1. Propionibacterium acnes-Reactive T Helper-1 Cells in the Skin of Patients with Acne Vulgaris 
Paul E. Mouser, Edward D. Seaton, Anthony C. Chu 
Journal of Investigative Dermatology 
Volume 121, Issue 5, Pages (November 2003)
DOI: /j _6.x
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Increased proliferative response to P. acnes extract by acne versus psoriatic TCL. TCL were established by culturing skin fragments (4 mm acne skin biopsies or psoriasis shave biopsies) in RMPI 1640 medium (Gibco BRL, Invitrogen Ltd, Paisley, UK) supplemented with 2 mM L-glutamine, 100 U per mL penicillin and 100 μg streptomycin per mL (Gibco BRL), and 10% human AB+ serum (complete medium) containing P. acnes extract (1:100 dilution), adding 20 U per mL human recombinant IL-2 (Roche Molecular Biochemicals, Lewes, UK) every 3 d. After 14 d culture, TCL (2.5 × 104 per well) plus an equal number of irradiated, autologous peripheral blood mononuclear cells were cultured for 3 d in the presence or absence of optimal concentrations of each of the microbial preparations in 96-well plates. Tritiated thymidine (4 mCi per mL, specific activity 5 Ci per mmol; Amersham International, Amersham, UK) was added for the last 6 h of culture. The cells were harvested on to fiberglass paper, and the incorporated radioactivity measured in a β scintillation counter. Proliferation was expressed as the mean cpm of three replicates in the presence of microbial preparation minus the mean cpm of three replicates in the absence of microbial preparation. Bars indicate the mean proliferative response of each group.
Journal of Investigative Dermatology  , DOI: ( /j _6.x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Acne TCL express a T helper 1 cytokine profile after PMA/ionomycin or P. acnes stimulation. TCL (0.2 × 106) were either stimulated in 96 well plates for 6 h in complete RPMI containing PMA (25 ng per ml) (Sigma-Aldrich Poole, UK), 1 mM ionomycin (Sigma-Aldrich), and 10 μg Brefeldin A per mL (Sigma-Aldrich) or for 16 h with P. acnes extract plus an equal number of irradiated peripheral blood mononuclear cells adding Brefeldin A 3 h after initiation of the culture. Basal production of cytokines was determined by culturing the cells in the absence of stimulant. Cells were washed with phosphate-buffered saline (PBS), fixed with 2% formaldehyde in hypertonic PBS for 20 min at room temperature and washed in PBS/0.2% bovine serum albumin (BSA). Cells were pretreated with PBS/BSA/saponin (Sigma-Aldrich) and then double stained with anti-IFN-γ-PE plus anti-IL-4-fluorescein isothiocyanate, or with corresponding isotype-matched controls (Becton Dickinson, Oxford, UK) diluted in PBS/0.2% BSA/0.1% saponin for 30 min at room temperature. Cells were then washed twice with PBS/BSA/saponin and finally with PBS/BSA. Insignificant amounts of cytokines were detected in the irradiated peripheral blood mononuclear cell populations with or without P. acnes stimulation in the absence of skin T cells. Samples were analyzed with two color staining using FACS-Scan flow cytometer (Becton Dickinson) and Cell Quest software. Results are expressed as the percentage of IFN-γ+ and IL-4+ T cells in acne TCL after PMA/ionomycin (n=9) and P. acnes (n=5) stimulation. Data are given as mean±SD frequency of positive cells.
Journal of Investigative Dermatology  , DOI: ( /j _6.x)
Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions