Abstracts 803

183.

ACTIVATION DYNAMICS OF THE NONRESPONSIVE CELLULAR STATE ESTABLISHED BY CYCLOSPORIN IN HUMAN BLOOD LYMPHOCYTES AND SIMILARITIES WITH OTHER IMMUNOSUPPRESSOR DRUGS. A. Aszalos,

J.L. Weaver and P.S. Pine, Food and Drug Administration, Division of'Research and Testing Washington, D.C. 20204

The exact mode of action of the immunosuppressor drugs cyclosporin (CsA), FK506 and mycophenolic acid (MPA) is not known yet. We have found that several similarities exist in their cell biological and biophysical behavior. CsA (i ug/ml), FK506 (0.5 ug/ml) and

MPA (0.i ug/ml) can all induce a state of nonresponsiveness during anti-CD3 (12 ng/ml) stimulation. This nonresponsive state is not elicited by the immunological inactive CsH or in the presence of anti-TCR. The established nonresponsive state is bypassed by the signal generated by PMA (12 ng/ml) and ionomycin (I00 ng/ml) but not by ConA (I0 ng/ml),

anti-CD3 (12 ng/ml) and anti-TCR. These drugs also cause a hyperpolarization of the plasma membrane as measured with the oxonol dye and flow cyt~metry but do not affect intracellular Ca 2+ levels alone or during activation with anti-CD3.

184.

Modulation of Transcription from Adenovin~ Maior Late Promoter by Cyclosporin A Pramod B. Mahajan* and E.A. Thompson, Department Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77550

We have used the Adenovirus Major Late Promotor [AdMLP] as a model gene to study regulation of transcription of Class II genes by Cyclosporin A [CsA]. Nuclear extracts from Lymphosarcoma P1798 cells support faithful transcription from AdMLP. Nuclear extracts prepared from P1798 cells treated wi th 1 ug/ml CsA for 24 hr fail to support transcription from AdMLP. Transcription of human histone H4 gene is unaffected by such treatment. Both extracts exhibit similar CAAT-box, TATA-box and Upstream Stimulatory Element binding patterns as judged by gel mobility shift assay. A partially pure protein fraction has been obtained from control cell extracts. This fraction by itself does not support transcription from AdMLP, but confers upon the CsA-treated extracts the ability to support transcription from AdMLP i n v i t r o . These results support our hypothesis tha t CsA regulates expression of a subset of Class [[ genes by modulating activity of t rans -ac t ing factor(s) specific for the target genes. Our results also provide, for the first time, a model for the biochemical analyses of this regulatory event i n v i t r o .

185.

CYCLOSPORIN AND THE RAT THYMUS: AN IMMUNOHISTOLOGIC STUDY Henk-Jan Schuurman z'2, Henk van Loveren z, Jan Rozing a, Aalt van Dijk 2, J Gerard Loeber 1, Joseph G Vos 1

ZNational Institute of Public Health and Environmental Protection, Bilthoven; 2University Hospital, Utrecht; 3TNO Institute for Aging and Vascular Research, Leiden, The Netherlands

During daily administration of 15 mg/kg Cyclosporin A (CsA) for two weeks in 6-week-old male PVG (n=8) and WAG/CPB (n=8) rats, whole blood CsA levels were around 6 mg/I. The thymus immediately after treatment showed the almost disappearance of the medulla, including the microenvironment made by medula-type epithelium, interdi- gitating dendritic cells (IDC, defined by their MHC-class II expression), and lymphocytes with a mature T-immun- ophenotype. After discontinuation of CsA, a rapid recovery occurred, with reappearance of medulla after 2 weeks. These areas differed from the normal medulla by the absence of medulla-type epithelium. This cell population reco- vered in its normal location in about 4 weeks. The reappearance of medullary IDC was associated with that of lymp- hocytes with a mature T-immunophenotype. In the regenerating thymus 'holes' were present in the microenviron- ment that lacked epithelium and IDC, and that were filled by lymphocytes with an immature cortex phenotype. These changes in thymus (immuno)histology may be associated with changes in shaping the T-cell repertoire (defec- tive negative selection), as can be concluded from the so-called syngeneic graft-versus-hust disease that occurs un- der special conditions after CsA treatment.