The antiviral activity of zinc salts against rhinovirus types 1A and 39 was assayed by yield reduction and inhibition of cytopathic effect in cell culture. The findings indicate that the zinc salts tested have low in vitro therapeutic indices and suggest that the possible beneficial effects of zinc lozenges in reducing cold symptoms may not be related to selective antirhinovirus activity.Zinc ions inhibit the in vitro replication of diverse viruses, including rhinovirus (4, 5), herpes simplex virus (7), and vaccinia virus (3), and other pathogenic organisms, including Chlamydia trachomatis (2). Zinc chloride (0.1 mM) inhibited plaque formation by eight of nine human rhinoviruses in HeLa cells and reduced the yield of rhinovirus 1A when added as late as 6 to 8 h after infection (5). The antirhinovirus activity of this zinc salt appears to be mediated by inhibition of posttranslational cleavage of precursor polypeptides (4). Zinc gluconate lozenges were reported to be effective in reducing the duration of symptoms in natural colds of undefined viral etiology (1). In contrast, another study, using a somewhat lower dosage of zinc acetate lozenges, found no clinical benefit compared with a placebo in the treatment of natural colds (R. Douglas, personal communication). Unpalatable taste, mouth irritation, and nausea have been reported side effects of zinc lozenge administration (1; B. M. Farr and J. M. Gwaltney, Jr., J. Chronic Dis., in press).The present studies were undertaken to determine the in vitro antiviral activity of a variety of zinc salts against two human rhinoviruses representing serotypes previously shown to be inhibited by zinc chloride (5). Serotype 1A (passage history: MRC-5/5, WI-38/1, fetal tonsil/1) and serotype 39 (passage history: WI-38/2, MRC-5/4) were used. The following zinc salts (molecular weight) were provided by R. G. Achari, Bristol Meyers Products, Inc., Hillside, N.J.: acetate (243.5), carbonate (125.4), chloride (136.3), gluconate (455.7), lactate (183.5), sulfate (161.4), and oxide (81.4). The salts were dissolved in sterile distilled water or 0.1 N HCl to make stock solutions of 10 mM concentration. Serial logl0 or 0.5 log10 dilutions of the stock solutions were made in cell culture maintenance medium appropriate to the cell type. MRC-5 and WI-38 human embryonic lung fibroblast cells (M.A. Bioproducts, Inc., Walkersville, Md.) at passage levels 23 to 25 were maintained in Eagle minimal essential medium supplemented with glutamine, 2% fetal bovine serum (HyClone Laboratories, Logan, Utah), vancomycin (20 jig/ml), gentamicin (50 ,ug/ml), and amphotericin (1 jig/ml). For cytopathic effect (CPE) inhibition and infectious virus yield reduction assays, monolayers of a particular cell type were inoculated with 0.2 ml of Hanks balanced salt solution (HBSS) containing 30 to 1,000 50% tissue culture infective doses (TCID50) of virus. After incubation at 35°C for 1 h, the inoculum was decanted, and the monolayers were rinsed once or twice with Hanks balanced salt solution. The monolayers were o...
The Immulok test performed at 24 h after inoculation had a 73% sensitivity and 97.5% specificity for detecting herpes simplex virus in clinical specimens, as compared with viral culture in human embryonic lung fibroblasts. The test increased the proportion of positive specimens reportable at 24 h by 48% (P < 0.01) but failed to detect 4 of 31 (13%) specimens positive for cytopathic effect at 24 h and 13 of 32 (41%) specimens positive at-48 h after inoculation.
Ambient nitrogen dioxide (NO2) and particulate matter (PM) concentrations are often found to be proportional, such that inhalation of one pollutant is expected to be accompanied by the inhalation of the other. Epidemiological evidence consistently demonstrates the health burdens associated with the inhalation of NO2 and PM, though has difficulty deciphering the inhalation hazard of either pollutant individually. Airway models can be exposed to NO2 and PM separately or as a co-exposure in vitro, allowing mechanisms of toxicity to be elucidated. Herein, NCI-H441 cells were exposed to either 780 ng/cm2 Printex 90 carbon black (CB) (a surrogate model of PM) using a VitroCell Cloud12, 5 ppm NO2 (within a modified Coy hypoxic cabinet) or a combination of NO2+CB over 24 hours. Cytotoxicity was not altered by NO2 or CB (trypan blue exclusion assay), however CB, and to a lesser extent CB+NO2 exposure resulted in a non-significant increase in membrane permeability (p>0.05). qPCR revealed no alterations in expression of superoxide dismutase 1 (SOD1) for any exposure condition. Both CB and NO2 exposures resulted in significantly increased IL6 and IL8 release (ELISA) (p<0.05), although co-exposure of the two pollutants did not augment the detected IL6 or IL8 levels. These data indicate NO2 and CB produce an inflammatory response in this model, though little evidence suggests that NO2 and CB act in an additive or synergistic manner via this pathway. Additional work remains ongoing to establish the mechanisms of toxicity utilising an anatomically relevant triple cell co-culture of the alveolar epithelial barrier.
In vitro lung models are a valuable tool to assess the inhalation hazard of air pollutants, bioaerosols, chemicals and nanomaterials. Though it is essential that these models are characterised and anatomically relevant to the area of the human lung they seek to represent. Previously within inhalation toxicology, focus has been placed on the use of monocultures of alveolar epithelial cells, however use of alveolar co-culture models have been presented as more realistic and sensitive models. Here, we present the development of a novel triple cell co-culture system, cultured at the air-liquid interface that is sensitive to LPS and particulate matter. This co-culture contains alveolar type 1 cells (hAELVi), alveolar type 2 cells (NCI-H441) and alveolar macrophages (differentiated THP-1 (dTHP-1) using PMA (20 nM)). Using CellTrackers™ with confocal microscopy, the ratio of hAELVi cells to NCI-H441 cells was confirmed to be a histologically relevant 16:1 ratio forming a tight monolayer shown through blue dextran membrane permeability assay. Macrophages were seeded on top of the epithelial co-culture at an anatomically relevant density of 1 macrophage per 18x103 μm2 (confirmed via confocal microscopy) to form the triple cell co-culture. The model, when challenged, released more interleukin-8 and TNF-α than the epithelial mono- and bi-culture systems when exposed to LPS (1 μg/mL for 24 hrs) (p<0.05), suggesting that the macrophages do mount the theoretical (pro-)inflammatory response. This anatomically relevant in vitro model forms the basis to investigate inhaled exogenous substances, such as the differential toxicology impacts of particulate matter and nitrogen dioxide.
When considering air pollution particles (AP), they are often assessed in isolation, when this is not how the population is exposed to them. Within the indoor environment, there are bioaerosols present, which contain various allergens including house dust mite (HDM) and lipopolysaccharides (LPS). This RESPIRE study aims to apply a well-characterised in vitro model of the alveolar barrier (type I and II epithelial cells (NCI-H441 and hAELVi at relevant in vivo ratios) and macrophage-like cells) to determine the effects of these bioaerosols on the toxicological and immunological responses to AP. To achieve this, we will investigate cytotoxicity, inflammatory responses, morphology, oxidative stress, membrane integrity and genetic toxicology of these models with specific focus on responses from human allergic responses and airway diseases (i.e., bronchiolitis-interstitial lung disease and sarcoidosis) to determine the additional health considerations the addition of bioaerosols to AP has on our models. We will utilise a physiologically relevant aerosolization exposure method) and a standardised indoor (NIST2583) particulate at an environmentally relevant concentration to mimic air pollution along with HDM and LPS to determine if the addition of these environmentally appropriate bioaerosols influence the responses to IAP. This project is funded by the recently initiated RESPIRE project (NE/W002264/1), as part of the SPF Clean Air Programme.
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