Animal models to assess the effects of air pollutants on allergic lung disease.

Animal models provide toxicologists with useful tools for assessing risks associated with respiratory allergy. Both the mouse and BN rat models described exhibit many of the features of human allergic asthma. It is clear that environmental contaminants can exacerbate the expression of these features. Work is under way to explore underlying mechanisms and to develop methods for applying these data to human health risk assessment.

Enhanced allergic responses to house dust mite by oral exposure to carbaryl in rats.

Epidemiological studies have demonstrated an association between use of carbamate insecticides, including carbaryl, and increased incidence of allergic asthma in farmers. In this study the effect of oral carbaryl exposure on the development of allergic responses to house dust mite (HDM) was examined in female Brown Norway rats. Rats were gavaged for 2 weeks with 0, 2, 10, or 50 mg/kg/day of carbaryl. They were sensitized with a subcutaneous injection of HDM in aluminum hydroxide adjuvant 3 days after the beginning of carbaryl exposure and challenged with antigen via the trachea 1 day after the final carbaryl ingestion. Two days after challenge, antigen-specific cell proliferation in pulmonary lymph nodes was significantly higher in the 50 mg/kg group than in controls, while antigen-specific splenocyte proliferation was decreased in groups dosed with 2, 10, and 50 mg/kg carbaryl. Total protein and lymphocyte number in bronchoalveolar lavage (BAL) fluid were also increased in the 50 mg/kg group. By 7 days after challenge, immune-mediated pulmonary inflammation (eosinophils), antigen-specific immunoglobulin (Ig) E level in serum, and antigen-specific IgE and IgA levels in BAL fluid were significantly elevated in the 50 mg/kg group. No apparent change was observed for lactate dehydrogenase and eosinophil peroxidase in BAL fluid, while the number of BAL macrophages were decreased in groups dosed with 10 and 50 mg/kg carbaryl. The results suggest that carbaryl may cause systemic immune suppression, while enhancing pulmonary allergic responses to house dust mite antigen.

Suppression of allergic immune responses to house dust mite (HDM) in rats exposed to 2,3,7,8-TCDD.

Exposure to various xenobiotics, including oxidant gases, diesel exhaust, and certain pesticides, has been reported to exacerbate pulmonary allergic hypersensitivity responses. Increased lymphocyte proliferative responses to parasite antigens or increased antibody responses to sheep erythrocyte have also been reported in rats exposed to TCDD before infection or immunization. As a result, these studies were conducted to test the hypothesis that TCDD exposure exacerbates the allergic response to house dust mite antigen. Brown Norway rats were injected, ip, with 0, 1, 10, or 30 microg TCDD/kg 7 days before intratracheal (it) sensitization to semipurified house dust mite allergen (HDM). Fourteen days later, rats were challenged with HDM and immediate bronchospasm was measured. At this time point, plus 2 and 7 days later, inflammatory cells in bronchoalveolar lavage fluid (BALF), HDM-specific IgE levels in serum, and HDM-driven cell proliferation in bronchial lymph nodes and spleen were evaluated. TCDD exposure decreased both immediate bronchoconstriction and specific IgE synthesis after the HDM challenge; 7 days later, HDM-specific IgE responses remained suppressed. Total serum IgE levels were similar in all groups. HDM challenge alone significantly increased cellular and biochemical indicators of lung injury, both of which were suppressed by TCDD exposure. The proliferative response of lymph node cells, but not of spleen cells, to HDM was also suppressed at the highest TCDD dose, although the splenic response to Concanavalin A was elevated. It appears that early events in the response to HDM are affected by TCDD exposure, since message for IL5 was dramatically reduced 2 days after sensitization, but not after challenge. We therefore conclude that TCDD exposure suppressed, rather than enhanced the development of allergic immune responses and the expression of immune-mediated lung disease.

Improvement of respiratory syncytial virus replication in actively growing HEp-2 cells.

The growth of respiratory syncytial (RS) and parainfluenza type III (PI3) viruses has been studied in actively growing versus relatively stationary HEp-2 cells. There was no effect on PI3 virus growth. RS virus synthesis was stimulated from 20- to 60-fold by growth in actively growing cultures when the cell density was approximately 1/3 that of a confluent culture. This stimulation was manifested by a greater yield of virus per cell, more virus-specific mRNA produced per cell, and a 50% decrease in the time before cytopathic changes appeared.

A simple method for increased recovery of purified paramyxovirus virions.

A simple method involving vigorous agitation of infected cell monolayers prior to collection of culture medium is described to greatly increase the recovery of purified virions of measles virus, respiratory syncytial virus and human parainfluenza virus. Vigorous agitation of the flasks containing monolayers of infected cells increased the recovery of purified virions by at least 3- to 10-fold as judged by the intensity of [35S]methionine labeled viral proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). These protein profiles also indicated that these virions were as clean as those purified from gently collected medium. Analysis of titers of infectious virus recovered from medium of agitated and non-agitated flasks showed similar increases. These results suggest that the cell associated nature of these viruses may be at least partly due to either partially budded virions or mature virions sticking to the cell membrane, since these both might be expected to be freed from the cell by mechanical shearing.

Enhanced Allergic Sensitization in Animals Exposed to Particulate Air Pollution.

Epidemiological studies have found an association between elevated levels of particulate matter (PM) air pollution and increased medication use and hospital visits by asthmatics. While it is known that asthmatics are generally more sensitive to airborne contaminants such as sulfur dioxide and tobacco smoke, it is difficult to test which components of air pollution may also contribute to the induction of pulmonary allergy (sensitization) because of the risk in creating disease. Recent studies in mice and rats, however, have demonstrated that pulmonary exposure to combustion particles such as diesel and residual oil fly ash (ROFA) can exacerbate immunological sensitization (in the form of immunoglobulin E antibody and lymphocyte reactivity) to experimental and natural allergens. Subsequent allergen challenge in these animals results in a greater allergen-induced bronchoconstriction, elevated numbers of eosinophils in the lung, and enhanced airway responsiveness to cholinergic agents compared to what occurs in similarly immunized animals pretreated with vehicle or "inert" particles. Although the mechanisms for these effects are not known, it has been demonstrated that the adjuvant effects of diesel and ROFA can be reproduced with hydrocarbons and soluble transition metals from diesel and ROFA, respectively. In addition, analysis of mediator expression and release over the sensitization phase has revealed that PM exposure can enhance production of Th2 cytokines such as interleukin-5 (IL-5) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). These experimental systems demonstrate the potential of particulate air pollutants to enhance allergic sensitization and can be further used to elucidate the mechanism for these effects.

Enhanced allergic sensitization by residual oil fly ash particles is mediated by soluble metal constituents.

Epidemiological studies have demonstrated an association between elevated levels of particulate matter (PM) air pollutants and exacerbation of asthma symptoms. We have shown in a Brown Norway (BN) rat model of house dust mite (HDM) allergy that preexposure to residual oil fly ash (ROFA) particles enhanced the sensitization phase such that the secondary immune response and associated lung injury were increased after allergen challenge. To determine whether the metals present in ROFA mediated this effect, BN rats were intratracheally instilled with either ROFA (1000 microg) or acidified saline + NiSO(4) (105.12 microg), VSO(4) (98.2 microg), FeSO(4) (58.49 microg), or a mixture (Mix) of each metal. HDM-specific IgE was higher in the serum of the ROFA, Ni, V, and Mix groups than in the HDM group after challenge, and antigen-induced bronchoconstriction responses were increased in the Ni group. Lymphocyte proliferation to antigen was increased in the ROFA, Ni, and V groups compared to controls. Total protein and eosinophil peroxidase levels were elevated in the Fe group, and eosinophil numbers in the bronchoalveolar lavage fluid (BALF) were increased in the ROFA and Fe groups compared to HDM control. IL-5 and IL-13 mRNA expression was also increased in the lung tissue of all metal and ROFA-treated groups, while BALF IL-10 was elevated in the Fe and Mix groups, and IL-6 and TNF-alpha were elevated in the metal and ROFA-treated groups compared to controls. These results suggest that ROFA's metallic constituents mediate enhancement of sensitization to HDM and that pulmonary inflammation may play a role in this adjuvant effect.

Residual oil fly ash exposure enhances allergic sensitization to house dust mite.

Epidemiological studies have shown an association between elevated levels of particulate matter air pollution and increased morbidity and hospital visits in asthmatics. Residual oil fly ash (ROFA) is a primary combustion particle containing sulfate and metals such as vanadium, nickel, and iron. In this study the effect of ROFA on sensitization to house dust mite (HDM) was examined in a Brown Norway rat model of pulmonary allergy. Rats were instilled via the trachea with 200 or 1000 micrograms ROFA 3 days prior to local sensitization with 10 micrograms HDM and were challenged with 10 micrograms HDM 14 days later. Immunological endpoints were examined at 2, 7, and 14 days after sensitization and at 2 and 7 days after challenge (16 and 21 days post-sensitization, respectively). Antigen-specific immunoglobulin E and associated immediate bronchoconstriction responses to antigen challenge were increased in the ROFA-treated groups compared with the HDM control group. Lymphocyte proliferation to antigen was enhanced at Days 7 and 21 in the bronchial lymphocytes of ROFA-treated groups. Bronchoalveolar lavage fluid (BALF) eosinophil numbers and lactate dehydrogenase were significantly increased in the 1000 micrograms ROFA group at Days 2 and 16, BALF total proteins were elevated at Days 2 and 7 in both ROFA-treated groups, and BALF interleukin (IL)-10 was elevated in the 1000 micrograms ROFA group at Day 2. These results suggest that ROFA has an adjuvant effect on sensitization to HDM.

TNF-alpha enhanced allergic sensitization to house dust mite in brown Norway rats.

We have recently demonstrated that pulmonary exposure to residual oil fly ash (ROFA) resulted in enhanced sensitization to house dust mite (HDM) and augmented the development of allergic lung disease after allergen challenge. This effect was associated with increased tumor necrosis factor alpha (TNF-alpha), a macrophage- and epithelial cell-derived cytokine that promotes granulocyte migration to the lung. The present study examined whether exogenous administration of TNF-alpha enhances sensitization to HDM. One day prior to pulmonary sensitization with 10 microg HDM (5 microg each on days 1 and 3), female Brown Norway rats were instilled via the trachea with either 2.0 microg recombinant rat TNF-alpha, 2.0 microg bovine serum albumin (BSA), or 1,000 microg ROFA, and were challenged with 10 microg HDM 14 days later. Antigen-induced immediate bronchoconstriction responses, antigen-specific immunoglobulin E (IgE) titers, lymphocyte proliferation, (cytokines (TNF-alpha and interleukin [IL]-13), and eosinophils were elevated in rats treated with ROFA or TNF-alpha compared with BSA-treated controls after HDM challenge. Intratracheal administration of anti-TNF-alpha monoclonal antibody during ROFA exposure did not reduce ROFA-enhanced lymphocyte proliferation or IgE titers, but had a trend for reduced pulmonary inflammation. This study demonstrates that TNF-alpha has similar adjuvant activity as ROFA, but other factors may fulfill this function when TNF-alpha activity is blocked.

Transfer of allergic airway responses with serum and lymphocytes from rats sensitized to dust mite.

House dust mite (HDM) antigen is one of the most common allergens associated with extrinsic asthma. In a model of allergic lung disease, Brown Norway (BN) rats sensitized to HDM with alum and Bordetella pertussis adjuvants produce high levels of IgE antibody and experience bronchoconstriction, increased airway hyperresponsiveness (AHR) to acetylcholine (ACh), and pulmonary inflammation after antigen challenge. The purpose of this study was to determine whether these asthmatic symptoms could be transferred from sensitized animals to naive recipients via humoral or cellular factors. Syngeneic recipient rats were injected (intraperitoneally with 4 x 10(7) cells (precultured overnight with either HDM or bovine serum albumin [BSA]) from lymph nodes of sensitized or control rats, respectively. Other groups received a tail-vein injection of serum from either HDM-sensitized or control rats. Antigen challenge in rats injected with sensitized cells caused increases in pulmonary inflammation and in AHR, but no changes in immediate bronchoconstriction as compared with control recipients. Antigen challenge in serum recipients resulted in immediate bronchoconstriction but had no effect on AHR or on pulmonary inflammation. These data show that immune-mediated lung inflammation and AHR are promoted by antigen-specific lymphocytes, whereas immediate allergic responses are caused by serum factors.

Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion.

The synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp4l, are stable and potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), we have identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp41 within the glycoproteins of other fusogenic viruses. Here we report on antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV). We screened crude preparations of synthetic 35-residue peptides, scanning the DP-178-like domains, in antiviral assays. Peptide preparations demonstrating antiviral activity were purified and tested for their ability to block syncytium formation. Representative DP-178-like peptides from each paramyxovirus blocked homologous virus-mediated syncytium formation and exhibited EC50 values in the range 0.015-0.250 microM. Moreover, these peptides were highly selective for the virus of origin. Identification of biologically active peptides derived from domains within paramyxovirus F1 proteins analogous to the DP-178 domain of HIV-1 gp4l is compelling evidence for equivalent structural and functional features between retroviral and paramyxoviral fusion proteins. These antiviral peptides provide a novel approach to the development of targeted therapies for paramyxovirus infections.