MXPA00000430A - Nitric oxide inhibits rhinovirus infection - Google Patents

Abstract

Nitric oxide generating compounds or compounds which induce in situ synthesis of nitric oxide can be used to inhibit rhinovirus infection. Nitric oxide has the ability to inhibit both viral replication as well as the synthesis of cytokines, in particular the proinflammatory cytokines. Thus the symptoms of rhinovirus infections can be ameliorated by treatments to increase nitric oxide in the respiratory tract.

Description

NITRIC OXIDE INHIBITS RINOVIRUS INFECTION This application claims the benefit of the application Serial No. 60 / 052,307, filed on July 11, 1997. This invention was made using the support of the United States government, under National Institutes of Health granting AI377163. Therefore, the government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION This invention relates to the field of virology. More particularly, it relates to the field of human rhinoviruses.

BACKGROUND OF THE INVENTION Rhinovirus infections are the predominant cause of the common cold (18), the acute respiratory disease very often experienced in humans. Recent evidence also implicates rhinovirus infections as an important precipitating factor for exacerbations of asthma (21, 22, 37), chronic bronchitis (35), sinusitis (19.50), and otitis media (3). Despite the high health care costs associated with pnovirus infections, the underlying process through which viral infection leads to symptomatology is poorly understood. The epithelial cell is the primary site of rhinovirus infection (6, 51). In contrast to other respiratory viruses, such as influenza, cytotoxic damage of infected epithelial cells does not seem to play an important role in the pathogenesis of rhinovirus infections, since cytotoxicity is not observed even in infected human epithelial cell cultures (49). nor in the nasal mucosa of infected individuals (53, 54). In view of this, a great emphasis has been placed on the concept that the symptoms may result from the actions of pro-inflammatory mediators that are generated as a consequence of rhinovirus infection. Support for this hypothesis has come from two lines of evidence: 1) studies of subjects with experimentally induced or naturally acquired colds have shown increased levels of several mediators, including kinins (36, 41), IL-1 (40), and IL-6 (55) in nasal secretions during symptomatic rhinovirus infections, and 2) infection of human respiratory epithelial cell populations purified with rhinovirus has been shown to induce the production of pro-inflammatory cytokines, including IL-8, IL-6 and GM-CSF (49.55), which may contribute to the pathogenesis of the disease. To date, however, the specific biochemical events involved in the production of each of these cytokines through rhinoviruses are incompletely understood, and the role of specific cytokines, and other mediators, in the pathogenesis of colds remains to be established. .

COMPENDIUM OF THE INVENTION It is an object of the present invention to provide methods for alleviating symptoms induced by a rhinoviral infection. It is another object of the present invention to provide methods for reducing rhinoviral replication. It is an object of the present invention to provide methods for reducing cytokine production induced by a rhinovirus. Still another object of the invention is to provide a method for classifying the compounds to identify candidate therapeutic or prophylactic agents for rhinoviral infection. These and other objects of the invention are achieved by providing a method for alleviating symptoms induced by a rhinoviral infection, which comprises administering a compound to a human being infected with a rhinovirus. The compound is related to nitric oxide (NO). An amount is administered, which is sufficient to alleviate one or more symptoms associated with the infection. According to another aspect of the invention, a method for reducing rhinoviral replication is provided. The method comprises contacting human respiratory epithelial cells, which are infected by a rhinovirus, with a compound. The compound is one that releases NO A sufficient compound is administered to inhibit rhinovirus referencing According to yet another aspect of the invention, a method for reducing cytokine production induced by a pnovirus is provided. The method comprises contacting human respiratory epithelial cells, which are infected by a rhinovirus, with a compound. The compound is one that releases NO. Sufficient compound is administered to inhibit cytokine production induced by the rhinovirus. Another embodiment of the invention is a method for relieving symptoms induced by a rhinovirus infection. The method comprises administering an effective amount of a compound to a human being infected with a rhinovirus. The compound induces nitric oxide synthase (NOS) in human respiratory epithelial cells, so the symptoms of infection are alleviated. Yet another embodiment of the invention is to provide a method for reducing rhinoviral replication. The method comprises contacting the human respiratory epithelial cells, which are infected by a rhinovirus, with a compound. The compound induces NOS in human respiratory epithelial cells. The compound is administered in an amount effective to inhibit rhinovirus replication. In still another aspect of the invention is a method for reducing cytokine production induced by a rhinovirus. The method comprises contacting human respiratory epithelial cells, which are infected by a rhinovirus, with a compound. The compound is one that induces NOS in human respiratory epithelial cells. An amount that is effective to inhibit cytokine production induced by the rhinovirus is administered. In yet another embodiment of the invention is a method for testing compounds for identifying candidate agents for the prophylactic therapeutic treatment of a common respirator or other disease associated with human rhinoviruses. The method comprises the step of: infecting human respiratory epithelial cells with a human rhinovirus; contacting the cells with a test compound; and measuring the amount of at least one pro-inflammatory or active cytokine in the eosinophil produced by the respiratory epithelial cells, wherein a test compound, which reduces the amount of the cytokine produced, is a candidate agent for therapeutic prophylactic treatment of infection by human rhinovirus. Another aspect of the invention is another method for testing compounds for identifying candidate agents for therapeutic or prophylactic treatment of a common cold or other disease associated with human rhinoviruses. The method comprises the steps of: infecting human respiratory epithelial cells with a human rhinovirus; contacting the cells with a test compound; and measuring the amount of rhinoviral genome replicated in respiratory epithelial cells, wherein a test compound, which reduces the amount of the replicated rhinoviral genome, is a candidate therapeutic or prophylactic agent for prophylactic or therapeutic treatment of human rhinovirus infection.According to another aspect of the invention, a pharmaceutical composition for testing rhinovirus infections is provided. The composition comprises a liquid formulation comprising a compound that releases NO. The liquid formulation is drops for the nose. Another embodiment of the invention is a spray bottle or nose dropper, for administering a pharmaceutical composition to the nose of a human being. The bottle comprises a liquid formulation containing a compound that releases NO. Yet another aspect of the invention is an inhaler or nebulizer for delivering an anti-rhinoviral composition to the respiratory tract of a human being. The device comprises a liquid formulation containing a compound that releases NO. The invention in this manner provides the art with compositions, devices and methods for treating rhinovirus infections, as well as methods for identifying additional candidate therapeutic and prophylactic agents.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Cytokine production from BEAS-2B cells, 24 hours after infection with each variant strain of human rhinovirus (HRV). The data represent +. SEM average from 3 experiments. Figure 1A shows IL-8, while Figure 1B shows the production of IL-6. to «to¡a¿Jafc¿» ag. , -._.

Figure 2: Time course of stable-state mRNA induction and protein for IL-8 (left), and IL-6 (right) of BEAS-2B cells infected with HRV-14. Figures 2A and 2B show Northern stains for each cytokine and for the driving gene, GAPDH. Figures 2C and 2D show densitometric relationships, while Figures 2E and 2F show protein levels produced at each of observed time points. The data is from a representative experiment (n = 3). Figure 3: Time course of induction of stable-state mRNA levels and protein for IL-8 (left) and IL-6 (right) from BEAS-2B cells infected with HRV-16. Figures 3A and B show representative Northern stains for each cytokine and for the driving gene, GAPDH. Figures 3C and 3D show the mean values of +. SEM of densitometric relationships for 4 experiments. Figures 3E and 3F show the mean values of SEM plus protein produced for four experiments at each time point. The asterisks indicate significant increases in each parameter in relation to the zero time control (p <0.05 in each case). Figure 4: Cycloheximide does not alter steady state mRNA levels for IL-8 (left) and IL-6 (right) measured 1 hour after infection with HRV-16. Figures 4A and 4B show a representative Northern stain, while Figures 4A and 4B show the mean values of + _ SEM of densitometric ratios for 4 experiments.

Figure 5: Effects of Budesonide (10 ~ 7 M) on levels of stable-state mRNA and protein for IL-8 (left) and IL-6 (right) from BEAS-2B cells infected with HRN-16. Figures 5A and 5B show representative Northern stains using mRNA extracted 1 hour after injection. Figures 5C and 5D show the mean values of +. SEM of densitometric relationships of 3 experiments. Figures 5E and 5F show the mean values of + SEM of protein produced 7 hours after infection in 3 experiments. Figure 6: Dose-dependent inhibition of cytokine production from BEAS-2B cells infected with HRV-16 through NONOato. Figure 6A shows the mean values of +. SEM of 4 experiments for the production of IL-8 4 hours and 24 hours after infection by HRV-16, while Figure 6B shows the data for IL-6 production. The asterisks indicate significant inhibition compared to the levels produced at the same time after infection in the absence of NONOate (p <0.05, in each case). Figure 7: Dose-dependent inhibition by NONOate of HRV-16 titers in BEAS-2B supernatants recovered 24 hours after viral exposure. The data represent the mean values of + _ SEM of 4 experiments. Asterisks indicate significant inhibition compared to levels produced in the absence of NONOate (p <0.05, in each case) Figure 8: Comparison of the effects of inactive NONOato, and of active NONOato added at different times during the infection procedure, on the production of cytokine induced by HRV-16 from BEAS-2B cells. The NONOate was used at a final concentration of 1000 μM, and protein levels were measured 4 hours after infection. Figure 8A shows the mean values of +. SEM for the production of IL-8 from 3 experiments. Figure 8B shows data for IL-6. The asterisks indicate significant inhibition compared to levels produced only by the virus (p < 0.05, in each case). Figure 9: Effects of NONOate (500 μM) on the levels of Stable-state mRNA and protein for IL-8 (left) and IL-6 (right) at different times after infection with HRV-16. Figures 9A and 9B show representative Northern stains for each cytokine for the driving gene, GAPDH. Figures 9C and 9D show the mean values of +. SEM of densitometric relationships for 3 experiments. Figures 9E and 9F show the mean values of + _ SEM of protein produced for 3 experiments at each time point. The asterisks indicate significant inhibition through NONOate compared to the levels produced only by the virus (p <0.05, in each case). Figure 10: Effects of NONOate (500 μM) on the production of GM-CSF (left) and RANTES (right) from BEAS-2B cells 24 hours after injection with HRV-16. The data represent the mean values of + _ SEM of 4 experiments. Figure 11: HRV infection of cultured primary human bronchial epithelial cells that induces mRNA expression for NOS. The cells were exposed only to the medium (lanes 2 and 4) or to HRV-16 (lanes 3 and 5). The total cellular RNA was extracted and subjected to RT-PCR for NOS at 24 hours (lanes 2 and 3) and 48 hours (lanes 4 and 5). The primers used for this PCR amplify a 500 bp product. Lane 1 contains the DNA ladder to indicate the molecular size.

DETAILED DESCRIPTION It has been discovered that human rhinoviruses induce the production of pro-inflammatory cytokines for human respiratory epithelial cells. In addition, nitric oxide has been found to markedly inhibit rhinovirus replication and virally induced cytokine expression without affecting mRNA levels for the cytokine. The administration of nitric oxide (NO) donors or nitric oxide synthase (NOS) inducers can therefore be used to obtain prophylactic and therapeutic objectives. Since replication and inflammation are affected by nitric oxide, these treatments lead to a shorter duration of infection as well as reduced symptoms. Preferably, amounts of NO donors or NOS inducers are administered to achieve significant inhibition of symptoms, pro-inflammatory cytokine synthesis, eosinophil-active cytokine synthesis, and / or viral replication. Said inhibition is at least 10%, preferably at least 20%, and most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 95%, or 99%. Symptoms that are usually associated with human rhinovirus include the common cold, asthma, sinusitis, otitis media and bronchitis. Any of these or other symptoms may be alleviated or inhibited in accordance with the present invention. Methods for treating in accordance with the present invention include any method by which active compounds can access human respiratory epithelial cells. Such methods include without limitation: topical, through nose drops, or through an inhaler, through an aerosol, through a spray, through gargles, or through washing. Compounds that generate nitric oxide in situ can be used. Such compounds include without limitation nitroglycerin, organic nitrates, linsidomine, molsidomine and N-acetylpenicillamine, 3-morpholinocydinimine (SIN-1), aspirin derivatives that release NO, NOC-18, sodium niropruside, GEA 3162, GEA3175, GEA5171, nicorandil , C873754, N) -naroxen, S-nitrosogestein, S-nitrosoglutathione, FR 144420 and FK409, NOR4, NOC-7, polymers [N (O) NO], pirsidomine, 2,2-diethyl-1-nitrexylhydrazine, and furoxanes . Preferably, the compounds comprise a portion N2027. Most preferably, the compound is 3- (2-hydroxy-2-nitroso-1-propylhydrazimino) -1 -propanamine (a member of the class of NONOates). It is known that said compounds are known for topical application to cardiac tissue in both pastes and patches.

These compounds can be applied to the nose, mouth, throat, bronchi, or any portion of the respiratory system. Intravenous administration can also be used, as well as direct pharmacological injection. Suitable doses will generally be from about 0.01 mg to about 10 mg per application, preferably from 0.1 to 5 mg, and most preferably from 1-3 mg. The compounds that can be used as NOS inducers include any that is known in the art. These include without limitation, interferon, TNF-a, IL-1β, and bacterial lipopolysaccharide. The devices for delivering the compositions and compounds to the respiratory or ear tract according to the methods of the present invention can be any that are conventionally used in the art for such purposes. These include inhalers, nebulizers, bottles of nose drops, droppers. Any formulation that is suitable to be delivered to the nose, mouth, throat, bronchi, lungs, ears and / or breasts, may be adequate. The above description generally describes the present invention. A more complete understanding can be had by referring to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLES Current studies were taken to further delineate the kinetics and mechanisms of cytokine generation induced by rhinoviruses through epithelial cells, and to evaluate the effects of potential therapeutic interventions on these trajectories. It focused on the viral production of IL-8 and IL-6, since these cytokines are produced in relatively large amounts after infection by rhinoviruses, and since they have biological properties that are of interest with respect to the pathogenesis of colds. IL-8 is a potent chemoattractant for, and activator of, neutrophils (5) and also has chemotactic activity for lymphocytes (28), the two predominant cell types in the nasal mucosa during rhinovirus infections (29, 54). IL-6 is not only capable of stimulate T cell activation, induce B cell differentiation and antibody production (1), but also stimulate mucosal IgA immune responses (42). In terms of potential interventions, two aspects have been used. Based on the broad scale anti-inflammatory and immunomodulatory effects of the glucocorticoids (44), including their ability to inhibit the production of several cytokines in a variety of cell types (45), have examined the effects of the potent glucocorticoid, budesonide, on rhinovirus infection in epithelial cells. As a novel alternative aspect, the research has also been investigated ability of a nitric oxide donor to inhibit replication aja & s ^ iMl. viral and virally induced cytokine production in epithelial cells. Studies have shown that nitric oxide vasodilator (NO) can exert modulatory effects on inflammation (39), and it has been shown that nitric oxide has antiviral effects in some animal models (7, 12, 20, 24, 32), but this property has not been examined in human respiratory epithelial cells. The studies herein show that budesonide modestly inhibits the generation of cytokine induced by rhinovirus without affecting viral replication. In contrast, rust Nitric oxide significantly inhibits rhinovirus-induced cytokine generation as well as viral replication and may play an important role in the therapeutic aspect of rhinovirus infections.

EXAMPLE 1 15 Effects of the cell passage: Preliminary studies indicate that there is a marked effect on the repeated cell passage in the production of cytokine from BEAS-2B cells. Although the data obtained were always qualitatively identical for each In the passage, there was a progressive effect of cell passage on absolute levels of cytokines produced. For example, in 4 experiments performed an identical protocol with consecutive cell passages was used, the production of IL-8 was reduced from 7690 pg / ml to 3090 pg / ml. For this reason, each type of experiment performed continuation was always carried out in matching experiments "Using the same cell passages Comparison of effects of several strains of rhinoviruses on cytokine production from BEAS-2B cells: The effects on cytokine production of equal infection doses of 4 different rhinovirus strains were compared in cultures of BEAS-2B cells Three of the strains used, types 14, 16 and 39, are members of the main group of rhinoviruses that use the intercellular adhesion molecule -1 (ICAM-1) as their receptor, while the type 1A is a member of the minor independent group of ICAM-1 All strains induced the production of IL-8 and IL-6 measured 24 hours after infection (Figure 10) .With all viral strains, the levels generated from IL- 8 were approximately 10 times more than for IL-6 Materials: The following reagents were purchased: Dulbecco Minimum Essential Medium (DMEM), Eagle Minimum Essential Medium (EMEM), Ham Medium F-12, HBSS, L-glutamine , penicillin / streptomycin / fungisone , trace elements, and retinoic acid (Biofluids, Rockville, MD); hydrocortisone, epithelial cell growth factor and epithelial cell growth supplement (Collaborative Research, Bedford, MA); fetal bovine serum (Gemini Bio Products, Inc. Calabasa, CA); transferrin and insulin (GIBCO BRI, Grand Island, NY); 3- (2-hydroxy-2-nitroso-1-propyrazine) -1 -propanamine (NONOate) from Cayman Chemical Company (Ann Arbor, Ml); RNAzol ™ B (Tel-Test, Inc., Friendswood, TX); agarose (FMC Bioproducts, Rockland, ME), Mops (Biohringer, < ", -, £ -, i.

Mannheim, Indianapolis, IN), and 32P-dCTP (Amersham, Arlington Heights, IL). Other chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Budesonide was generously provided by vectors Drs. Per Anderson and Ralph Brattsand (Astra Pharmaceutical Production, Lund, Sweden). The following supply pH regulators were used: 10X Mops (0.2 M Mops, 0.05 M sodium acetate, 0.01 MEDTA); 50X Denhardr's (1% ficoll, 1% poly in ilpirro lidina, 1% bovine serum albumin); and 20X SSPE (175.3 g (NaCl, 27.6 g NaH2PO4.H2O, 7.4 g EDTA in 1 liter of H2 =, pH 7.4) Virus and Cell Lines: Human rhinovirus types 14 (HRV-14), 16 (HRV) -16), 39 (HRV-39) and 1A (HRV-1A), WI-38 cells and HeLa cells were purchased from the American Type Culture Collection (Rockville, MD), additional viral supplies were generated for HRV-14 and HRV- 16 through the passage in HeLa or WI-38 cells, respectively, as previously described (49) It was not possible to generate equivalent supplies of these two viral strains using the same host cell line, since the two strains exhibited marked preferences In terms of ability to infect and replicate in these cell lines, this variable sensitivity of host cells to different strains of rhinovirus has been previously documented (11). For some experiments, HRV-16 was purified to remove ribosomes and soluble factors from WI-38 origin by centrifugation through draw bear, according to published methods (16) Inactivation of HRV-16 was performed through UV exposure for 30 minutes as previously described (49). For the experiments using HRV-39 and 1A, viral supplies were used directly as obtained from the supplier. The supply of HRV-39 provided was prepared in WI-38 cells, while the HRV-1A supply obtained was generated in HeLa cells. The BEAS-2B cell line (43) was generously provided by Dr. Curtis Harris (National Cancer Institute Bethesda, MD). Epithelial Cell Culture: Primary human tracheal epithelial cells were obtained through digestion of human tissue protease as previously described (10). Both primary cells and BEAS-2B cells were grown in the culture medium consisting of Ham's F-12 nutrient medium with penicillin (100 U / ml), streptomycin (100 U / ml), fungizone (250 ng / ml), L-glutamine (2 mM), phosphoethanolamine / ethanolamine (0.5 mM), transferrin (10 μg / ml), epithelial cell growth supplement (3.75 μ / ml), epidermal growth factor (12.5 ng / ml), insulin ( 5 μg / ml), hydrocortisone (10"7 M), cholera toxin (10 ng / ml), 3,375-triodothyronine (3 x 10" 9 M), retinoic acid (0.1 ng / ml), and trace elements. This medium is hereinafter referred to as F12 / 10X. The cells were incubated at 37 ° C in an atmosphere of 95% air and 5% CO2. For the experiments, cells were plated between passages 35 and 50 on 6-well plates or 75 cm2 flasks (Costar, Cambridge, MA) at a density of 2.5 × 10 4 cells / cm 2.

Viral infection of BEAS-2B cells: washed 3 times with HBSS monolayers of BEAS-2B cells (confluent 70-80%). Rhinovirus (strain 14, 16, 39 or 1A) was added to the cells at a concentration of 104 TCID56 units / ml HBSS. This is equal to an infection dose of 0.01 TCID50 units / BEAS-2B cell, although it is not clear that this is represented in terms of multiplicity of infection (MOI-infectious units per cell) for BEAS-2B cells, since the capacity of the rhinovirus to infect different host cells is quite variable (see above). The cells were incubated with the virus at 34 ° C for 1 hour, washed 3 times with F12 / 10X, and then a fresh medium of F12 / 10X was added to the cells. Supernatants were removed from the cells at various times after infection and stored at -80 ° C for further analysis of cytokine protein production and viral content. In some experiments, the total cellular RNA was extracted from the cells at various times after infection and stored at -80 ° C for further analysis. Quantification of IL-8 and IL-6: Cytokine levels in cell supernatants were determined using specific ELISAs. Measurements of IL-8 were performed using the previously described ELISA analysis sensitive to 30 pg / ml of cytokine (49), while the levels of IL-6 were analyzed using commercial equipment sensitive to 15 pg of IL-6 / ml (Biosource International, Camarillo, CA) Neither the culture medium nor the vehicles for the drugs used in the experiments caused some non-specific interference effects in any of the trials. Statistical Analysis: The data are expressed as the + _ SEM average. Comparisons of the kinetics of RNA expression, protein secretion and viral titers were made using a one-way ANOVA analysis. The effects of cycloheximide and glucocorticoid on RNA expression and protein secretion were compared using the Student's test for even samples. Comparisons of the effects of NONOate on cytokine production, viral titers, and RNA expression were made through two-way ANOVA analysis with a repeated measurement, except for the experiments comparing the active and inactive NONOato that were analyzed through one-way ANOVA. When significant variant relationships were obtained, pairwise comparisons of the means were made with the Multiple Significant Difference Multiple scale test (47). The differences were considered significant for p <values; 0.05.

EXAMPLE 2 Kinetics of protein secretion cytokine mRNA expression: Figure 2 demonstrates that mRNA for IL-8 and IL-6 was significantly elevated within 1 hour after infection by HRV-14. Maximal expression occurred at 3 hours, but mRNA levels remained higher than non-infected controls 24 hours after infection. The mRNA induction was followed by significant elevations of the IL-8 and IL-6 proteins in the supernatants. The increased production of cytokine occurred at 3 hours after infection, and reached maximum concentrations at 24 hours. Interestingly, the time course of production of IL-8 and IL-6 after infection by HRV-16 was faster than that observed for HRV-14 (Figure 3). Maximal mRNA expression occurred one hour after infection and maximum protein production occurred at 7 hours. According to the data shown above (Figure 1), the magnitude of cytokine generation was approximately 4 times higher after infection by HRV-16 than the response after infection of HRV-14 (Figure 3 vs. Figure 2) . Since HRV-16 produced a more rapid and robust production of cytokines and is the strain that will be used for subsequent in vivo studies, subsequent experiments were performed on the mechanism of virus-induced cytokine generation using HRV-16. To confirm the specific character of the effects of HRV-16, 3 coincident experiments were performed comparing the generation of IL-8 through active viral preparations and inactivated with UV. The active virus generated 3307 ± 1156 pg / ml of IL-8, whereas the virus inactivated by UV produced only 520 ± 90 pg / ml (uninfected, control cells produced 345 ± 110 pg / ml). The specific character was further confirmed by demonstrating that similar amounts of IL-8 were generated, in matching experiments, when the cells were infected with the standard viral preparation or with an equal infection dose of the same purified HRV-16 supply material through of sucrose density centrifugation (2080 ± 340 pg / ml and 1750 ± 500 pg / ml, respectively, n = 3). The time course of the viral induction of IL-8 and IL-6 was also identical for the standard and purified preparations of HRV-16 (not shown).

EXAMPLE 3 Viral titers after HRV-16 infection: The supernatants were collected at various times after infection were analyzed for viral titers in the WI-38 cell cytotoxicity assay for HRV-16. The virus was detected in the culture medium starting approximately 7 hours after infection and progressively increased between 7 and 24 hours after infection (Table 1). The supernatants were collected during a second period of 24 hours after infection and contained virus levels similar to those seen 24 hours later (see Table 2 below). This pattern of viral titers is virtually identical to that previously observed with HRV-14 (39). 7? TABLE 1 Viral Content of Cell Supernatants BEAS-2B a Different Times After Infection with Human Rhinovirus-16 Time After Viral Titration Infection (hours) (Units of Registry TCID50) * 0 ND 1 ND 3 ND 7 1 25 ± 025 16 2 25 ± 0 25 24 24 ± 0 13 The data represent ± SEM average of 4 experiments TABLE 2 Effects of NONOate on Reductions of Viral Titrations with Time Viral Titration (TCID50 Units of Registry) * Treatment 0-24 h 24-48 h HRV-16 2 8 + 0 1 2 9 + 0 1 HRV-16 + 300 μM NONOate 25 + 03 2 8 ± 0 1 HRV-16 + 1000 μM NONOate 0 5 + 0 5 * 24 + 0 2 * The data represent ± SEM average for 3 experiments * p < 0 05 vs HRV-16 only EXAMPLE 4 Effects of cycloheximide on the expression of IL-8 and IL-6 mRNA: The mRNA levels for IL-8 and IL-6 of HRV-16-infected cells treated with cycloheximide (10 μg / ml) were not different from the infected control cells (Figure 4). Comparisons were made 1 hour after the viral infection, the time of peak mRNA expression in the kinetic studies described above. Effect of Cicioheximide on the expression of IL-8 and IL-6 mRNA induced by HRV-16: BEAS-2B cells were treated with cycloheximide (10 μg / ml), or control medium, 1 hour after viral infection. The drug was present during and after infection with HRV-16. One hour after infection, the RNA was harvested for Northern analysis. This concentration of cycloheximide was used since previously it was shown to inhibit TNFa and the expression induced by IFNg of RANTES mRNA in this cell line (48).

EXAMPLE 5 Effect of glucocorticoid pretreatment on cytokine production and viral titers: Cells were treated with 107 M budesonide or vehicle control 24 hours before viral infection. Comparisons of RNA expression and protein production were made at the times of maximum response as determined in the kinetic experiments described above; 1 hour for mRNA and 7 hours for cytokine protein production. Expression of IL-8 and IL-6 mRNA in BEAS-2B cells infected with HRV-16 was not significantly altered by budesonide (Figure 5). In any experiment, however, the production of the IL-8 and IL-6 proteins from BEAS-2B cells treated with budesonide was lower than that of the control infected cells (p <0.05 for the comparison in pairs of standardized data). Viral titers (2.2 ± 0.6 TCID50 record units) were not altered by exposure to budesonide. Effect of budesonide on cytokine production and viral replication: Budesonide was prepared as a 10 ~ 2 M supply solution in DMSO. Since the BEAS-2B cells were usually maintained in a growth medium containing low levels of hydrocortisone, the cells for these experiments were placed in a hydrocortisone medium for 24 hours before the glucocorticoid treatment. The cells were then treated with 10"7 M budesonide or vehicle control appropriately diluted for 24 hours before the viral infection.The budesonide was again included in the medium after the viral infection.The concentration of budesonide used was selected. since previously it was shown to inhibit maximally the production of RANTES induced by TNFa from BEAS-2B cells (48) .Supernatants from cells with and without budesonide were removed at different times after viral infection and stored at -70 ° C for the ., ^ feg¿- determination of the content of IL-8 and IL-6 and viral protein. In some experiments, Northern analysis was used to compare the RNA extracted 1 hour after infection of the budesonide treated cells with that extracted from the control infected cells.

EXAMPLE 6 Effect of a nitric oxide donor on cytokine production and viral titers: Supernatants were collected at 4 and 24 hours after infection with HRV-16 of BEAS-2B cells incubated in the absence or presence of NONOate and analyzed for viral content and cytokine levels. The NONOate significantly inhibited the production of IL-8 and IL-6 in a dose-dependent manner (Figure 6). The production of IL-6 was significantly inhibited through doses of NONOate as low as 100 μM. The levels of cytokine generated were more inhibited at 4 hours than at 24 hours, presumably due to the levels of NO decrease at 24 hours. The viral titers were also significantly inhibited through NONOato (Figure 7). The viral content in the supernatant collected at 24 hours was almost completely eliminated through 1000 μM of NONOato. Supernatants from a second 24-hour collection, however, contained similar amounts of virus, whether the cells were treated with NONOate or not (Table 2). Parallel studies, the effects of NONOato on the epithelial cell viability and cell numbers were analyzed. There was no significant effect of NONOate on cell viability at any dose or time. There was a small, but important, reduction in the number of cells with 1000 μM of NONOato only at 24 hours (1.7 ± 0.4 x 106 cells / cavity without NONOato against 1.2 ± 0.4 x 106 cells / cavity with NONOato, n = 3, p <0.05). No effect was observed at lower doses of NONOate. The effects of NONOato were also confirmed using purified HRV-16 (not shown). The inhibitory effects of NONOato were not limited to infection by HRV-16. Cytokines produced from BEAS-2B cells infected with another main strain, HRV-14, or a minor strain, HRV-1A, were also significantly inhibited through NONOato. In the presence of 500 μM of NONOato, the production of IL-8 induced by virus in BEAS-2B cells was inhibited by approximately 60% at 4 hours (350 ± 51 to 117 ± 59 pg / ml for HRV-14). and 1857 ± 58 to 670 ± 64 pg / ml for HRV-1A, n = 3, p <; 0.01). In addition, NONOato inhibited viral titers in supernatants collected from BEAS-2B cells 24 hours after infection with HRV-14 (data not shown). The ability of NONOate to inhibit cytokine production inhibited by rhinovirus was also observed in primary human cells. In one experiment, 1000 μM of NONOato reduced actuainduced levels of IL-8, measured 4 hours after infection, from 1400 pg / ml to 366 pg / ml, whereas, in a second experiment, IL-8 was reduced from levels of 3420 pg / ml in cells virainfected at 1980 pg / ml and 1030 pg / ml in cells treated with 500 μM and 1000 μNONOato, respectively To further examine the effects of NONOato, additional experiments were conducted where the NONOato was added only during or after the viral infection. Figure 8 demonstrates that NONOate present only during virus challenge, or only after infection by the virus, inhibited the production of IL-8 and IL-6 by 50-60%. Complete inhibition of protein production was observed if the NONOato was present as well as during after viral exposure. To determine if the inhibition observed was specificadue to nitric oxide, experiments were conducted with active NONOato and with NONOato that released all the available NO. Figure 8 shows that the inactive compound did not inhibit the production of IL-8. Effect of NONOate on cytokine production and viral replication: The NONOato was prepared in an alkaline solution (0.01 M NaOH as a 100 mM supply solution, the which was kept at 4 ° C until used.New NONOate supply solutions were prepared for each experiment and were used after 1 hour of preparation.The defined half-life of the NO release from NONOate is 76 minutes at a time. pH 7.4 and 22 ° C (Cayman Chemical Co., Ann Arbor, Ml.) Under alkaline conditions, NONOate does not release nitric oxide. i _.- ft.jaas. directly aliquots of the alkaline supply solution to the BEAS-2B culture medium (pH 7.4) in a final concentration scale of 100 μM to 1000 μM. For most experiments, NONOate was present both during and after exposure to the virus. In some experiments, NONOate was added only during exposure to the virus or only after exposure to the virus. Supernatants from BEAS-2B cells incubated with or without NONOate were removed at various times after viral infection and stored at -70 ° C for subsequent determination of IL-8 and IL-6 protein and viral content. In some cases, the RNA extracted at various times after infection of the cells treated with NONOato and the infected control cells was compared through Northern analysis. To control the non-specific effects of the NONOate compound, an experiment was performed in which the cells were treated with an inactive solution of NONOato. Inactivation was achieved by placing a 1000 μM solution of NONOate in a medium at a pH of 1.4 at room temperature for 24 hours to allow the NONOate to release all available NO before adding it to the cell cultures. Kinetics and mechanism of NONOate inhibition of IL-6 and IL-8 production: To examine the time course of inhibition by NONOato, BEAS-2B cells were studied in the presence and absence of 500 μM of NONOate at various times after infection with HRV 16. The inhibitory effect of NONOato was more j8aü. ^, a_, < yi¿ »pronounced at the first points of time with a 60-70% reduction in protein levels at 1 hour, 50% at 3 hours, and 30-40% at 7 hours (Figure 9). These results probably reflect the declining concentration of nitric oxide in the medium as the NONOate degrades. Interestingly, NONOate did not alter cytokine levels in mRNA expression. As shown in Figure 9, mRNA levels for BEAS-2B cells infected with HRV-16 in the presence or absence of NONOato were not significantly different. There was a tendency during the 3 hours and 7 hours for the IL-8 mRNA that was higher in the cells treated with NONOato. In additional control studies, NONOato alone had no effect on the expression of mRNA for IL-8 or IL-6, nor did the inactive NONOato alter the virally induced expression of mRNA for IL-8 or IL-6 in BEAS- cells. 2B (data not shown). Probes for Northern Staining: A full-length cDNA for IL-8 was obtained through a reverse transcription polymerase chain reaction (RT-PCR) using RNA extracted from the human BEAS-2B cell line. The full-length cDNA was cloned into a pCRI1 vector (Invitrogen Corp., San Diego, CA) between two EcoRI sites grown in competent E. coli XL1-blue cells (Stratagene, La Jolla, CA). The sequence of the cDNA probe used for Northern analysis was identical to the published sequence of IL-8 (31, 34) through di-deoxy sequencing. A full-length cDNA for IL-6 was provided by Dr. Steven Gillis (Immunex, Seattle, WA). The full-length cDNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Clontech (Palo Alto, CA). Probes for IL-8, IL-6 and GAP were labeled for a highly specific activity through the random primer method (15) 5 using 32P dCTP and a random primer DNA labeling kit (Boehringer Mannheim, Indianapolis, IN ). Unincorporated nucleotides were separated using Nuctrap Push columns (Stratagene). RNA extraction and Northern analysis: RNA was extracted cell-cell total BEAS-2B using RNAzol B (1 ml / 10 cm2) in a modification of the Chomczynski and Sacchi method (9). Briefly, cell monolayers were used with RNAzol B and transferred to a 13 ml polypropylene tube to which chloroform (0.1 ml / 1 ml RNAzol) was added. After cooling on ice for 5 minutes, the samples were centrifuged at 7900 x g for 30 minutes at 4 ° C. The aqueous phase was precipitated with an equal volume of 95% ethanol cooled with ice at -20 ° C overnight. After repeated centrifugation, the RNA pellet was washed twice in 75% ethanol, dried and dissolved in 50 μl of water treated with 0.2% diethylpyrocarbonate. The integrity of each RNA was analyzed by electrophoresis in an aliquot (0.5 μg) on a 1% agarose gel with a pH regulator of 0.5 μg ethidium bromide / ml. The RNA was stored at -80 ° C. For Northern analysis, equal amounts were electrophoresed (15-20 μg) of RNA of each experimental condition in a gel of L2 8 8??? Ja ja ja agar agar agar 1% / 2.2 M formaldehyde agarose in a Mops pH regulator system. The RNA was transferred to a nylon membrane (GeneScreen Plus, New England Nuclear Research Products, Wilmington, DE). The membranes were intertwined through exposure to ultraviolet light and then prehybridized in 10 ml of pH buffer containing 4.5 ml of formamide, 2.5 ml of 10X Denhardt's, 2 ml of 20X SSPE, 1 ml of 20% SDS, and 100 μg of denatured salmon sperm DNA / ml in a hybridization oven for 2 hours at 42 ° C. Immediately after prehybridization, the appropriate a22P labeled cDNA probe was added to the prehybridization solution and the mixture was rotated for a further 18 hours at 42 ° C. The stains were washed at a final severe concentration of 0.2 x SSC / 0.2% SDS at 60 ° C and exposed to a film (Biomax MS, Kodak, New Haven, CT) using Lightening Plus Screens at -70 ° C. The films were revealed as done routinely for variable times to ensure that the band intensities analyzed by the densitometry were within the linear scale for the film. Densitometry was performed using a scanning densitometer (UVP gel documentation system, San Gabriel, CA) and a densitometric analysis was performed using NIH Image software.

EXAMPLE 7 This example demonstrates the effect of NONOate on active cytokines with eosinophil in rhinoviral infection.

Since increased eosinophils in the lower respiratory tracts play an important role in asthma, the effects of NONOate on the virally induced production of cytokines that affect eosinophil function were examined. These cytokines include the macrophage and granulocyte colony stimulation factor (GM-CSF), which promotes survival and improves the activation of eosinophils, and RANTES, which is a potent chemotactic factor for eosinophils, memory T lymphocytes, monocytes and basophils. Figure 10 shows the protein levels for GM-CSF and RANTES produced by epithelial cells in the presence and absence of NONOato, 24 hours after infection by rhinovirus (HRV-16). The addition of NONOato significantly inhibited the viral induction of both cytokines , suggesting that nitric oxide plays an important role in the regulation of cytokine production of eosinophilic activation, during virally induced asthma attacks. It has been hypothesized that nitric oxide is an important part of the host antiviral response to rhinovirus. In recent studies, it has been examined whether rhinovirus infection of epithelial cells alters the expression of the inductive nitric oxide synthase (iNOS) gene, the enzyme that produces nitric oxide. Using RT-PCR, ¡NOS gene expression has been analyzed in RNA isolated from primary human bronchial epithelial cells uninfected and infected with HRV-16 at 24 and 48 hours. As shown in Figure 11, the viral infection induced mRNA expression for NOS both 24 and 48 hours after viral infection. These data support the concept that expression of the NOS gene is induced as part of the host response to viral infection. It has previously been shown that HRV-14 induces the production of IL-8 and IL-6 from BEAS-2B cells (49), and now it is shown that other major group strains (HRV-16 and HRV-39), and type 1A of the minor group all share stability, suggesting that the induction of pro-inflammatory cytokines can occur with many, if not all , the rhinoviruses. It has already been shown that the production of cytokine via HRV-14 can be blocked both through ICAM-1 antibodies and through the UV inactivation of the virus (49). These studies not only showed that the effects of HRV-16 can be abolished through UV inactivation, but also that a purified preparation of HRV-16 induces cytokine production. Together, these data indicate that the induction of cytokine specifically it is due to the virus, and not to some contaminant of the viral supply solutions. In addition, the common nature of this response implies that the induction of epithelial cell cytokine production plays an important role in the pathogenesis of upper respiratory viral infections in humans, a concept that is further supported by the fact that other viruses, such as influenza virus and respiratory syncytial virus (RSV) also induces epithelial cytokine production before occasions manifest cytotoxicity (2, 8, 33, 38). The importance of the differences in levels of cytokine production across each strain is difficult to interpret due to the titration of viral strains that are determined in several different cell lines and may not be exactly comparable. However, it is clear that the kinetics of cytokine mRNA expression and protein secretion vary among strains of rhinoviruses. Infection with HRV-14 leads to a time-dependent accumulation of mRNA for IL-8 and IL-6, with levels observed being maximum at 3 hours after infection and remained elevated 24 hours after infection. Consistent with the previous report (49), protein production for each cytokine was increased up to 24 hours after infection, but production during a second 24-hour period was not different from uninfected control cells (not shown). This time course of protein production was similar to that observed with this viral strain in A549 type II epithelial cells (55), although the time course for mRNA accumulation differs somewhat, presumably reflecting differences of the two cell populations . Interestingly, the time course of mRNA expression and cytokine production was faster, and the magnitude of the response was greater, for cells infected with HRV-16 than with HRV-14. Not only were the levels of mRNA and protein that were achieved faster, but more passengers by nature, being essentially complete in 7 hours. As for HRV-14, cytokine production during a second 24 hour period after infection with HRV-16 was not different from the control infected cells (not shown). The reasons for the difference in initial production rates of IL-8 and IL-6 through HRV-14 and HRV-16 are unknown, but may be related to a difference in recognition, consumption or discovery of the two viral strains in cells BEAS-2B. Despite the different rates of cytokine production, no differences were observed in viral replication rates between the two strains. In each case, the virus was detected in the supernatants of BEAS-2B cells 7 hours after infection and reached maximum levels at 24 hours. A second collection at 24 hours, produced titers similar to the first 24-hour sample, preventing viral proliferation and release to the culture medium occurred at a constant speed. The transient induction of IL-8 and IL-6 through both viral strains in the binding of continuous viral replication suggests that a primary event in the viral infection, and not the same viral replication, stimulates the production of proinflammatory cytokines. This rapid production of cytokines raises the speculation that this relatively primary event in the pathogenesis of colds may be important in initiating rapid inflammatory cell infiltration. To further elucidate the biochemical mechanisms of virus-induced cytokine generation, the effects of the selected drug on virus-induced expression of mRNA and protein for cytokines were examined. The protein synthesis inhibitor, cycloheximide, did not alter mRNA levels for IL-8 or IL-6, again suggesting that protein synthesis was not required for the expression of rhinovirus-induced mRNA. This is consistent with recent observations in A549 cells, indicating that the induction of IL-6 through HRV-14 occurs through a pathway dependent on the nuclear factor kB that is not affected by cycloheximide (55). It has been shown that glucocorticosteroids inhibit the production of several cytokines in patients with allergic inflammatory diseases (46, 52), as well as the cell culture system (45, 48). For the first time, the effects of a potent glucocorticoid on viral replication and mRNA expression induced by IL-8 and IL-6 and protein production in rhinovirus-infected epithelial cells were evaluated. Budesonide had no effect on mRNA expression for any cytokine, but caused a modest inhibition of secreted protein levels. This reduction in protein secretion in the absence of changes in mRNA levels may reflect an ability of glucocorticoids to alter the post-transcriptional events involved in the production or secretion of cytokine protein. Previously it was reported that glucocorticoids, at best, modestly inhibit the production of mRNA and protein, IL-8, from cultured epithelial cells exposed to cytokines (27, 30), but it has been reported that dexamethasone inhibits RNA production and protein, IL-6 induced by TNFa from BEAS-2B cells (30). The lack of the effect of budesonide in viral titers, and the modest inhibition of IL-8 and IL-6 secretion, are consistent with, however, in vivo studies of experimental rhinovirus infections where glucocorticoids have little or no effect effect on viral spreading and symptoms (14, 17). It is now clear that nitric oxide can not exert a wide range of actions, serving as a vasodilator, neurotransmitter, antimicrobial and immune regulator (39). In recent years, it has also been shown that NO has antiviral properties in murine cell lines and in a mouse model in vivo. The replication of several viruses, including vaccine virus (20), herpes simplex-1 (12, 24), vesicular stomatitis virus (7), Coxsackie virus (32), and poliovirus (26) was inhibited through the induction of nitric oxide synthase, the enzyme that generates NO, or through the addition of the NO donor, S-nitroso-1-acetyl penicillamine. Since NO levels are increased in the exhaled air of humans with upper respiratory viral infections (25), it was examined whether nitric oxide can inhibit rhinovirus replication, and these extended studies evaluated the effects of NO on the production induced by rhinovirus. rhinovirus of IL-6 and IL-8. Although it has been shown that normal human respiratory epithelial cells express both constitutive and inducible forms of NO (4) synthase, the expression of these enzymes is markedly reduced in the BEAS-2B cell line (data not shown). For this reason, and to ensure a controlled level of nitric oxide exposure, NONOato was used, a donor that releases nitric oxide with a defined half-life. The data show that, for the first time, nitric oxide can inhibit both rhinovirus replication and the production of IL-8 and IL-6 induced by rhinovirus in human respiratory epithelial cells. These effects were dose dependent and occurred in the absence of any effect on epithelial cell viability. Inhibition of cytokine production was more pronounced 4 hours after injection than 24 hours after injection, while viral diffusion from epithelial cells was also recovered to normal levels during a second 24-hour collection period. These data are consistent with the ability of NONOate to cause an inhibition only when it is capable of releasing significant amounts of NO, and indicate that both viral replication and cytokine production are summarized as the compound degrades. Additional support is provided for the key to NO release through data that the inactivated NONOate had no effect on viral titration or cytokine production. The ability of NONOate to cause partial inhibition of cytokine production. when present only after the period of viral infection suggests that the NONOato is not inhibiting directly by killing the virus, or by inhibiting the virus from entering the BEAS-2B cells. This is also supported by the ability of the viral titers to recover after the degradation of NONOato. Rather, it seems likely that nitric oxide inhibits one or more of the primary events in the process of viral infection. The failure of NONOate to inhibit the printing of cytokine mRNA at any point of time examined suggests that nitric oxide may be functioning through a post-transcriptional mechanism, but other studies are needed to confirm this. However, there is the ability of NO to inhibit protein synthesis in other cell types (13, 23). In summary, it has been shown that multiple strains of rhinoviruses induce the production of pro-inflammatory cytokines from human respiratory epithelial cells, but that there are variations in terms of the levels and kinetics of cytokine production through different strains. Although glucocorticoids modestly inhibit cytokine secretion induced by rhinovirus infection, it does not alter the expression of cytokine mRNA or viral replication. In contrast, nitric oxide markedly inhibits rhinovirus replication and virally induced cytokine expression, without affecting mRNA levels for this cytokine. Although other studies are necessary to clarify the mechanisms through which NO inhibits viral replication and cytokine production, the present data indicate that topical application of nitric oxide donors can provide a novel therapeutic aspect for the treatment. of colds induced by pnovirus and its complications. ..--.-._ REFERENCES 1. Akiro, S., T. Hirano, T. Taga, and T. Kishimoto. 1990. Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and 5 TNF). FASEB J. 4: 2860-2867. 2. Arnold, R., B. Humbert, H. Werchau, H. Gallati, and W. Konig. 1994. lnterleukin-8, interleukin-6, and soluble tumor receptor necrosis type I relay from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus. Immunology 82: 126-133. 3. Aróla, M., T. Ziegler, H. Puhakka, O. P. Lehtonen, and O. Ruuskanen. 1990. Rhinovirus in otitis media with effusion. Ann. Otol. 15 Rhinol. & Laryngol. 99: 451-4534. Asano, K, C. B. E. Chee, B. Gaston, C. M. Lilly, C. Gerard, J. M. Drazen, and J. S. Stamler. 1994. Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc. Nati Acad. Sci. USA-91: 10089-10093.

S. Baggiolini, M., A. Walz, and S. L. Kunkel. 1989. Neutrophil-activating peptide-1 / interleukin 8, a novel cytokine that activates neutrophils. J Clin. Invest. 84: 1045-1049 25 6. Bardin, P. G., S. L Johnston, G. Sanderson, S. Robinson, M. A. Pickett, D. J. Fraenkel, and S. T. Holgate. 1994. Detection of rhinovirus infection of the nasal mucosa by oligonucleotide in situ hybridization. Am. J. Respir. Cell Mol. Biol. 10: 207-213. 7. Bi, Z., and C. S. Reiss. 1995. Inhibition of vesicular stomatitis virus infection by nitric oxide. J. Virol. 69: 2208-2213. 8. Choi, A.M. K., and D.B. Jacoby. 1992. Influenza virus A infection induces interleukin-8 gene expression in human airway epithelial cells. FEBS Lett. 309: 327-329. 9. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolate by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159.

. Churchill, L., F. Chilton, J. H. Resau, R. Bascom, W. C. Hubbard, and D. Proud. 1989. Cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells. Am. Rev. Respir. Dis. 140: 449-459. 11. Couch, R. B. 1996. Rhinoviruses, pp. 713-734. In B. N. Fields and D. M. Knipe and P. M Howley (ed.), Fields Virology, 3rd edition Lippincott-Raven, Philadelphia. 12. Croen, KD 1993. Evidence for an antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication. j. Clin. Invest. 91: 2446-2452. 13. Curran, R. D., F. K. Ferrari, P. H. Kispert, J. Stadler, D. J. Stuehr, R. L. Simmons, and T. X Billiar. 1991. Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis. FASEB J. 5: 2085-2092. 14. Farr, B. M., J. M. Gwaitney, Jr., J. O. Hendley, F. G. Hayden, R. M. Naclerio, T. McBride, W. J. Doyle, J. V. Sorrentino, D. K. Riker, and D. Proud. 1990. A randomized controlled trial of glucocorticoid prophylaxis against experimental rhinovirus infection. J. Infecí. Dis. 162: 1173-1177. 15 15. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. 16. Gern, J. E., R. Vrtis, E. A. B. Kelly, E. C. Dick, and W. W.

Busse. 1996. Rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism. J. Immunol. 157: 1605-1612. 17. Gustafson, L M., D. Proud, J. O. Hendley, F. G. Hayden, and J. 25 M. Gwaitney, Jr. 1996. Oral prednisone therapy in experimental TIT2te_ *. rhinovirus infections. J. Allergy Clin. Immunol. 97: 1009-1014. 18. Gwaitney, J. M., J. O. Hendley, G. Simon, and W. S. Jordan. 1966. Rhinovirus infections in an industrial population. I. The occurrence of illness. N. Engl. J. Med. 275: 1261-1268. 19. Gwaitney, J. M., Jr., C, D. Philips, R. D. Miller, and D. K. Riker. 1994. Computed tomographic study of the common cold. N. Engl. J. Med. 330: 25-30.

. Harris, N., R.M. L, Buller, and G. Karupiah. 1995. Gamma interferon-induced, nitric oxide-mediated inhibition of vaccinia virus replication. J. Virol. 69: 910-915. 21. Johnston, S.L., P.K. Pattemore, G. Sanderson, S. Smith, M.J. Campbell, L., K. Josephs, A. Cunningham, B. S. Robinson, S.H. Myint, M.E. Ward, D.A.J. Tyrrell, and S. T. Holgate. 1996. The relatioship between upper respiratory infections and hospital admissions for asthma: a time-trend analysis. Am. J. Respir. Crit. Care Med. 154: 654-660. 22. Johnston, S. U, P. Pattemore, G. Sanderson, S. Smith, F. Lampe, L. Josephs, P. Sympington, S. O'Toole, S. H. Myint, D. A. Tyrrell, and S. T. Holgate. 1995. Community study of the role of viral infections in exacerbations of asthma in 9-11 year old children Br.

Med. J. 310: 1225-1228. 23. Karupiah, G., and N. Harris. 1995. Inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites. J. Exp. Med. 181: 2171-2179. 24. Karupiah, G., Q.-w. Xie, R. M. L. Buller, C. Nathan, C. Duarte, and J. D. MacMicking. 1994. Inhibition of viral replication by interferon-g-induced nitric oxide synthase. Science. 261: 1445-1448. 10 25. Kharitonov, S. A., D. Yates, and P. J. Barnes. 1995. Increased nitric oxide in exhaled air of normal human subjects with upper respiratory tract infections. Eur. Respir. J. 8: 295-297. . 26. Komatsu, T., Z. Bi, and C. S. Reiss. 1996. Interferon-g induced type 1 nitric oxide synthase activity inhibits viral replication in neurons. J. Neuroimmunol. 68: 101-108. 27. Kwon, 0. J., B. T. Au, P. D. Collins, J. N. Baraniuk, L M. 20 Adcock, K F. Chung, and P. J. Barnes. 1994. Inhibition of? Nterleukin-8 by dexamethasone in human cultured airway epithelial cells. Immunology 81: 389-394. 28. Larsen, C. G., A. 0. Anderson, E. Appella, J. J. Oppenheim, and 25 K Matsushima. 1989. The neutrophil activating protein (NAP-1) is also chemotactic for T lymphocytes. Science. 243: 1464-1466. 29. Levandowski, R.A., C.W. Weaver, and G.G. Jackson. 1988. Nasal secretion leukocyte populations determined by flow cytometry 5 during acute rhinovirus infection. J. Med. Virol. 25: 423-432.

. Levine, S. J., P. Larivée, C. Logun, C. W. Angus, and J. H. Shelhamer. 1993. Corticosteroids differentially regulate secretion of IL-6, IL-8, and G-CSF by a human epithelial cell line. Am. J. Physiol. 10 265: L360-L368. 31. Lindley, L, H. Aschauer, J. M. Seifert, C. Lam, W. Brunowsky, E. Kownatskj, M. Thelen, P. Peveri, B. Dewald, V. von Tscharner, A. Walz, and M. Baggiolini. 1988. Synthesis and expression in 15 Escherichia coli of the gene encoding monocyte-derived neutrophil-activating factor: biological equivalence between natural and recombinant neutrophil-activating factor. Proc. Nati Acad. Sci. USA. 85: 9199-9203. 32 Lowenstein, C.J., S.L. Hill, A. Lafond-Walker, J. Wu, G. Alien, M. Landavere, N.L. Rose, and A. Herskowitz. 1996. Nitric oxide inhibits viral replication in murine myocarditis. J. Clin. Invest. 97.1837-1843. 33. Matsukura, S, F Kokubo, H. Noda, H. Tokunaga, and M. 25 Adachi. 1996. Expression of IL-6, EL-8, and RANTES on human bronchial epithelial cells < ? | # CI-H292, induced by influenza virus A. J. Allergy Clin. Immunol. 98: 1080-1087. 34. Matsushima, K., K. Morishita, T. Yoshimura, S. Lavu, Y. Kobayashi, W. Lew, E. Appella, H. F. Kung, E. J. Leonard, and J. J. Oppenheim. 1988. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF by interleukin 1 and tumor necrosis factor. J. Exp. Med. 167: 1883-1893.

. McHardy, V. U., J. M. Inglis, M. A. Calder, J. W. Crofton, L. Gregg, D. A. Ryland, P. Taylor, M. Chadwick D. Coombs, and R. W. Riddell. 1980. A study of infectious and other factors in exacerbations of chronic bronchitis. Br. J. Dis. Chest. 74: 228-238. 36. Naclerio, R.M., D. Proud, M. M. Lichtenstein, A. Kagey-Sobotka, J. O. Hendley, J. Sorrentino, and J. M. Gwaitney. 1988. Kinins are generated during experimental rhinovirus colds. J. Infecí. Dis. 157: 133-142. 37. Nicholson, K.G., J. Kent, and D.C. Ireland. 1993. Respiratory viruses and exacerbations of asthma in adulls. Br. Med. J. 307: 982-986. 38. Noah, T. L, and S. Becker. 1993. Respiraíory syncylial virus-induced cytokine production by a human bronchial epithelial __ ._ m ^ ^ cell line. Am. J. Physiol. 265: L472-L478. 39. Nussler, A. K., and T. R. Billiar. 1993. Infltion, immunoregulation, and inducible nilric oxide synlhase. J. Leukocyie 5 Biol. 54: 171-178. 40. Proud, D., J. M. Gwaitney, Jr., J. O. Hendley, C. A. Dinarello, S. Gillis, and R. P. Schleimer. 1994. Increased levéis of interleukin-1 are detected in nasal secretions of volunteers during experimental rhinovirus colds. J. Infecí. Dis. 169: 1007-1013. 41. Proud, D., R. M. Naclerio, J. M. Gwallney, and J. O. Rendley. 1990. Kinins are generated in nasal secretions during naïve rhinovirus colds. J. Infecí. Dis. 161: 120-123. 15 42. Ramsay, A.J., A.J. Husband, L.A. Ramshaw, S. Bao, K.I. Maílhaei, G. Koehler, and M. Kopf. 1994. The role of nterleukin-6 in mucosal IgA antibody responses in vivo. Science. 264: 561-563. 43. Reddel, R. R., Y. Ke, B. I. Gerwin, M. G. McMenamin, J. F. Lechner, R. T. Su, D. E. Brash, J.-B. Park, J. S. Rhim, and C. C. Harris. 1988. Transformation of human bronchial epithelial cells by infection, SV40 or adenovirus-12 SV40 hybrid virus, or transfection via serum phosphate coprecipilation, wilh to plasmid conisaining SV40 early region genes. Cancer Res. 48: 1904-1909. i «E-« lÍTIFÍÍÍÍÍCICI-í-MlM 44. Schleimer, R. P. 1993. Glucocorlicosleroids: the mechanism of action and use in allergic diseases, p. pp. 893-925. In E. Middlelon and C. E. Reed and E. F. Ellis and N. F. Adkinson, Jr. and J. W. Yunginger and W. W. Busse (ed.), Allergy: Principies and Praclice, 4th. ed. Mosby, St. Loius. 45. Schwiebert, L. A., L. Beck, C. Slellaio, C. A. Bickel, B. S. Bochner, and R. P. Schleimer. 1996. Glucocoríicosíeroid inhibilion of 10 cyclokine production; relevance ío anliallergic acíions. J. Allergy Clin. Immunol. 97: 143-152. 46. Sim, T. C, L M. Reece, K A. Hilsmeier, J. A. Grani, and R. Alam. 1995. Secretion of chemokines and oiher cylokines in 15 allergen-induced nasal responses: inhibilion by lopical steroid íreatmenl. Am. J. Respir. Crit. Care Med. 152: 927-933. 47. Sleel, R. G. D., and J. H. Torrie. 1980. Principies and Procedures of Stalistics, a Biometrical Approach, 2nd ed. 20 McGraw-Hill, New York. 48. Stellalo, C, L. A. Beck G. A. Gorgone, D. Proud, T. J. Schall, S. J. Ono, L. M. Lichinenstein, and P. P. Schleimer. 1995. Expression of the chemokine RANTES by a human bronchial epilhelial cell line. 25 Modulalion by cyíokines and glucocorlicoids. J Immunol 155: 410-418. 49. Subausle, M.C., D.B. Jacoby, S.M. Richards, and D. Proud. 1995. Infection of a human respiratory epithelial cell line with rhinoviruses. Induction of cytokine relay and modulation of susceplibilily to infection by cytokine exposure. J. Clin. Invesl. 96: 549-557. 50. Turner, B. W., W. S. Cail, J. O. Hendley, F. G. Hayden, W. J. l. Doyle, J. V. Sorrenino, and J. M. Gwaitney, Jr. 1992. Physiologic abnormalities of the paranasal sinus during experimental rhinovirus colds. J. Allergy Clin. Immunol. 90: 474-478. 51. Turner, R. B., J. O. Hendley, and J. M. Gwailney, Jr. 1982. 15 Shedding of infected epithelial cells in rhinoviruses colds. J. Infect. Dis. 145: 849-853. 52. Wang, J. H., C. J. Trigg, J. L. Devalia, S. Jordan, and R. J Davies. 1994. Effecl of inhaled beclomelhasone dipropionale on 20 expression of proinflamniaíory cylokines and aclivaled eosinophils in íhe bronchial epilhelium of patienís wilh mild aslhma. J. Allergy Clin Immunol. 94: 1025-1034. < t? j &sünay? = a¡¿ Mrf. - -ft? * J ^ át «£ &C ._» ^ 53. Winther, B., S. Brofeldt, B. Chrislensen, and N. Mygind 1984. Light and scanning electron microscopy of nasal biopsy material from palienís wilh nafurally acquired common colds. Acia. Ololaryngol. (Stockh). 97: 309-318. 54. Winther, B., B. Farr, R. B. Turner, J. O. Hendley, J. M. Gwaitney, Jr., and N. Mygind. 1984. Histopayhologic examination and enumeration of polymorphonuclear leukocyles in the nasal mucosa during experimental rhinovirus colds. Acia. Oiolaryngol. Suppl. (Slockh) 413: 19-24. 55. Zhu, Z., W. Tang, A. Ray, Y. Wu, O. Einarsson, M. Landry, J. M. Gwailney, Jr., and J. A. Elias. 1996. Rhinovirus simulation of interleukin-6 in vivo and in vilro. Evidence for nuclear factor kB-dependent transcriptional activation. J. Clin. Invest. 97: 421-430.

Claims (42)

1. - A method for alleviating the synapses induced by a rhinoviral infection comprising: administering an effective amount of a compound to a human being infected with a rhinovirus, wherein the compound releases nitric oxide (NO).

2. The method according to claim 1, wherein the rhinovirus induces common colds.

3. The method according to claim 1, wherein the rhinovirus induces asthma.

4. The method according to claim 1, wherein the rhinovirus induces sinusitis.

5. The method according to claim 1, wherein the rhinovirus induces otitis media.

6. The method according to claim 1, wherein the rhinovirus induces bronchitis.

7. The method according to claim 1, wherein the compound releases NO in a conrolled form.

8. The method according to claim 1, wherein the compound releases NO at a palphysiological pH.

9. The method according to claim 1, wherein the compound comprises a portion N2O2. "

10. The method according to claim 1, wherein the compound is 3- (2-hydroxy-2-nitroso). -1-prop? Lhydrazino) -1 -

z s? s? s? á &?, s ^^^^^^ i ^ ra! ^ em ^^^ 7M? M7a7: 'j ^' AA.

propanamine.

11. The method according to claim 1, wherein the compound is administered through drops for the nose.

12. The method according to claim 1, wherein the compound is administered topically.

13. The method according to claim 1, wherein the compound is administered through an inhalant.

14. The method according to claim 1, wherein the compound is administered in a spray.

15. A method for reducing viral replication, comprising: contacting human respiratory epithelial cells that are infected by a rhinovirus, with a compound that releases NO in an amount effective to inhibit rhinovirus replication.

16. A method for reducing cytokine production induced by a rhinovirus comprising: contacting the human respiratory epithelial cells, which are infected by a rhinovirus, with a compound that releases NO in an effective amount to inhibit the production from

20 cytokine induced by the rhinovirus.

17. The method according to claim 16, wherein the cytokine is a pro-inflammatory cytokine.

18. The method according to claim 16, wherein the cytokine is interleukin-8.

19. The method according to claim 16, wherein

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^

20. The method according to claim 15, wherein the composition comprises a portion of N2O27

21. The method according to claim 15, wherein the compound is 3- (2-hydroxy-2-nitroso-1) -propylhydrazino) -1 -propanamine.

22. The method according to claim 16, wherein the compound comprises a portion N2O27

23. The method according to claim 16, wherein the compound is 3- (2-hydroxy-2-nitroso-1-) propylhydrazino) -1 -propanamine.

24. The method according to claim 1, wherein the compound is S-nitroso-1-acetyl penicillamine.

25. The method according to claim 15, wherein the compound is S-nitroso-1-acetyl penicillamine.

26. The method according to claim 16, wherein the compound is S-nitroso-1-acetyl penicillamine.

27. A method for alleviating the symptoms induced by a rhinoviral infection, comprising: administering an effective amount of a compound to a human being infected with a rhinovirus, wherein the compound induces nitric oxide synthase (NOS) in respiratory epithelial cells human 28.- A method to reduce rhinoviral replication that includes:

n ~? i «¿^ ._......« * ^ * «^ - put in the human respiratory cells epithelial cells, which are infected by a rhinovirus, with a compound that induces NOS in the epileliales cells human respiratory in an amount effective to inhibit rhinovirus replication.

29 - A method to reduce cytokine production induced by a rhinovirus, which comprises: counted human respiratory epithelial cells, which are infected by a rhinovirus, with a compound that induces NOS in human respiratory epithelial cells in an amount effective to inhibit the production of cytokine induced by rhinovirus.

30. The method according to claim 27, wherein the compound is interferon.

31. The method according to claim 28, wherein the compound is interferon y.

32. The method according to claim 29, wherein the compound is inlerferon ?.

33. The method according to claim 27, wherein the compound is lipopolysaccharide (LPS).

34. The method according to claim 28, wherein the compound is lipopolysaccharide (LPS).

35. The method according to claim 29, wherein the compound is lipopolysaccharide (LPS).

36.- A method for testing compounds to identify candidate agents for the prophylactic therapeutic treatment of a

common cold or other disease associated with human rhinovirus, which comprises the steps of: infecting human respiratory epithelial cells with a human rhinovirus; counting the cells with a test compound; and measuring the amount of at least one proinflammatory or eosinophilic cytokine produced by respiratory epithelial cells, wherein a test compound, which reduces the amount of the cytokine produced, is a candidate agent for the prophylactic treatment of idiopathic the infection by human rhinovirus.

37. The method according to claim 36, wherein the cytokine is a pro-inflammatory cytokine.

38. The method according to claim 36, wherein the cytokine is an active cytokine by eosinophil.

39.- The method according to claim 36, wherein the step of contacting is done before the step of infecting.

40.- A method for testing the compounds to identify candidate agents for the idiopathic or prophylactic trafficking of a common cold or other disease associated with human pnovirus, which comprises the steps of: infecting human respiratory epilemal cells with a human rhinovirus, putting into contact the cells with a test compound, and measure the canineity of the pnoviral genome replicated in the cells

Respiratory epilelials, wherein a test compound that reduces the quality of the replicated rhinoviral genome, is a candidate therapeutic or prophylactic agent for the prophylactic or therapeutic treatment of human rhinovirus infection.

41. The method according to claim 40, wherein the step of counting is performed before the infection step.

42. - A pharmaceutical composition for bringing rhinovirus infections, comprising: a liquid formulation comprising a compound that releases NO, wherein the liquid formulation is golas for the nose. 43.- A spray bottle or nose drops for administering a pharmaceutical composition to the nose of a human being, comprising a liquid formulation comprising a compound that releases NO. 44.- An inhaler or nebulizer to supply an ani-rinoviral composition to the respiratory fluid of a human being, comprising a liquid formulation comprising a compound that releases NO.