Effect of Cyclosporine on Peripheral Blood and Lesional Skin in Psoriatic Patients: MATERIALS AND METHODS

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Patients

Eight patients (5 females, 3 males, mean age 42.5 years) with severe chronic plaque psoriasis were Antibodies and reagents for staining Primary unconjugated and fluorescein isothio- cyanate (FITC)-, phycoerythrin (PE)-, or biotin- conjugated monoclonal antibodies (mAbs)/reagents used in this study are as follows; anti-cutaneous lymphocyte antigen (CLA, Becton-Dickinson, Lin­coln Park, NJ), anti-E-selectin (CD62E, R&D Systems, Minneapolis, MN), anti-CD3 (Dako, Kyoto, Japan), anti-CD4 (Dako) and anti-CD8 (Dako). Irrelevant monoclonal antibodies of the appropriate immunoglobulin isotype were used as negative controls. mAbs were titrated and diluted in staining buffer (5% FBS, IVGG, NAN3).

Immunohistochemistry

Five adult patients (2 females and 3 males, mean age 42.5) were biopsied. 9 paraffin-embedded, formalin-fixed skin samples were subject to each time-sequential skin biopsies (at baseline, 3 weeks, 6 weeks, 18 weeks following initiation of treatment, recurrence after treatment termination). Two nor­mal skins were used for comparison. Paraffin- embedded, formalin-fixed skin samples were used in immunohistochemical staining by the standard streptavidin-biotin peroxidase method. Results were recorded as the mean number of each marker- positive cells in 10 high power fields (x 400) by 2 dermatologists and 1 pathologist. Anti E-selectin antibody (CD62E, Novocastra, Vector Laboratories, Burlingame, U.S.A.) was used as a primary antibody in immunohistochemical staining using standard streptavidin-biotin peroxidase method. Results were interpreted as follows; (3 +): strong intensity on more than 90% of upper dermal vessels, (2 +): moderate intensity on more than 30% and less than 90% of upper dermal vessels, (+): weak intensity on less than 30% of upper dermal vessels. viagra plus

FACS

The lymphocytes expressing CD3, CD4, CD8, or CLA were detached briefly with 0.02% trypsin and incubated for 20 min in ice with each monoclonal antibody (CD3, CD4, CD8, or CLA) or normal human IgG, followed by FITC-conjugated goat anti-mouse IgG or anti-human secondary Abs. After fixation in 3.7% formaldehyde, cells were analyzed by flow cytometry using a Becton Dickinson FACS machine (San Jose, CA) and the data was analyzed using CellQuest software (Becton Dickinson).

Statistical analysis

The results were expressed as mean ± standard deviation. Statistical analysis of the difference between each group was made by Wilcoxon rank sum and signed rank test.