Gene expression of proinflammatory cytokines (IL-1ß, IL-6, TNF-α) and iNOS in BMDMs treated with soluble tannin hydrolysate.

<p>mRNA levels of IL-1ß (a), IL-6 (b), TNF-α (c), and iNOS (d) were determined by real-time PCR. BMDMs were pre-treated with soluble tannin hydrolysate (0, 30, and 100 μg/ml) for 1 hour followed by stimulation with three different strains of MAC (MAC-1, MAC-2, and MAC-3) for 6 hours. mRNA was extracted from BMDMs, reverse-transcribed into cDNA and qPCR performed. The values are presented as means ± SEM (n = 3–4). * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared with no treatment with soluble tannin hydrolysate (0 μg/ml).</p

Compositions of diets (%).

<p>Compositions of diets (%).</p

ORAC values of soluble tannin powder and soluble tannin hydrolysate.

<p>Soluble tannin powder was hydrolyzed by heating at 90°C for 3 hours with 5 ml of a 1.2 N HCl–50% methanol solution, followed by dilution to 10 ml using 1.2 N HCl–50% methanol solution. The values are presented as means ± SEM (n = 3). *** <i>P</i> < 0.001 compared with non-hydrolyzed soluble tannin powder.</p

Proinflammatory cytokine (IL-1ß, IL-6, TNF-α) secretion by BMDMs treated with soluble tannin hydrolysate.

<p>BMDMs were pre-treated with soluble tannin hydrolysate (0, 30, and 100 μg/ml) for 1 hour followed by stimulation with three different strains of MAC (MAC-1, MAC-2, and MAC-3) for 6 hours. The supernatant was harvested and IL-1ß (a), IL-6 (b), and TNF-α (c) protein concentration was determined by ELISA. The values are presented as means ± SEM (n = 4). * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001 compared with no soluble tannin hydrolysate (0 μg/ml).</p

Analysis of soluble tannin and soluble tannin hydrolysate bacteriostatic activity against MAC.

<p>MAC strains (2×10² CFU each) were inoculated in culture medium with soluble tannin (hydrolysate). Soluble tannin (a) and soluble tannin hydrolysate (b) was added at 0, 30, and 100 μg/ml soluble tannin powder to each of three tubes and the CFU determined. The values are presented as means ± SEM (n = 4). * <i>P</i> < 0.05 when compared with no treatment with soluble tannin hydrolysate (0 μg/ml).</p

The effect of soluble tannin in a MAC-infected pulmonary granuloma model.

<p>Diets were started 1 week before MAC infection. All mice were analyzed at 8 weeks after MAC infection. (a) Microscopic morphology of granulomas in the H&E-stained lungs of MAC-infected BALB/c mice fed control and 2% soluble tannin diets. Dotted lines indicate the border of granulomas. Original magnification, ×40 and ×200. (b) Analysis of the size of lung granulomas between control and 2% soluble tannin diet. (c) MAC CFU counts in one lobe of lungs using Middlebrook 7H10 agar plates as described in the Materials and Methods. The values are presented as means ± SEM (uninfected mice, n = 5; MAC-infected mice, n = 8). * <i>P</i> < 0.05, *** <i>P</i> < 0.001 compared with MAC-infected mice fed the control diet.</p

Viral loads in coughs of unvaccinated influenza patients.

<p>Data from days 2 and 3 of symptom onset were plotted together in respect to the status of receiving antiviral treatment, including treatment time before cough collection. The open circle, closed circle, and closed triangle represent subjects untreated, and treated with oseltamivir or zanamivir, respectively. Viruses isolated in oseltamivir-treated cases were all subtype A(H1), which is considered to be oseltamivir-resistant.</p

Viral loads in coughs of influenza patients.

<p>Cough samples were collected from individual patients and subjected to viral quantification. The viral load/cough was plotted on the ordinate along with days of illness during sample collection on the abscissa. The onset day of any influenza symptom was defined as day 1. Viral subtypes are presented using different symbols: open circle, closed circle, and open triangle represent infections with A(H1), A(H3), and A(H1N1)pdm09 virus, respectively. Daggers indicate cases in which the active virus was detected using a conventional plaque assay.</p

Viral loads of samples from patients with or without cough symptom.

<p>Viral loads in cough and throat swab samples from cases with and without a symptom of coughing were compared. The viral load in the throat swab was calculated from the viral load of the transport medium in which the swab fluid was eluted and the volume of the eluted swab fluid, which was tentatively estimated by the increase of the weight of the cotton swab used for sample collection. Circle and triangle represent cases with and without symptoms of coughing. Closed circles indicate cases with higher viral loads among cough or throat swab samples, and identical numbers associated with the closed circles indicate that the data were from the same patient.</p

Viral recovery rate of collection and quantification system.

a<p>The experiments were performed in triplicate for the high amount experiment controls and the number is the average and standard error (SE) of the results.</p>b<p>The low dose experiment was performed once to confirm the reproducibility of the recovery rate, as determined by the high dose experiment.</p>c<p>Average copy number of the influenza A M1 gene in the gelatin membrane.</p>d<p>Percentage of viral load on the gelatin membrane to that of collection when the nebulizer was set close to the membrane.</p>e<p>The nebulizer was set 10 cm from the gelatin membrane filter through which the viral mist was suctioned.</p>f<p>The cone-shaped, megaphone-like device of 50 cm in length was connected with the gelatin membrane filter and the nebulizer was set at the entrance of the device.</p>g<p>Percentage of viral load in the product of each step to those at the start of each step.</p>h<p>Viral load in the viral fluid poured into the gelatin membrane solution.</p>i<p>Viral load in the solution of the gelatin membrane filter dissolved in 10 mL of MEM.</p>j<p>The gelatin solution containing the virus was digested with 10 µg/mL of collagenase.</p>k<p>The digested fluid was ultra-centrifuged at 125,000 g for 100 min and the viral RNA was extracted from the precipitate and concentrated by freeze-drying and vacuum centrifugation.</p