Inhibition of embryo implantation in mice through vaginal administration of a proprotein convertase 6 inhibitor.

Uterine proprotein convertase 6 (PC6) plays a critical role in embryo implantation in both mice and women. It was hypothesized that inhibiting uterine PC6 could prevent pregnancy. Vaginal administration of a PC6 inhibitor presents the ideal route for local drug delivery. A peptide-based PC6 inhibitor, C-30k-PEG Poly R that was previously shown to have properties of increased vaginal absorption and penetration was tested for its contraceptive potential in mice following vaginal administration. The study demonstrated that this approach could inhibit embryo implantation in some mice (24% completely and 47% partially inhibited).

Circulating nucleic acids in type 1 diabetes may modulate the thymocyte turnover rate.

The autoimmunity of type 1 diabetes is associated with T-cell hyperactivity. Current study was designed to examine the effect of circulating ribonucleic acids (RNAs), isolated from type 1 diabetic patients on proliferative, apoptotic and inflammatory potential of rat thymocytes. Rat thymocytes were assayed for proliferating nuclear cell antigen (PCNA), Bcl-2, Bax and NF-κB level, using the flow cytometric and fluorometric assays. Cells were allocated into groups, treated with RNAs purified from plasma of juvenile diabetics, adult type 1 diabetic patients, control healthy children, healthy adult persons, nucleic acids and polynucleotide standards (RNA, polyC, PolyA, PolyIC, and CpG). The upregulation of PCNA and Bcl-2 protein and downregulation of Bax protein and NF-κB was shown when the thymocytes where incubated with RNA purified from plasma of juvenile type 1 diabetic patients. The dysregulation of inflammatory cascade and central tolerance may be a defect in autoimmune diseases related to innate immunity leading to corresponding alteration in adaptive immune response.

Oligonucleotides and polyribonucleotides: a review of antiviral activity.

Current antiviral therapies are insufficient for treating emerging, re-emerging and established viral diseases. In an effort to find new therapeutics, oligo- and polyribonucleotides are being studied for their antiviral capabilities. Studies have shown that uniquely modified single- and double-stranded nucleic acid constructs are effective in inhibiting viral proliferation by various mechanisms. This review gives a brief history and highlights the development of oligo- and polyribonucleotides as antiviral agents primarily in the fields of interferon induction, mRNA complementation and reverse transcriptase inhibition.

[Theoretical validation of local express auto-cytokine therapy]. / K voprosu o teoreticheskom obosnovanii metodiki lokal'noi ékspress-autotsitokinoterapii.

Theoretical validation for local express autocytokine therapy (LEACKT), used in recent years with good results for the treatment of viral and nonviral diseases of the eyes, is provided. In vitro experiments showed that autologous cytokine complex appearing after stimulation of patient's peripheral whole blood with poludan (polyA:polyY complex, 200 micrograms) includes all three interferon types (alpha, beta, and gamma) and interleukin-8 (IL-8) and tumor necrosis factor (TNF). It was shown for the first time that poludan stimulated the production of IL-8, the main chemotactic cytokine inducing chemotaxis, neutrophil activation, angiogenesis stimulation, and regeneration. This was not paralleled by an increase in the production of TNF, the key mediator of inflammation, whose level in whole blood did not increase. Addition of poludan to cell culture led to a 2-fold decrease of lymphocyte apoptosis in comparison with that after treatment with dexamethasone alone. The results of experiments indicate that LEACKT with poludan is characterized by pronounced antiinflammatory, interferonogenic, regeneratory, and antiapoptosis effects in various diseases of the anterior segment of the eye and is a simple and safe method.

"Senseless" antiviral polyribonucleotides: poly (1-propargylinosinic acid).

Previous work has shown that novel amphipathic oligo and polyribonucleotides exhibiting secondary structure in solution are potent inhibitors of HIV and HCMV replication and cytopathicity in tissue culture. It was hypothesized that the mechanism(s) of action for these compounds might be inhibition of retroviral reverse transcriptase (RT) and/or viral uptake by cells. Pursuit of the essential pharmacophore has led to the discovery of poly (1-propargylinosinic acid) (10), an HIV and HCMV-active polyribonucleotide lacking the secondary structure previously thought to be essential for the observed antiviral activity.

Immunomodulators in the prevention of acute respiratory viral infections.

The polyethiology of the respiratory diseases and the lack of effective vaccines to prevent them (except for influenza) underline the need for clinical trials and the practical application of immunomodulatory preparations that could become part of the group of anti-viral drugs with a wide range of activity. It has been demonstrated that amixin and poludan, the drugs made in our country, cause an increase of the serum IFN level, enhance the ability of leukocytes and lymphocytes to produce IFN-alpha and IFN-gamma, and lead to the activation of NK and peripheral blood phagocytes. In other words, they have an immunostimulating activity. The number of CD3(+), CD4(+), CD8(+), CD19(+) cells, the level of the Ig A, IgG, IgM and several other parameters of the immune system were not affected by these drugs. It has been demonstrated that amixin and poludan show good drug tolerance, without side effects on the hemogram in individuals who were taking these drugs just for disease prevention. The studied immunomodulators have a significant prophylactic activity in the cases of the polyethiologic group of acute respiratory viral infection during the seasonal peak of the disease, with a coefficient of efficiency of 3.6 (amixin) and 2.1 (poludan), and corresponding protection indices of 72.1% and 52.7%. Having the same chance of getting infected, individuals protected with these drugs often have the disease in a milder or asymptomatic form.

Antiviral oligo- and polyribonucleotides containing selected triazolo[2,3-a]purines.

Several amphipathic (hydrophobic base, hydrophilic backbone) polyribonucleotides have recently been shown to have potent antiviral activity against HIV and human cytomegalovirus (HCMV). The working hypothesis developed during these studies was that the ability to form an ordered, non-hydrogen-bonded array in solution was an important criterion for activity. To explore further the role of structure and molecular size on the inhibition of virus replication, one new polynucleotide and two 32-mer oligonucleotides based on the triazolo[2,3-a]purine ring system have now been prepared. High-molecular-weight polynucleotide 4a (PTPR) and sulfur-containing 32-mer 5b (TTPR) were moderately active against HIV but showed greater potency against HDMV than ganciclovir. Both 4a and 5b gave clear evidence of cooperative melting behavior, whereas inactive 32-mer 5a showed no such behavior.

Inhibition of immunoglobulin production stimulating activity of glyceraldehyde-3-phosphate dehydrogenase by nucleotides.

We have previously identified glyceraldehyde-3-phosphate dehydrogenase as an immunoglobulin production stimulating factor (IPSF) which facilitated immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of this enzyme was suppressed by the coexistence of some sorts of nucleotides. We now report that the IPSF effect of GAPDH was suppressed by the coexistence of DNA, the inhibiting effect of degraded DNA being inferior to that of long-chain DNA. Both single-stranded and double-stranded synthetic polyribonucleotides also inhibited the IPSF activity of GAPDH. Moreover, nicotinamide adenine dinucleotide (NAD+) repressed the IPSF effect.

SUG1, a component of the 26 S proteasome, is an ATPase stimulated by specific RNAs.

SUG1 is an integral component of the 26 S proteasome. Belonging to a novel putative ATPase family, it shares four conserved motifs characteristic of ATP-dependent DNA/RNA helicases. Recombinant rat SUG1 (rSUG1) produced in Escherichia coli was highly purified and characterized in terms of its biochemical properties. The rSUG1 exhibited a Mg2+-dependent ATPase activity. The Km for ATP and Vmax of rSUG1 were 35 microM and 7 pmol of ATP/min/microg of protein, respectively. Both ATPase activity to release [32P]monophosphate and [32P]ATP-labeling activity were coordinately affected by cold ATP severely, GTP and UTP moderately, and CTP little. Interestingly, the rSUG1 ATPase activity was stimulated by poly(U) and poly(C), but not by poly(A), poly(G), or by any forms of DNAs tested. A UV cross-linking assay also indicated poly(U)- and poly(C)-stimulated labeling of rSUG1 with [alpha-32P]ATP. Moreover, the ATPase activity was facilitated by cellular poly(A)+ RNA, but not by poly(A)- RNA. RNA transcribed in vitro from cDNA encoding a b-Zip protein could stimulate the ATPase activity. This is the first report to demonstrate a specific RNA requirement for ATPase with respect to the proteasomal ATPases. Our present work suggests that SUG1 can specifically interact with protein-coding RNA (mRNA) and play some roles in mRNA metabolism.

Unusual single-stranded polyribonucleotides as potent anti-HIV agents.

Polyribonucleotides (PTMG and PMTI) containing 1-methyl-6-thioguanosine or 1-methyl-6-thioinosine, respectively, as the sole nucleoside component are shown to be potent inhibitors of various strains of HIV-1 and HIV-2 in a number of human lymphocyte and macrophage cell lines in tissue culture as well as in fresh human peripheral blood lymphocytes and macrophages. PMTI and PMTG exhibit potencies in the range of 10(-7)-10(-8) M in these systems. The polynucleotides are active against virus strains resistant to AZT and pyridinone derivatives. Both PMTI and PMTG are synergistic with AZT and with ddI, and both inhibit HIV reverse transcriptase at nanomolar concentrations. The polymers show little or no toxicity in human cell lines at the highest doses tested (100 micrograms/mL, or about 0.2-1 microM). This class of compounds represents a new lead in AIDS therapeutic drug discovery.

Protective effect of LPS and poly A:U against immune oxidative injury: role of thiols released by activated macrophages.

Oxidative injury of immune cells has been observed both at inflammatory sites and in pathologic situations, such as human immunodeficiency virus infection. We used an ex vivo model of immune oxidative injury to test the antioxidant effect of two immunomodulating agents administered to C57B1/6 mice. Lipopolysaccharide (LPS) and the synthetic polyribonucleotide poly A:U preserved the ConA-induced proliferative response of spleen T cells against oxidative injury ex vivo. The glutathione and thiol contents of fresh spleen T cells from LPS- and poly A:U-treated mice were significantly higher than control values. In addition, spleen T cells from LPS- and poly A:U-treated mice were protected against the oxidative injury-induced decrease in glutathione content after 48 h of ConA stimulation. Because LPS and poly A:U both activate macrophages, we sought an antioxidant effect of macrophage-released compounds. Neither rhIL-1 alpha nor rhTNF alpha protected against oxidative injury in vitro. In contrast, LPS and poly A:U induced macrophages to release acid-soluble thiols, which have been reported to participate in the regulation of glutathione levels in lymphocytes and could therefore protect against immune oxidative injury.

Short, terminally phosphorylated oligoriboguanylic acids effectively inhibit cytopathicity caused by human immunodeficiency virus.

Various synthetic ribonucleic acids were evaluated for inhibition of HIV-induced cytopathicity of cultured cells; only poly and oligoguanylic acids, but not other homopolymers, showed potent inhibitory activity. Phosphorylation of either the 5'- or 3'-end of oligoribonucleotides converted short inactive oligomers, such as dimers to effective anti-HIV agents. The efficacy of the 3'-phosphorylated phosphorothioate trimer of guanylic acid was comparable to that of other longer oligonucleotides so far reported. Phosphorothioate oligoriboguanylic acids were superior to the corresponding oligodeoxyguanylic acids in their capacity to prevent HIV cytopathicity.

Molecular adjuvants and immunomodulators: new approaches to immunization.

Epitopes on microbial antigens responsible for protective immunity have begun to be identified and isolated, and their chemical structures have been determined. Ensuing knowledge of their weak immunizing capacity per se has led to an appreciation of the need for adjuvants to increase the immunogenicity of these low-molecular-weight synthetic structures. As such, a recent surge in adjuvant research has emerged. Accordingly, this review will highlight a number of those adjuvant substances whose activity in animals indicates a potential use in human vaccines. In addition, the potential of several well-defined substances, termed immunomodulators, which nonspecifically stimulate resistance of animals to multiple 50% lethal doses of microbial challenge is described. Among the most extensively characterized adjuvants of microbial origin discussed in detail are (i) the lipopolysaccharides isolated from gram-negative bacteria and their nontoxic analogs, (ii) the synthetic muramyl dipeptides and their multiple analogs, and (iii) the synthetic polyribonucleotide complexes, mimicking the interferon-inducing capacity of viruses. Discussed also are the heat-labile enterotoxin of Escherichia coli, the nonionic block copolymers, the saponins, a quinolamine derivative, and the hormone dihydroepiandrosterone.

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.

RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties.

The catalytic properties of electrophoretically homogeneous RNA-directed RNA polymerase (RdRP, EC 2.7.7.48) from tomato leaf tissue were studied with the aid of oligonucleotides of defined sequence. It was found that RdRP catalyzes in vitro the transcription of short single-stranded RNA and DNA molecules into precisely complementary RNA copies up to the full length of these templates. The transcription of RNA- and DNA-oligonucleotide templates was equally effective. Differences in transcription efficiency were found to depend on nucleotide sequence rather than on the RNA or DNA nature of the single-stranded nucleic acid. Double-stranded nucleic acids such as poly(A).poly(U) and a double-stranded DNA 14-mer were not transcribed. The RdRP-directed transcription could be primed because RNA and DNA dinucleotides and trinucleotides complementary to the 3'-terminal nucleotides of the template were extended by the enzyme. The unprimed transcription was shown to start preferentially at the 3'-terminal nucleotides of the template. RdRP is capable of adding a single noncomplementary nucleotide to the 3' terminus of about 50% of the runoff transcripts. AMP was preferred over GMP, whereas CMP and UMP were terminally added at very low frequency.

Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices.

Macrophage scavenger receptors exhibit unusually broad, but circumscribed, polyanionic ligand-binding specificity. For example, the polyribonucleotides poly(I) and poly(G) are ligands but poly(A) and poly(C) are not. To further investigate the molecular basis of this polynucleotide-binding specificity, we tested the capacity of various oligodeoxyribonucleic acids to inhibit the scavenger receptor-mediated degradation of 125I-labeled acetylated low density lipoprotein by Chinese hamster ovary cells expressing the type I bovine scavenger receptor. A series of short oligodeoxyriboguanines (dGn, where 5 < or = n < or = 37) were effective inhibitors. The dG6, dG12, and dA5G37 members of this series were shown by circular dichroism and UV spectroscopy to be assembled into four-stranded helices stabilized by G-quartets. [32P]dA5G37 bound directly to scavenger receptors. Partial or complete denaturation of the quadruplex structures of these oligonucleotides by boiling destroyed their inhibitory activity. Receptor activity was also inhibited by d(T4G4)4, a telomere-like oligonucleotide which forms an intramolecular quadruplex. In addition, conversion of the four-stranded potassium salt of poly(I) to the single-stranded lithium salt dramatically reduced its inhibitory activity. Addition of KCl to the Li+ salt resulted in the reformation of poly(I)'s quadruplex structure and restoration of its inhibitory activity. A variety of single-stranded and double-stranded oligo- and polydeoxyribonucleotides (e.g. dA37, HaeIII restriction fragments of phi X174) exhibited very little or no inhibitory activity. Thus, a base-quartet-stabilized four-stranded helix appears to be a necessary structural determinant for polynucleotide binding to and inhibition of scavenger receptors. This conformational requirement accounts for the previously unexplained polyribonucleotide-binding specificity of scavenger receptors. The spatial distribution of the negatively charged phosphates in polynucleotide quadruplexes may form a charged surface which is complementary to the positively charged surface of the collagenous ligand-binding domain of the scavenger receptor.

Beta-adrenergic agonists that down-regulate receptor mRNA up-regulate a M(r) 35,000 protein(s) that selectively binds to beta-adrenergic receptor mRNAs.

In an effort to explore the molecular basis for agonist-induced destabilization of beta-adrenergic receptor mRNA, we investigated the nature of RNA-binding proteins both in untreated and agonist-treated DDT1-MF2 smooth muscle cells. Messenger RNAs for the alpha 1b-, beta 1-, and beta 2-adrenergic receptors as well as for beta-globin were transcribed in vitro, incubated with cytosolic fractions, covalently cross-linked by short-wave UV light, and analyzed by SDS-polyacrylamide gel electrophoresis. A prominent M(r) 35,000 radiolabeled protein(s) with the following characteristics was identified: (i) binds selectively to beta 1- and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind to either alpha 1b-adrenergic receptor mRNA, which does not undergo agonist induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A), -(C), or -(G) RNA; and (iv) displays binding to receptor mRNA that can be competed by RNA harboring destabilizer sequences that are AU-rich and AUUUA pentamer-rich. The abundance of the M(r) 35,000 RNA-binding protein selective for beta-adrenergic receptor message, a factor we term beta ARB protein, varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA.

VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis.

Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities.

Cyclic AMP mediates the direct antiproliferative action of mismatched double-stranded RNA.

Previous experiments have demonstrated that double-stranded RNAs (dsRNAs) can exert an antiproliferative effect on human tumor cells, independent of interferon (IFN) induction. However, the mechanism by which dsRNAs inhibit tumor growth has not been elucidated. As a first step in determining the molecular events responsible for growth arrest, we have explored the role of signal transduction through the cAMP system in the antiproliferative effect of the mismatched dsRNA, r(I)n.r(C12,U)n (Ampligen). These studies utilized the human glioma cell line A1235, which does not produce detectable levels of IFN-alpha, -beta, or -gamma in response to mismatched dsRNA treatment. Treatment of A1235 cells with mismatched dsRNA in combination with either 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), which inhibits cAMP-dependent protein kinase and protein kinase C, or N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), which preferentially inhibits the cAMP-dependent protein kinase, yielded an antagonism of the mismatched dsRNA-induced antiproliferative effect. Measurement of adenylate cyclase activation showed a dose-dependent increase in activity at antiproliferative mismatched dsRNA concentrations, but not at lower, nonantiproliferative doses. This increase in activity was rapid, seen as early as 30 sec after initiation of treatment, and it was sustained at peak levels for 1-2 hr. Analysis of the intracellular cAMP concentration gave similar kinetics of induction. Exposure of cells to the stable cAMP analogue dibutyryl cAMP yielded dose-dependent inhibition of cell growth. The cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also inhibited proliferation. In contrast, neither H-7 nor HA1004 had an effect on growth inhibition induced by human natural IFN-alpha treatment. In addition, antiproliferative doses of IFN-alpha did not increase cAMP concentrations. These results indicate that the cAMP system is utilized by mismatched dsRNA as an early signal transduction mechanism for growth control. Furthermore, the antiproliferative effects induced by mismatched dsRNA and IFN can occur by different mechanisms of action.