Cytoplasmic-delivery of polyinosine-polycytidylic acid inhibits pancreatic cancer progression increasing survival by activating Stat1-CCL2-mediated immunity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective therapies and with poor prognosis, causing 7% of all cancer-related fatalities in the USA. Considering the lack of effective therapies for this aggressive cancer, there is an urgent need to define newer and more effective therapeutic strategies. Polyinosine-polycytidylic acid (pIC) is a synthetic double-stranded RNA (dsRNA) which directly activates dendritic cells and natural killer cells inhibiting tumor growth. When pIC is delivered into the cytoplasm using polyethyleneimine (PEI), pIC-PEI, programmed-cell death is induced in PDAC. Transfection of [pIC]PEI into PDAC cells inhibits growth, promotes toxic autophagy and also induces apoptosis in vitro and in vivo in animal models. METHODS: The KPC transgenic mouse model that recapitulates PDAC development in patients was used to interrogate the role of an intact immune system in vivo in PDAC in response to [pIC]PEI. Antitumor efficacy and survival were monitored endpoints. Comprehensive analysis of the tumor microenvironment (TME) and immune cells, cytokines and chemokines in the spleen, and macrophage polarization were analyzed. RESULTS: Cytosolic delivery of [pIC]PEI induces apoptosis and provokes strong antitumor immunity in vivo in immune competent mice with PDAC. The mechanism underlying the immune stimulatory properties of [pIC]PEI involves Stat1 activation resulting in CCL2 and MMP13 stimulation thereby provoking macrophage polarization. [pIC]PEI induces apoptosis via the AKT-XIAP pathway, as well as macrophage differentiation and T-cell activation via the IFNγ-Stat1-CCL2 signaling pathways in PDAC. In transgenic tumor mouse models, [pIC]PEI promotes robust and profound antitumor activity implying that stimulating the immune system contributes to biological activity. The [pIC]PEI anti-PDAC effects are enhanced when used in combination with a standard of care (SOC) treatment, that is, gemcitabine. CONCLUSIONS: In summary, [pIC]PEI treatment is non-toxic toward normal pancreatic cells while displaying strong cytotoxic and potent immune activating activities in PDAC, making it an attractive therapeutic when used alone or in conjunction with SOC therapeutic agents, potentially providing a safe and effective treatment protocol with translational potential for the effective therapy of PDAC.

Allergen Immunotherapy Enhances Airway Epithelial Antiviral Immunity in Patients with Allergic Asthma (VITAL Study): A Double-Blind Randomized Controlled Trial.

Rationale: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. Objectives: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. Methods: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. Measurements and Main Results: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid-induced expression of IFN-ß at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. Conclusions: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT04100902).

Tricking melanoma to self-digest: a deal of a meal!

Melanoma cells acquire multiple genetic and epigenetic alterations that promote their metastasis and resistance to available therapies. In this issue of Cancer Cell, Soengas and colleagues reveal that the induction of endosome-mediated autophagy results in efficient melanoma cell death, thereby offering new potential means for treatment of this devastating cancer.

Interferon inducer, polyriboguanylic.polyribocytidylic acid, inhibits experimental hepatic metastases in mice.

The antitumour activity of an interferon inducer, the double-stranded complex of polyriboguanylic.polyribocytidylic acid was studied on murine lymphosarcoma LS/BL. The antitumour effect was determined with the aid of an experimental liver-colony model and compared to that exhibited by another synthetic RNA, polyriboinosinic.polyribocytidylic acid. We found that both polynucleotide complexes decreased the number of liver colonies and prolonged the survival of the tumour-bearing mice. This effect was only observed if the complexes were applied in an appropriate dose schedule which included the administration of the drug prior to tumour cell inoculation and subsequent continuous treatment. We have also verified that the polynucleotide complexes did not exert their antitumour effect by a direct action on tumour cells.

[Dependence of the antiviral activity of the poly(G).poly(C) complex on the size of the continuous poly(C)segments]. / Zavisimost' protivovirusnoi aktivnosti kompleksa poli(G).poli(Ts) ot razmera nepreryvnykh uchastkov poli(Ts).

Modification of poly(C) by various frequency treatment with adenosine non-complementary to guanosine has produced poly(G) X poly (C.A) complexes with continuous double-stranded areas the length of which is determined by C/A ratio. Studies of the antiviral activity of poly(G).poly(C,A) complexes with C/A from 10:1 to 90:1 and poly(G).poly(C) in vesicular stomatitis virus-infected chick embryo cell cultures and in experimental tick-borne encephalitis of mice demonstrated that the maximum activity is achieved at an average lengths of double-stranded areas of 90 nucleotide pairs. At the same time, a low but statistically significant antiviral activity is observed at a length of double-stranded areas of 10-30 nucleotide pairs.

[Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex]. / Otsenka razmera nepreryvnogo uchastka poli(G), neobkhodimogo dlia biologicheskoi aktivnosti kompleksa (G).poli(Ts).

On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area.

[Effect of virazole on the antiviral activity of poly(G) X poly(C) and other polyribonucleotide interferonogens]. / Vliianie virazola na protivovirusnuiu aktivnost' poli(G) X poli(Ts) i drugikh poliribonukleotidnykh interferonogenov.

The effect of virazole on the antiviral activity of poly (G) X poly (C), poly (G, A) X X poly (C) and poly(G, I) X poly (C) was studied in cell cultures and on mice. It was shown that virazole in concentrations not sufficient for significant inhibition of the development of vesicular stomatitis virus or Sindbis virus in chick embryo cell cultures markedly increased the antiviral effect and allowed decreasing the minimum effective doses of the synthetic polyribonucleotide complexes with respect to the above viruses. Combined administration of poly (G) X poly (C) and virazole to mice 1-2 or 24 hours after infection with tick-borne encephalitis virus provided a much more pronounced decrease in the death rate of the animals than the use of the interferonogen alone. Virazole per se was little active and had no significant effect on the intensity of interferonogenesis promoted by the use of poly (G) X poly (C). A possibility of successful therapy of viral infections with polyribonucleotide interferonogens in combination with virazole or other chemotherapeutic drugs with broad antiviral spectrum is discussed.

[Combined antiviral effect of synthetic polyribonucleotide interferonogens and specific antiviral antibodies in arbovirus infections]. / Kombinirovannyi protivovirusnyi éffekt sinteticheskikh poliribonukleotidnykh interferonogenov i spetsificheskikh protivovirusnykh antitel pri arbovirusnykh infektsiiakh.

A model of tick-borne encephalitis in BALB/c mice was used to investigate the protective anti-viral effect of an interferon inducer, poly(G).poly(C), and specific gamma-globulin administered to the animals together or separately in small doses 24 hours before or after virus inoculation. Administration to the animals of poly(G).poly(C) alone or gamma-globulin alone was shown to produce a poor protective effect. Simultaneous administration of both preparations resulted in a significant decrease of mouse mortality after infection. As a result of the pretreatment of chick embryo cell cultures with poly(G).poly(C) before inoculation and the addition of specific immune serum to the agar overlay after the Sindbis virus inoculation, its multiplication was inhibited much more than after treatment of the cells with interferon inducer alone or antibody alone. Possible mechanisms of the observed additive antiviral effects of the interferon inducer and antibody, including those associated with the influence on the virus-induced interferon production, as well as the possibility of their combined use for the prevention and treatment of viral infections are discussed.

[Protective action of double-stranded complexes--interferon inducers in encephalomyocarditis in mice]. / Zashchitnoe deistvie dvunitevykh kompleksov--induktorov interferona pri éntsefalomiokardite myshei.

Interferon inducers, dsRNA and poly (G) X poly (C) ( polyguacyl ), injected into mice intraperitoneally induced interferon formation, the maximal activity being equal to 640-1280 U/ml. Antiviral effect of the inducers was studied upon intraperitoneal and intracerebral injections. Both polyguacyl and dsRNA exerted a marked antiviral action against infection induced by murine encephalomyocarditis virus. The most powerful effect was attained when the inducers were injected 24 or 4 h before challenge. The protective action was produced by polyguacyl and dsRNA even after intracerebral infection. There is a good agreement between the interferon-inducing and antiviral activity of interferon inducers. The data obtained demonstrate the fitness of experimental encephalomyocarditis of mice for testing interferon inducers.

[Immunostimulating effect of interferon inducers]. / Immunostimuliruiushchii éffekt induktorov interferona.

The effect of interferon inducers on the immune response to vaccination was determined. The prophylactic effect of interferon inducers in combination with vaccines was determined with regard to experimental influenza, the therapeutic effect with regard to rabies and tick-borne encephalitis. Despite the differences in the experimental design (administration of the inducers before, after, or together with vaccines), the additive or synergistic effect was regularly observed with a 2-6-fold increase in the level of protection of the animals infected with the appropriate viruses as compared with the use of vaccines or interferon inducers separately. The protective effect depends a lot on the concentration, site and time of inoculation of the preparations, multiplicity of infection, and some other conditions.

[Thermoactivation of poly(G).poly(C): a description of the effect and its hypothetical mechanism]. / Termoaktivatsiia poli(G).poli(Ts): opisanie éffekta i ego predpolagaemyi mekhanizm.

Heating of poly(G).poly(C) complex solutions at a temperature about 100 degrees C was shown to overcome a decrease in the antiviral and interferon-inducing activity of the preparations which were obtained at relatively high concentrations of polynucleotides from poly(G) stored in solution, or were stored frozen themselves. These unfavourable conditions contributed to stabilization of the poly(g) secondary structure and decrease in the degree of regularity of the complex molecules. The results suggest that thermal activation of such poly (G). poly(C) preparations occurred in 2 stages by melting residual free regions of poly(G) and their subsequent interaction with poly(C) with formation of a more regular complex.

Antiviral effects of single-stranded polynucleotide inhibitors of the influenza virion-associated transcriptase against influenza virus infection of hamsters and ferrets.

Administration of a single-stranded polynucleotide copolymer containing 9% cytidine residues and 91% 4-thiouridine residues [poly(C,S4U10)], a known potent inhibitor of the virion transcriptase of influenza viruses, suppressed the amount of virus recoverable from the nasal washes of influenza virus-infected hamsters and ferrets. The incidence of sneezing and nasal discharge in infected ferrets was also reduced. In hamsters, poly(C,S4U10) was more effective than amantadine-HCl or Virazole. Polyinosinic acid in combination with poly-5-hydroxy cytidylic acid also had anti-influenza effects. Poly(C,S4U10) annealed to polyadenylic acid was not effective, nor was the double-stranded polymer (polyinosinic acid) . (polycytidylic acid) even when complexed with carboxymethylcellulose and polylysine. No toxic effects of poly(C,S4U10) were apparent in the treated hamsters and ferrets, and high doses (greater than or equal to 2.86 g/kg) administered intraperitoneally to mice produced no adverse effects.

[Trial of polyguacyl safety and tolerance]. / Ispytanie bezvrednosti i perenosimosti poliguatsila.

The results of studies of the antiviral and interferon-inducing activity of the synthetic interferon inducer polyguacyl in white mice as well as the results of the study of safety and tolerance of this drug given to human subjects as aerosol and intranasally are presented. Both modes of administration to mice induced production of endogenous interferon, although after intranasal inoculation high interferon titres in the blood serum of the animals were observed for longer periods of time, whereas after aerosol administration interferon disappeared more rapidly. Significant antiviral protection was achieved only by the intranasal administration of the inducer resulting in 84.0% survival of the animals challenged with the mouse-adapted influenza A/Aichi virus. Clinical trials of polyguacyl in human volunteers demonstrated the safety and good tolerance of this drug given both as aerosol and intranasally.

[Interferon-inducing and protective action of polyguacyl in an experimental influenza infection]. / Izuchenie interferonindutsiruiushchego i zashchitnogo deistviia poliguatsila pri éksperimental'noi grippoznoi infektsii.

A comparative study of the interferon-inducing and anti-influenza activity of polyguacyl, an interferon inducer, given to human volunteers intranasally and as an aerosol, was carried out. The induction of endogenous interferon in the blood to 100-127 units/ml was observed only after intranasal administration of 5 mg polyguacyl. By both routes of administration the inducer did not diminish the implantation of the virus and development of postvaccination reactions in volunteers to intranasal administration of live influenza A (H1N1) vaccine.

Studies on the mode of action of single-stranded polynucleotides which are antiviral against encephalomyocarditis virus infection of mice.

Poly(dI) and poly(dC) administered separately or sequentially show no antiviral effects against EMC virus infection of mice, whereas poly(rI) and poly(rC) are antiviral in such treatment regimens without evidence of interferon induction. The antiviral effects of poly(rI) and poly(rC) appear to depend on single-strandedness because their antiviral effects are decreased by annealing to poly(dC) and poly(dI) respectively. This decrease in antiviral effect would not seem to be due to an adverse effect of polydeoxyribonucleotides on EMC virus infection because the polydeoxyribonucleotides have no effect on the antiviral activity of another single-stranded RNA, E. coli tRNA.

[Clinical trials with mercapto polycytidylic acid in the treatment of acute childhood leukemia (author's transl)]. / Behandlungsversuche mit Mercaptopolycytidylsäure bei akuten Leukämien des Kindes.

Mercapto Polycytidylic acid (MPC) is a potent inhibitor of oncornaviral reverse transcriptase. The fact that this enzyme has been found in human leukemic cells, has let to the clinical trials of MPC in the treatment of childhood acute leukemia. This report discribes the first pilot clinical trial on 23 patients. The results are encouraging, and have let to a second pilot study including other clinical centers on the clinical efficacy of MPC in the treatment of leukemia.