Oromucosal interferon therapy: marked antiviral and antitumor activity.

Oromucosal administration of murine interferon-alpha/beta (IFN-alpha/beta) or individual recombinant species of murine IFN-alpha, IFN-beta, or IFN-gamma or recombinant human IFN-alpha1-8, which is active in the mouse, exerted a marked antiviral activity in mice challenged systemically with a lethal dose of encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV), or varicella zoster virus (VZV). The effects observed were dose dependent and similar in magnitude to those observed following parenteral administration of the same dose of IFN. No antiviral activity was observed after oromucosal administration of murine IFN-alpha/beta in animals in which the IFN receptor had been inactivated by homologous recombination. In contrast to parenteral treatment, oromucosal IFN therapy was found to be ineffective when IFNs were administered before virus infection. Oromucosal administration of IFN-alpha also exerted a marked antitumor activity in mice injected i.v. with highly malignant Friend erythroleukemia cells or other transplantable tumors, such as L1210 leukemia, which has no known viral etiology, the EL4 tumor, or the highly metastatic B16 melanoma. These results show that high doses of IFN can be administered by the oromucosal route apparently without ill effect, raising the possibility that the oromucosal route will prove to be an effective means of administering high doses of IFN that are clinically effective but poorly tolerated.     

Nucleic acid as interferon inducer. Its antiviral and antitumor activity.

The results of the present study have demonstrated that dsRNA obtained in the system E. coli--bacteriophage is an interferon inducer and possesses antiviral and antitumoral activities.     

Oromucosal interferon therapy: relationship between antiviral activity and viral load.

Intraperitoneal (i.p.) administration of 20,000 IU recombinant murine IFN-alpha (rMuIFN-alpha) was highly effective in protecting mice challenged i.p. with doses of encephalomyocarditis virus (EMCV) ranging from 44 to 440 LD(50) (p<0.001). Oromucosal (o.m.) IFN therapy was also found to be effective in protecting mice challenged with a lethal dose of EMCV. Thus, 40% of animals infected with 44 LD(50) of EMCV and treated o.m. with 20,000 IU rMuIFN-alpha survived infection with a mean survival time of 12.0 +/- 2.46 days relative to a mean of 6.11 +/- 0.38 days in the control group (p<0.05). Oromucosal IFN therapy was found to be ineffective, however, in animals infected with higher doses of EMCV (88-440 LD(50)), even though intraperitoneal administration of the same dose of rMuIFN-alpha resulted in the survival of 90%, 50%, and 60% of animals infected with 88, 220, and 440 LD(50) of EMCV, respectively. These results suggest that oromucosal IFN therapy is effective at relatively low viral load only and that the mechanism of action of oromucosal IFN therapy may be different from that of parenterally administered IFN. Our results suggest that oromucosal IFN therapy may be most effective in chronic viral infections as an alternative to parenterally administered IFN, which is clinically effective but poorly tolerated.     

Interferons in enteroviral heart disease: modulation of cytokine expression and antiviral activity

Interferon (IFN)- has a more than 120-fold higher antiviral activity than the closely related IFN- in human myocardial fibroblasts infected with the cardiotropic enterovirus coxsackievirus B3 (CVB3). CVB3 replication induces interleukin (IL)-6 and IL-8 expression in myocardial fibroblasts, and suppresses the expression of monocyte chemoattractant protein-1 (MCP-1). We investigated whether the higher antiviral activity of IFN- compared to IFN- was a result of a suppression of IL-8 expression by IFN- since previous studies had indicated that IL-8 stimulates enterovirus replication. Human myocardial fibroblasts were treated with either IFN-, IFN- or IFN- (0, 10, 100, or 1,000 IU/ml) and the concentrations of IL-6, IL-8 and MCP-1 were measured in culture supernatants by immunoassays. Both IFN- and IFN- reduced IL-6 and IL-8 expression significantly. In addition, neutralization of IL-8 in culture supernatants of myocardial fibroblasts using a monoclonal antibody demonstrated a significant reduction of CVB3 titers. Antiproliferative effects of all three IFNs were very low (<30% with 1,000 IU/ml), indicating that the suppression IL-6 and IL-8 was not related to cytotoxicity. MCP-1 expression was increased only by high concentrations of IFN- (1,000 IU/ml). By contrast, IFN- had no significant effect on IL-6, IL-8 and MCP-1 expression. In conclusion, suppression of IL-8 expression is an "immuno-modulating" feature of IFN- in human myocardial fibroblasts, which is similar to the activity of IFN-. This feature of IFN- contributes to its high antiviral activity against CVB3 and may be useful in the treatment of enteroviral heart disease.     

Granzyme B: pro-apoptotic, antiviral and antitumor functions

Granzyme B is a caspase-like serine protease that is released by cytotoxic lymphocytes to kill virus-infected and tumor cells. Major recent advances in our understanding of granzyme B biochemistry, biology and function include an appreciation of its uptake into and trafficking within target cells, a thorough dissection of how cell death is triggered, and the identification of the serpin protease inhibitor PI-9, which regulates its function in lymphocytes and in other cells. The roles that granzyme B plays in human pathologies, such as transplant rejection, viral immunity and particularly tumor immune surveillance, remain a topic for vigorous debate and conjecture. The recent discovery of a triply mutated human granzyme B allele, whose product is predicted to possess a reduced capacity to induce cell death, opens the way for major progress in these areas in coming years.     

Macrophage antiviral activity: extrinsic versus intrinsic activity.

Peritoneal exudate cells from strains of mice both resistant and susceptible to challenge with mouse hepatitis virus strain JHM were examined for extrinsic and intrinsic antiviral activity. Thioglycolate-elicited and resident peritoneal cells from uninfected mice were able to suppress viral growth in a permissive cell. The active cell in both populations is an adherent, radiation-resistant, Thy-1.2 antigen- and Ia antigen-negative cell. The suppression of virus replication was not related to nonspecific cellular cytotoxicity directed against the permissive host cell, and no interferon was detected. The expression of extrinsic antiviral activity was not related to the ability of the host to resist mouse hepatitis virus infection by virtue of either age or genetic background. The expression of intrinsic antiviral activity, on the other hand, correlated with the ability of the host to resist virus challenge, indicating a characteristic distinction between these two in vitro mechanisms of macrophage-mediated antiviral activity with regard to host resistance to viral infection. Further, the ability of a macrophage to support viral replication itself was independent of the ability of the macrophage to suppress virus growth in another cell.     

2.8 A resolution crystal structure of human TRAIL, a cytokine with selective antitumor activity.

TRAIL is a newly identified cytokine belonging to the large tumor necrosis factor (TNF) family. TRAIL is a novel molecule inducing apoptosis in a wide variety of tumor cells but not in normal cells. To help in elucidating its biological roles and designing mutants with improved therapeutic potential, we have determined the crystal structure of human TRAIL. The structure reveals that a unique frame insertion of 12-16 amino acids adopts a salient loop structure penetrating into the receptor-binding site. The loop drastically alters the common receptor-binding surface of the TNF family most likely for the specific recognition of cognate partners. A structure-based mutagenesis study demonstrates a critical role of the insertion loop in the cytotoxic activity of TRAIL.     

Pegylation of IFN-alpha and antiviral activity.

The development of pegylated interferons (PEG-IFN) has significantly improved the eradication rates in patients with chronic hepatitis C. Two forms of PEG-IFN have been developed, based on two pegylation chemistries: the 12-kDa linear PEG-IFN-alpha2b and the 40-kDa branched PEG-IFN-alpha2a. We compared the in vitro antiviral activity of linear and branched PEG-IFN using the vesicular stomatitis virus (VSV) cytopathic effect (CPE) reduction assay. The specific antiviral activity of branched PEG-IFN was 7% of that of linear PEG-IFN. A given quantity of linear and of branched PEG-IFN does not represent the same biologic activity. A bioassay could give new insights to compare the pharmacokinetic profile of linear PEG-IFN and of branched PEG-IFN.     

Recent advances in antiviral therapy.

In the early 1980s many institutions in Britain were seriously considering whether there was a need for specialist departments of virology. The arrival of HIV changed that perception and since then virology and antiviral chemotherapy have become two very active areas of bio-medical research. Cloning and sequencing have provided tools to identify viral enzymes and have brought the day of the "designer drug" nearer to reality. At the other end of the spectrum of drug discovery, huge numbers of compounds for screening can now be generated by combinatorial chemistry. The impetus to find drugs effective against HIV has also stimulated research into novel treatments for other virus infections including herpesvirus, respiratory infections, and hepatitis B and C viruses. The need to understand the function of the immune system during HIV infection has brought virologists and immunologists together into new partnerships. The huge increase in activity in antiviral research is reflected in the frequency with which these drugs are now being licensed: in 1985 there were two licensed antiviral drugs for systemic use. Since then approximately 20 compounds have been licensed and more are being submitted to the regulatory authorities on a regular basis.     

The antiviral activity of dipyridamole.

Dipyridamole, a coronary vasodilatator, was found to possess antiviral activity against representatives of different families. The antiviral properties were studied in chick embryo, human diplid and FL cell cultures by the agar diffusion plaque inhibition and plaque reduction tests and one-step growth cycle experiments. The inhibition of the virus-induced cytopathic effect was estimated quantitatively. Dipyridamole significantly inhibited the yield of members of the viral families Picornaviridae, Togaviridae, Orthomyxoviridae, Paramyxoviridae, Herpetoviridae and Poxviridae, as well as of Chlamydiaceae (sheep abortion agent).     

Mucosal immunoadjuvant activity of liposomes: role of alveolar macrophages.

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s-IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasally to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome-mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facilitating local and systemic antibody responses.     

Anti-inflammatory activity of salmeterol: down-regulation of cytokine production.

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a beta 2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1 beta production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-alpha) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50s of approximately 0.1 microM. This inhibition was effectively reversed by the beta 2-antagonist oxprenolol, indicating that the inhibition was mediated through the beta 2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-alpha production, and was not reversed by a beta 2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-alpha inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0.1 mg/kg. This inhibition could be abrogated by dosing orally with the beta-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3 h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-alpha production. Though salmeterol inhibited serum TNF-alpha levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-alpha localized in tissues may not be accurately reflected by serum levels.     

Human recombinant myeloperoxidase antiviral activity on cytomegalovirus.

In vitro incubation of human cytomegalovirus (Towne strain) with 8 U/ml human recombinant myeloperoxidase plus sodium chloride and glucose nearly abolished viral infectivity. To assay the effect on intracellular infection, cell toxicity of the enzymes was first studied. Even the high dose of 16 U/ml of recombinant myeloperoxidase plus 10 mU/ml glucose oxidase did not decrease MRC5 cell growth. By contrast, this dose reduced proliferation of activated THP1 cells. Even half of the myeloperoxidase dose proved slightly toxic to these cells. Non-cytotoxic concentrations of the reagents were used to monitor their effect on cytomegalovirus infection. In MRC5 cells, even the low dose of 4 U/ml myeloperoxidase plus glucose oxidase inhibited synthesis of cytomegalovirus early antigens, as tested by immunofluorescence. Viral release in the supernatant was decreased by 4 logs. In THP1 cells, which produce endogenously hydrogen peroxide, myeloperoxidase alone (8 U/ml) decreased the formation of early and late antigens by 53 and 44%, respectively.     

Bivalphor: a myelopeptide possessing antitumor activity

It is shown that the bone marrow-derived hexapeptide bivalphor restores the functions of human T lymphocytes inhibited by products of HL-60 leukemia cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 119, N ^o 5, pp. 530–532, May, 1995     

Metabolism and antiviral activity of ribavirin

Thirty years after its synthesis, the mechanism of action of ribavirin is still not completely understood. Although much is known about the metabolism and biochemical effects of ribavirin in human cells, there is still much to be learned about the precise mechanism of action of ribavirin with the various viruses. New information about its ability to induce mutations in viral genomes has led to new questions about its mechanism of action. There is considerable evidence that indicates that ribavirin triphosphate (RTP) can interact with the various viral RNA polymerases, and it seems likely that this interaction is important to the mechanism of action of ribavirin. It seems likely that ribavirin will not have one universal mechanism of action, but will inhibit different viruses in different ways. In some cases, inhibition of IMP dehydrogenase may be sufficient for antiviral activity. Whereas, in other cases, inhibition of viral RNA polymerases by RTP may be more important. It is also likely that RTP will interact with the different viral RNA polymerases in different ways leading to different mechanisms of actions. More comprehensive studies are needed that address all aspects of ribavirin metabolism and biochemical actions to gain a thorough understanding of the activity of this agent. Finally, the differences in the metabolism and biochemical actions of ribavirin, selenazofurin, and tiazofurin indicate that small structural changes can have profound effects on biological activity. This observation is well known by investigators familiar with nucleoside analogs, but indicate that one should not assume that agents of similar structure have identical activities.     

Mucosal IL-12 gene delivery inhibits allergic airways disease and restores local antiviral immunity

Allergic asthma strongly correlates with airways inflammation driven by interleukin (IL)-4 and IL-5 secreted by allergen-specific CD4+ T cells. It is possible that over-production of these factors in the lungs may render asthmatic individuals less able to resolve virus infection of the respiratory tract by down-regulating type 1 cytokine-driven immune responses. IL-12 is important for the establishment of cell-mediated immunity (CMI) and may also inhibit responses driven by type 2 cytokine production. Sustained expression of IL-12 in the airways may, therefore, represent an effective preventive treatment or therapy for allergic asthma and any adverse consequences of excessive production of type 2 cytokines for the development of local CMI. Here, we show that allergic responses in airways profoundly inhibit the development of antiviral CMI in mice following local immunization with vaccinia virus (VV) leading to persistent lung infection. However, mucosal gene transfer of IL-12 in the lung, via a VV vector, inhibited local type 2 cytokine production, both prevented the development of allergic disease and airways hyperreactivity in a manner largely dependent on endogenous interferon-γ expression and suppressed established allergic disease, and reversed the suppression of local antiviral CMI responses resulting in rapid resolution of virus infection. Our study provides the first direct demonstration that allergic conditions, particularly in airways, may inhibit immune responses to concomitant virus infection and suggests that transient mucosal IL-12 gene therapy represents an effective approach to both the prevention and treatment of allergic airways disease and associated immunosuppression of CMI.