In Vitro Model of LPS-induced Inflammation

We hypothesized that the antiinflammatory activity would be an important component of the therapeutic effect of TEL on respiratory infections. Thus, we designed the present study to test the antiinflammatory activity of TEL in an in vitro model of LPS-induced airway inflammation, and to determine whether this antiinflammatory activity occurs in vivo, in a mouse model of acute, LPS-induced lung injury.

Materials and Methods

Cells and Culture Conditions

Murine lung epithelial (MLE) cell 12 tine was provided by Dr. S. Roman-Roman (Prostrakan Pharmaceuticals; Romainvine, France). MLE-12 cells were grown in Roswell Park Memorial Institute 1640 medium supplemented with 5% heat-inactivated fetal calf serum (FCS), 1 mmol/L sodium pyruvate, and 2 mmol/L L-glutamine (Sigma; St Louis, MO). Macrophage cell line RAW 264.7    was provided by Dr. F. Gamarro (Lopez Neyra Institute of Parasitology and Biomedicine, CSIC; Granada, Spain). RAW 264.7 cells were cultured in Dulbecco minimal essential medium (DMEM) [Sigma] supplemented with 10% FCS, 1 mmol/L sodium pyruvate, and 2 mmol/L L-glutamine. Both MLE-12 and RAW 264.7    cells were cultured at 37°C in 5% CO2.

In Vitro Model of LPS-induced Inflammation

RAW 264.7 cells were grown to confluence in 24-well plates, and stimulated with 1 pg/mL LPS. TEL (Aventis Pharma; Neuville-sur-Saone, France) was diluted in the culture medium and added to cultures at final concentrations of 2.5, 5, and 10 pg/mL, respectively. After incubation for 6 or 24 h, cell-free supernatants were collected and stored at — 20°C until assayed.

MLE-12 cells were grown to confluence in 24-well plates and From the Microbial Immunology Research Group, Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain.

Support for this research was provided by Junta de Andalucia (Research Group CVI201). Dr. Leiva was the recipient of a grant from the Ministerio de Educacioin, Cultura y Deporte.

The authors have reported to the ACCP that no significant conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

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