IFN-γ and FasL expression in LTBMC stromal layers.

Total mRNA was extracted from adherent cells of confluent LTBMCs from patients and controls and subjected to RT-PCR analysis for IFN-γ and FasL expression using specific primers. PCR products were electrophoresed on a 1.5% agarose gel and visualized under ultraviolet light by ethidium-bromide staining. As positive controls for IFN-γ and FasL expression, cDNA was obtained from peripheral blood mononuclear (IFN-γ) or Jurkat (FasL) cells stimulated with phorbol myristate acetate (PMA;100 ng/mL) plus ionomycin (1 μM) for 4 hours. (A) RT-PCR detection of IFN-γ and FasL in cell cultures derived from positive controls and representative patients with CIN. Three samples derived from patients with cyclic, severe congenital (Kostmann disease) and familial neutropenia, respectively, were negative for IFN-γ and FasL and are also shown. GAPDH was used as a control for cDNA amplification. (B) Hybridization of RT-PCR products derived from the positive controls, the 2 CIN patients, and the 3 patients with cyclic, severe congenital, and familial neutropenia was performed using gene-specific primers to confirm the nature of the amplified products. MWM indicates molecular weight marker.