In Vivo Effect of Innate Immune Response Modulating Impurities on the Skin Milieu Using a Macaque Model: Impact on Product Immunogenicity.

Unwanted immune responses to therapeutic proteins can severely impact their safety and efficacy. Studies show that the presence of trace amounts of host cells and process-related impurities that stimulate pattern recognition receptors (PRR) can cause local inflammation and enhance product immunogenicity. Here we used purified PRR agonists as model impurities to assess the minimal level of individual innate immune response modulating impurities (IIRMIs) that could activate a local immune response. We show that levels of endotoxin as low as 10 pg (0.01 EU), 1 ng for polyinosinic:polycytidylic acid (PolyI:C), 100 ng for synthetic diacylated liopprotein, thiazoloquinolone compound, or muramyl dipeptide, 1 µg for flagellin or ß-glucan, or 5 µg for CpG-oligodeoxynucleotide increased expression of genes linked to innate immune activation and inflammatory processes in the skin of rhesus macaques. Furthermore, spiking studies using rasburicase as a model therapeutic showed that the levels of PRR agonists that induced detectable gene upregulation in the skin were associated with increased immunogenicity for rasburicase. This study underscores the need for testing multiple IIRMIs in biologics, strengthening the connection between the local mRNA induction in skin, innate immune activation, and antibody development in primates, and provides an indication of the levels of IIRMI in therapeutic products that could impact product immunogenicity.

Hippocampal Aß expression, but not phosphorylated tau, predicts cognitive deficits following repeated peripheral poly I:C administration.

Alzheimer's disease is marked by the accumulation of the amyloid-beta (Aß) peptide, and increases in phosphorylation of the microtubule associated protein, tau. Changes in these proteins are considered responsible, in part, for the progressive neuronal degeneration and cognitive deficits seen in AD. We examined the effect of repeated consecutive peripheral poly I:C injections on cognitive deficits, central Aß, and phosphorylated tau accumulation, following three treatment durations: 7, 14, and 21 days. Forty-eight hours after the final injection, animals were trained in a contextual fear-conditioning paradigm, and tested 24h later. Immediately after testing, the hippocampus was collected to quantify Aß and phosphorylated tau accumulation. Results showed that, although poly I:C-induced Aß was significantly elevated at all time points examined, poly I:C only disrupted cognition after 14 and 21 days of administration. Moreover, elevations in phosphorylated tau were not seen until the 14-day time point. Interestingly, phosphorylated tau expression then declined at the 21-day time point. Finally, we demonstrated that Aß levels are a stronger predictor of cognitive dysfunction, explaining 37% of the variance, whereas phosphorylated tau levels only accounted for 0.2%. Taken together, these results support the hypothesis that inflammation-induced elevation in Aß disrupts cognition, independently of phosphorylated tau, and suggest that long-term administration of poly I:C may provide a model to investigate the contribution of long-term inflammation toward the development of Alzheimer's-like pathology.

Schizophrenia associated sensory gating deficits develop after adolescent microglia activation.

Maternal infection during pregnancy is a well-established risk factor for schizophrenia in the adult offspring. Consistently, prenatal Poly(I:C) treatment in mice has been validated to model behavioral and neurodevelopmental abnormalities associated with schizophrenia. By using the Poly(I:C) BALB/c mouse model, we investigated the functional profile of microglia by flow cytometry in relation to progressive behavioral changes from adolescence to adulthood. Prenatal Poly(I:C) treatment induced the expected sensory gating deficits (pre-pulse inhibition (PPI) of the acoustic startle response) in 100day-old adult offspring, but only in female not in male descendants. No PPI-deficits were present in 30day-old adolescent mice. Sensory gating deficits in adult females were preceded by a strong M1-type microglia polarization pattern during puberty as determined by flow cytometric analysis of multiple pro- and anti-inflammatory surface markers. Microglia activation in females did not persist until adulthood and was absent in behaviorally unaffected male descendants. Further, the specific activation pattern of microglia was not mirrored by a similar activation of peripheral immune cells. We conclude that prenatal Poly(I:C) treatment induces post pubertal deficits in sensory gating which are specifically preceded by a pro-inflammatory activation pattern of microglia during puberty.

Involvement of natural killer cells in PolyI:C-induced liver injury.

BACKGROUND/AIMS: The roles of T cells, natural killer T cells (NKT) and macrophages in autoimmune hepatitis have been well documented. However, the roles of natural killer (NK) cells in liver injury remain obscure. Here we examined the effect of Polyinosinic: polycytidylic acid (PolyI:C)-activated NK cells on liver injury. METHODS: Mice were intraperitoneally injected with PolyI:C at a dose of 20mug/g body wt. The percentage and absolute number of NK cells in the liver were analyzed with flow cytometry. Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) assay and H-E staining were used to evaluate the liver injury. RESULTS: Following PolyI:C injection, NK cells accumulation and activation occurred in the liver. Meanwhile, slight elevation of ALT/AST in the serum, mild inflammation and focal necrosis in the liver were also observed. Depletion of NK cells markedly attenuated PolyI:C-induced liver injury. Neutralization of endogenous Interleukin-12 produced by Kupffer cells abrogated the accumulation of NK cells in the liver and subsequent liver injury. The liver injury was also alleviated by neutralization of vascular cell adhesive molecule-1. CONCLUSIONS: These findings suggest that PolyI:C preferentially recruits and activates hepatic NK cells, which may be responsible for the mild hepatitis.

[Effect of non-steroidal anti-inflammatory agents on interferon induction]. / Vliianie nesteroidnykh protivovospalitel'nykh sredstv na induktsiiu interferona.

Nonsteroid antiinflammatory agents (NAIA) such as antipyrine, butadion, acetylsalicylic acid, sodium salicylate, stampyrine and 4-iodantipyrine are not interferonogenic. Still, they stimulated interferonogenic action of poly(G).poly(C) in studies on animals. Relation between the interferon-stimulating action of the NAIA and their effect on activity of prostaglandin and the influence on the immune system was suggested.

[Characteristics of interferon production from the enteral administration of inducers]. / Osobennosti produktsii interferona pri énteral'nom vvedenii induktorov.

When interferon inducers are administered to animals, autonomous local production of interferon by organs (intestine, liver) occurs alongside with the production of serum interferon. After oral administration of a low molecular interferon inducer, tilorone, the intestine is the main site of interferon synthesis. Here the peak of interferon production precedes that in the serum by 20 hours and exceeds it 16-fold. The sequence of events follows the scheme: intestine--liver--serum. Upon oral administration of high molecular inducers incorporated into liposomes (polyguacyl, lafarin) the predominant site of interferon production is the liver where over 80% of the administered inducer is localized and interferon production is 2-4-fold higher than in the intestine. The sequence of events follows the scheme: liver--intestine--serum.

[Interferon induction via peroral administration of high-molecular polynucleotides]. / Induktsiia interferona pri peroral'nym vvedenii vysokomolekuliarnykh polinukleotidov.

High molecular polynucleotides, poly(I).poly(C) and poly(G).poly(C), incorporated into liposomes may be used for serum interferon induction when administered orally. Titres of interferon induced by this method are sufficiently high (320-640 units/ml) and close to those induced by the same preparations administered parenterally (640-1280 units/ml). The use of liposomal polynucleotides results in prolonged (up to 24 hours) circulation of interferon in the blood of mice at a sufficiently high level. Liposomal polynucleotides are low-toxic upon oral intake. The liposome-polynucleotide complex is sufficiently stable on storage, and retains the interferon-inducing activity, if given orally, after 6 months of storage at 4 degrees C in the liquid form.

[Possibilities of overcoming hyporeactivity to interferon induction]. / Vozmozhnosti preodoleniia giporeaktivnosti k induktsii interferona.

The possibilities of overcoming hyporeactivity to interferon production in mice were studied. Hyporeactivity developing in response to the first inoculation of the inducer was shown not to be overcome by changes of the inoculation sites of the same inducer. Choice of the inducer pairs is of primary importance in overcoming the refractoriness. The maximum effect was found with combination of intraperitoneal injections of Tash-3 followed in 3 days by polyguacyl.

[Effect of amphotericin B on the interferonogenic activity of poly(G) . poly(C) and poly(G,I) . poly(C) in mice and their resistance to infection by the tick-borne encephalitis virus]. / Vliianie amfoteritsina B na interferonogennuiu aktivnost' poli(G) poli(Ts), poli(G,I) poli(Ts) v organizme myshei i ikh rezistentnost' k zarazheniiu virusom kleshchevogo éntsefalita.

It was shown that amphotericin B, a polyenic macrolide markedly potentiated in mice the interferonogenic activity of the two-strand synthetic polyribonucleotide complexes, Poly (G) . Poly (C) and Poly (G, I) . Poly (C). At the same time amphotericin B used in high or low doses lowered or somewhat increased respectively the protective effect of Poly (G) . Poly (C) and Poly (G, I) . Poly (C) which was not adequate to the antibiotic effect on their interferonogenic activity. It was found that amphotericin B stimulated in the mice the infection caused by the forest spring encephalitis virus, accelerated the period of its manifestation and increased the death rate. This effect correlated with the concentration of amphotericin B and the dose of the virus. The relationship between the differential effect of amphotericin B on the interferonogenic and antiviral activity of polyribonucleotide interferonogenes and the stimulation of the viral infection by them is discussed.

[Optimization of the schemes for using interferon inducers]. / Optimizatsiia skhem ispol'zovaniia induktorov interferona.

The results of study on 2 Soviet interferon inductors, i.e. the synthetic polyguacyl polynucleotide and natural double-strand phage RNA or dsRNA were studied. It was shown that the time course of accumulation and period of circulation of interferon depended on the route of the inductor administration. The antiviral activity of polyguacyl and dsRNA in experimental influenza and tick-borne encephalitis is described. The maximum protective effect with respect to experimental influenza was observed with intranasal administration of the drugs 4 hours before inoculation. A pronounced protective effect with respect to tick-borne encephalitis was observed with intraperitoneal administration of the inductors or their use in the form of aerosols. Direct correlation between interferon production and the final protective effect was found.

[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.

[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.

[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.

[Laboratory and clinical study of biological activity of poly G-poly C complex]. / Laboratornoe i klinicheskoe issledovanie biologicheskoi aktivnosti kompleksa poli(G).poli(Ts).

Poly(G).poly(C) inoculated intravenously to mice in a dose of 100 microgram induced interferon in the blood in amounts comparable to those induced by poly(I).poly (C). In contrast to rapid accumulation (within 2 hours after induction) and rapid disappearance of interferon in response to poly(I).poly(C) inoculation, the interferon induced by poly(G).poly(C) reached the maximum titer by 6 hours and remained at a high level for 24 hours after inoculation. When given to human volunteers intranasally in a dose of 6 mg, the poly(G).poly(C) complex induced interferon in the blood serum in 70% of the subjects in a titer of 85 units/ml within 24 hours.

Anti-viral activity against encephalomyocarditis virus and Semliki Forest virus and acute toxicity of poly I and poly C administered sequentially to mice.

Mice are protected against lethal intraperitoneal and intravenous infection by encephalomyocarditis virus and Semliki Forest virus by sequential treatment with poly I followed by either polyC or poly5-hydroxyC without production of interferon when the treatments are 4 or more hours apart and by the intraperitoneal or intravenous routes. Maximum protection occurs around 4 hours before infection and is still significant 20 hours after infection. Treatments with combinations of other homoribopolynucleotides were not found to be anti-viral. Protection by sequential polyI, polyC treatment of mice is relatively short-lived and does not 'hypo-reactivate' the protective effect of polyI:C and shows approximately half the protective effect of polyI:C. The toxicity of sequential polyI, polyC treatment is lower than that of polyI:C particularly if poly5-hydroxyC is substituted for polyC. Silica treatment of mice indicates that stationary macrophages are required for protection by polyI followed by polyC but an effect on humoral or cell mediated immune responses does not appear to be involved. The effect appears to be a synergism between the protection conferred by polyI or polyC alone.