized with poly(A).poly(dT) and myeloma cells. The antibody was purified from ascitic fluid by the formation of immune complexes with poly(A). The antibody reacted with poly(A) and some homopolymers (poly(dT), poly(I) and poly(dC)) which was most probably due to its reaction with the ribose-phosphate part of the molecules, but not with cellular RNAs and DNA. Poly(A) tailed 5 S rRNA (about 20 nucleotides) and tobacco mosaic virus RNA (1060 nucleotides) were precipitated quantitatively by the antibody. Dot hybridization and in vitro translation confirmed that the RNA precipitated by the antibody from mouse spleen total RNA contained biologically active mRNA.

Inhibition of in vitro aminoacylation and translation by RNA reactive antibodies.

Antibodies were raised against guanosine-BSA, GMP-BSA and tRNA-mBSA conjugates separately in rabbits. Binding characteristics of these antibodies to various RNAs were studied using a sensitive avidin-biotin micro ELISA. These antibodies inhibited in vitro aminoacylation of tRNA in a dose dependent manner. This inhibition was reversed by the addition of the respective homologous haptens thereby showing the specificity of these antibodies. In vitro translation of endogenous mRNAs in rabbit reticulocyte lysate was also inhibited by these antibodies in a dose dependent manner.

[Effects of exogenous poly(A)+ mRNA on antigenic properties and growth of Krebs-2 tumor cells]. / Vliianie ékzogennoi poli(A)+ mRNK na antigennye svoistva i rost kletok opukholi Krebs-2.

The treatment of Krebs-2 ascitic tumor cells (TC) with total RNA from the liver of Wistar rats (2 mg/ml) altered their antigenicity. As a result, the growth of treated TC in contrast with untreated TC was inhibited when transplanted i. m. to mice preimmunized with rat liver homogenate. Investigations of poly(A+)mRNA, rRNA and tRNA isolated from the same tissue established that the alteration of antigenicity is due to mRNA (8-24 micrograms/ml). In the cytotoxicity assay with antisera against rat Wistar antigens, the expression of rat antigens in TC treated mRNA was observed in the next cell generations.

Serologic evaluation of patients receiving procainamide.

This controlled study examined the characteristics of serologic abnormalities in 52 patients receiving procainamide for cardiac arrhythmias, who had no symptoms of a connective tissue disease. Antinuclear antibodies occurred in 43 patients (83%). Significant elevation of antibody binding to single-stranded DNA (mean +/- SEM 30 +/- 2.6%), double-stranded DNA (13 +/- 1.1%), Z-DNA (optical density 0.54 +/- 0.06), and poly A (7.2 +/- 0.6%) was seen (P less than 0.001). Thirty-four patients (65.4%) had antibodies to total histones, most frequently, the H2A/2B dimer. IgG antibodies to H2A/2B correlated with the cumulative procainamide dose. One patient subsequently developed drug-related lupus.

Amino acid sequence of the FV region of a human monoclonal IgM (NOV) with specificity for the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1, which cross-reacts with polynucleotides and with denatured DNA.

The complete amino acid sequences of the VH and VL regions of a biologically significant Ig, IgMNOV, were determined. IgMNOV is reactive with the capsular polysaccharide of the group B meningococcus and of Escherichia coli K1. As reported earlier, it cross-reacts completely with polynucleotides poly(A) and poly(I) and to a lesser extent with denatured DNA and protects newborn rats against infection with E. coli K1, and is equal in potency to the standard horse anti-group B meningococcal serum. The reduced and alkylated chains were sequenced directly, identifying the L chain as lambda-subgroup II and the mu-H chain as subgroup III. The complete sequence of the VL region was determined by sequencing peptides generated by cleavage with Staphylococcus aureus protease, chymotrypsin, and trypsin. The H chain was cleaved with cyanogen bromide followed by enzymatic cleavages to obtain a large part of the VH region sequence. The structure was completed by sequencing tryptic peptides of the Fab fragment and by mass-spectrometric analysis.

The relationship between autoantibodies and intrauterine growth retardation in hypertensive disorders of pregnancy.

Abnormal levels of autoantibodies have recently been demonstrated in patients with hypertensive disorders of pregnancy and patients whose fetuses have intrauterine growth retardation. We determined total immunoglobulin levels (immunoglobulin G, immunoglobulin M, and immunoglobulin A) and a broad panel of autoantibodies (six antiphospholipid, four antihistone, and four antipolynucleotide antibodies) in 50 normotensive pregnant females, 19 patients with preeclampsia, and 18 patients with chronic hypertension to examine the relationship to intrauterine growth retardation. Mothers who were delivered of infants with intrauterine growth retardation demonstrated significantly more autoantibody abnormalities in the two hypertensive groups and in the normotensive control group as compared with patients delivered of appropriately grown infants. The most frequently observed autoantibody abnormalities were antiphospholipid antibodies and the most frequently observed among those were immunoglobulin G isotypes. Total immunoglobulin levels in both hypertensive and normotensive groups were identical. These results suggest a close association between the degree of B-cell activation and both severity of hypertensive diseases and development of intrauterine growth retardation in their offspring.

Immunological memory for HIV-1 induced in rabbits by immune poly(A)+ RNA.

New Zealand rabbits were used to demonstrate the in vitro and in vivo transfer of reactivity, including immunological memory, to a synthetic peptide corresponding to residues 586-606 of the gp-160 protein of human immunodeficiency virus (HIV-1). The transfer were mediated by immune poly (A)+ RNA from lymphoid organs (spleen and mesenteric nodules) harvested after immunization of a sheep with the peptide (8 subcutaneous injections plus glucan and complete Freund's adjuvant using a total of 1750 micrograms peptide). Immunological reactivity was detected by the leukocyte adherence inhibition (LAI) test for cellular immunity. A dose of 150 micrograms poly(A)- RNA ml-1 10(7) leukocytes-1 or 2.0 micrograms poly(A)+ RNA ml-1 10(7) leukocytes-1 was used for in vitro transfer. For in vivo transfer the recipient rabbits received 3,000 micrograms poly(A)- RNA or 20 micrograms poly(A)+ RNA. The mean non-adherence index (NAI) obtained in vitro was 10 +/- 7 for leukocytes treated with poly(A)-RNA and 60 +/- 10 leukocytes treated with poly(A)+ RNA. The poly(A)+ RNA fraction induced a primary-like response and memory cells in vivo. The poly(A)-RNA fraction had no effect. Since sheep are refractory to, and rabbits are sensitive to HIV-1, we suggest the use of this animal model for testing the immunomodulating effect of anti-HIV-1 immune poly(A)+ RNA.

Immunological memory for HIV-1 induced in rabbits by immune poly(A)+ RNA

New Zealand rabbits were used to demonstrate the in vitro and in vivo transfer of reactivity, including immunological memory, to a synthetic peptide corresponding to residues 586-606 of the gp-160 protein of human immunodeficiency virus (HIV-1). The transfers were mediated by immune poly (A)+ RNA from lymphoid organs(spleen and mesenteric nodules) harvested after immunization of a sheep with the peptide (8subcutaneous injections plus glucan and complete Freund's adjuvant using a total of 1750 *g peptide). Immunological reactivity was detected by the leukocyte adherence inhibition (LAI) test for cellular immunity. A dose of 150 *g poly (A) RNA ml-1 10 *******7 leukocytes -1 or 2.0 *g poly(A)+ RNA ml-1 10*******7 leukocytes-1 was used for in vitro transfer. For in vivo transfer the recipient rabbits received 3,000 *g poly(A)- RNA or 20*g poly(A)+ RNA. The mean non-adherence index(NAI) obtained in vitro was 10ñ7 for leukocytes treated with poly (A)- RNA and 60ñ10 leukocytes treated with poly(A)+ RNA. The poly(A)+ RNA fravction induced a primary-like response and memory cells in vivo. The poly (A)- RNA fraction had no effect. Since sheep are refractory to, and rabbits are sensitive to HIV-1, we suggest the use of this animal model for testing the immunomodulating effect of anti-HIV-1 immune poly(A)+ RNA

Monoclonal antibodies to cerebellar pinceau terminals obtained after immunization with brain mRNA-injected Xenopus oocytes.

A method was developed to produce monoclonal antibodies to brain cell antigens by using Xenopus oocytes as immunological vectors. The method consists in injecting Xenopus oocytes with rat brain mRNA to express foreign proteins and using the oocytes for immunization. Immunizations were preceded by immunotolerization of mice to antigens of native oocyte membranes. With this approach we generated a set of monoclonal antibodies that are specific markers for the cerebellar "pinceau"--a unique complex synapse formed between basket cell terminals and the initial segment of the Purkinje cell axon. Our findings reveal an immunoreactivity highly localized at the pinceau and its late expression beginning at postnatal day 19 during cerebellar development.

Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.

3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.

Evidence for an association between U1 RNA and interspersed repeat single-copy RNAs in the cytoplasm of sea urchin eggs.

Psoralen crosslinking of RNA-RNA intermolecular duplexes in sea urchin egg extracts reveals that some maternal poly(A)+ RNA molecules are complexed with U1 RNA, a cofactor in somatic nuclear pre-mRNA splicing. Reaction of egg extracts with a monoclonal antibody specific for U1 snRNP selects, in addition to U1, RNAs that contain repeated sequences interspersed with single-copy elements. Antibody-selection experiments with nucleate and anucleate egg halves demonstrate that most of the U1 RNA-interspersed RNA complexes are cytoplasmic, as is the egg's store of total U1 snRNP. These results raise the possibility that maternal interspersed RNAs include unprocessed pre-messenger RNA molecules in arrested complexes with splicing cofactors.

Immunochemical approaches to gene probe assays.

Antibodies specific for helical nucleic acids can be applied to assays for hybridized DNA and/or RNA. The assays can use either radioactive or nonradioactive detection systems. Antibodies specific for RNA-DNA hybrids are applicable to assays for measuring hybrid helices that are immobilized on plastic or nitrocellulose, whether the helices are preformed in solution or are formed on the solid-phase support. Alternatively, anti-RNA-DNA hybrid antibodies can be immobilized and used to capture hybrids formed in solution, resulting in an assay with a high signal-to-noise ratio. It has been applied to a test for the presence of ribosomal RNA of Campylobacter jejuni in biological samples.

In vivo transfer of delayed hypersensitivity to Trypanosoma cruzi antigens with polysomal immune RNA.

The polysomal immune RNA (iRNA) was extracted from the spleens of mice infected with Trypanosoma cruzi. The RNA isolated from normal (nRNA) animals served as control. We found that the polysomal iRNA is able to transfer delayed-type hypersensitivity (DTH) in vivo to T. cruzi antigens as assessed by the inhibition of cell migration assay and skin test. We also demonstrated that this phenomenon is antigen specific. The polysomal iRNA preparations were fractionated by affinity chromatography on oligo (dT)-cellulose column. Our results suggest that the poly (A)-containing iRNA is the fraction responsible for transferring DTH in vivo to T. cruzi antigens.