Gripp-Heel® (stock 24335-05.2011) and Engystol® (stock 22981-02.2001) solution were supplied as sterile ampoules (1.1 ml H2O) from Biologische Heilmittel Heel (Baden-Baden, Germany). Gripp-Heel® contains Aconitum (D4), Byronia (D3), Lachesis (D11), Eupatorium perfoliatum (D2) and phosphorus (D4). Engystol® contains Vincetoxicum hirundinaria (D6), Vincetoxicum hirundinaria (D10), Vincetoxicum hirundinaria (D30), sulphur (D4) and sulphur (D10). The test preparations were diluted in cell culture medium before addition to the cell culture. Final concentrations in the assays ranged from 1:4 to 1:320.
Cell culture and viruses
Human rhinovirus B serotype 14 (HRV-14) was obtained from the Institute for Virology (University of Jena, Germany). Influenza A virus (FluA), Chile 1/83 (H1N1), herpes simplex virus 1 (HSV-1, strain Thea), vesicular stomatitis virus (VSV, FL1), respiratory syncytial virus (RSV, strain Long), parainfluenza type 3 (Para3) and adenovirus type 5 (Ad5) were obtained from the Friedrich-Löffler Institute (FLI) Tübingen and the Department of Medical Virology and Epidemiology of Virus Diseases of the Hygiene Institute at University of Tübingen, respectively.
For IFN assays
HeLa cells (University of Jena, Germany) were incubated with UV-inactivated HRV-14, Hep-2 cells (CCL-23, ATCC) with UV-inactivated HSV-1 and Madin-Darby canine kidney cells (MDCK) with UV-inactivated FluA.
For cultivation of viruses
Hep-2 and HeLa cells were cultivated in MEM with Hanks' buffered saline solution containing 2% fetal calf serum (FCS, PAA, Pasching, Austria), 25 mM MgCl2, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin). Cells were incubated in serum-free MEM containing 1 μg/ml trypsine, 2 mM L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin.
Determination of virus titre
The respective cells were incubated in 12-or 24 well tissue culture dishes with serially diluted serum-free virus stock solutions for 1 h at 34°C as described elsewhere .
Isolation of PBMCs
Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors (Wiener Rotes Kreuz, Wien, Austria). Healthy donors were identified by diagnostic parameters (negative haemogram, infection serology). The blood samples were treated with heparin (Sigma) and subjected to Ficoll-Hypaque (1,077 g/l PAA) density gradient centrifugation (30 min. 2100 rpm). The PBMCs were isolated from the interphase, subsequently washed twice with medium (RPMI 1640, 10% FCS) and counted using the trypan blue exclusion test.
The cells were incubated for 2 to 6 days with the test preparations before being tested for IFN release (96-well round bottom microtiter plates, Greiner, Bio-one, Kremsmünster, Austria, 2 × 105 cells/well, RPMI 1640, 10% FCS). In an alternative approach, cells were co-stimulated with HSV-UV to induce IFN synthesis.
Virus titration for costimulation experiments
To induce IFN release in PBMCs and virus-susceptible cell lines, virus suspensions of inactivated HSV-1, HRV-14 and FluA were employed.
High titre virus suspensions (1 × 108 PFU/ml) of the respective viruses were produced and inactivated with an UV-GS linker. The virus titre inducing 20-30% IFN synthesis (calculated with an IFN-α standard) was determined in serial dilutions and kinetic experiments.
Plaque formation assays and virus-specific ELISA
The antiviral activity of Gripp-Heel® against influenza, Para3, RSV, HRV-14, HSV-1 and Ad5 was determined by means of a plaque formation assay or cytopathogenic effect (CPE), respectively as described elsewhere . For Ad5 a virus specific ELISA was employed. Briefly, the virus permissive cell lines were incubated with the test medications at different concentrations for 2h, 24h and 48h. The test medication was removed and the cells were infected with a multiplicity of infection (MOI) of 0.00037 (Flu A), 0.00044 (Para3), 0.00046 RSV, 0.00042 (HRV-14), 0.0004 (HSV-1) and 0.004 (Ad5). The virus inoculum was removed and subsequently 1) cells were overlaid with solid medium only ("prophylactic" protocol) or 2) cells were overlaid with solid medium containing the test substances ("therapeutic" protocol) and cultivated until in the control plaques or CPE appeared. The percentage of inhibition was calculated in reference to the untreated control (100% inhibition) and expressed as relative inhibition (n = 2 in duplicates).
Interferon-α specific ELISA
A commercial IFN-α ELISA (Biosource) was used to determine the quantity of IFN-α in the cell culture supernatant. Briefly, cells (96-well round bottom microtiter plates, Greiner, Bio-one, Kremsmünster, Austria, 2 × 105 cells/well, RPMI 1640, 10% FCS) were seeded and incubated with the test preparations for the indicated time points. Additional samples were co-stimulated with the appropriate UV-inactivated virus. After incubation for the indicated time points, the supernatant was collected and the IFN-α content was quantified according to the manufacturer instructions. The quantity of IFN-α is proportional to the extinction values at OD450 nm and is calculated in pg/ml using an IFN-α standard curve. To calculate an increase or decrease in percentage terms, the MEM control or co-stimulated control, respectively, was defined as 100% IFN-α and thereby served as relative values compared to control. The data represent the mean values +/- SD (n = 3).