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Phenotypic and functional analysis of antigen presenting cells in a murine model of cutaneous carcinogenesis

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Rist, Michael John 1999 , 'Phenotypic and functional analysis of antigen presenting cells in a murine model of cutaneous carcinogenesis', PhD thesis, University of Tasmania.

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Abstract

To elucidate the nature of the cells involved in the induction of
immunosuppression when antigen is applied through Langerhans cell depleted skin, due
to prior exposure to 9, 10- dimethyl benzanthracene (DMBA) or
2,4,6-trinitrochlorobenzene (TNCB) the phenotype of migrating antigen bearing cells
was analysed. The initial experiments were aimed at the identification of the antigen
bearing cells migrating from murine skin previously treated with DMBA or TNCB to
deplete Langerhans cells. This involved application of the fluorescent
contact sensitiser fluorescein isothiocyanate (FITC) to DMBA or TN.CB treated skin,
enrichment of low density cells from draining lymph nodes by metrizamide
gradient centrifugation and the phenotypic characterisation of the FITC
bearing cells by flow cytometry. Two distinct distinct populations were identified on
the basis of FITC fluorescence intensity. These were classified as FITChi or FITCl0.
intensities of FITC positive cells were detected in draining lymph nodes of
acetone-FITC treated mice. DMBA or TNCB treatment prior to FITC application
substantially reduced the number of FITChi cells intensity positive (FITChi)
cells within the draining lymph nodes, whereas the number of FIT0° was slightly
reduced This demonstrates that the cells present in DMBA or TNCB treated skin
had an impaired ability to trap antigen and migrate to the draining lymph node.

Single colour phenotypic analysis of FITC positive cells that did migrate from
DMBA or TNCB treated skin revealed populations that expressed markers
consistent with dendritic cells, γδ-TCR+ cells, CD4+ cells, CD8a+ cells or B cells.
Markers for dendritic cells, CD4+ cells and CD8a+ cells were detected upon both
FITChi and FITC'0 populations whereas populations of cells expressing γδ-TCR and B cell
markers were restricted to the FITC'0 population. Both FITChi and FITCl0
populations expressed accessory and adhesion molecules at levels consistent
with phenotypically mature dendritic cells.

Further analysis of the FITC bearing populations by multiple colour flow
cytometry revealed that FITChi cells consisted primarily of mature
dendritic cells, while the FITCl0 population was more heterogenous. FITCl0 cells
expressed CD11c, CD11b, MHC 11, CD8α, CD80 and CD86. Analysis of FITC positive cells
revealed FITChi dendritic cells from DMBA-FITC and TNCB­ FITC treated mice were
defective in the ability to activate FITC specific responder cells.

To determine the identity of the effector population induced by DMBA an in vitro
assay was developed. The effector cell was identified as a nylon wool adherent cell,
that expressed CD11b. This population was found to act in an antigen nonspecific
manner in vitro.

Item Type: Thesis - PhD
Authors/Creators:Rist, Michael John
Keywords: Carcinogenicity testing, Carcinogens, Antigen presenting cells, Skin
Copyright Holders: The Author
Copyright Information:

Copyright 1999 the author - The University is continuing to endeavour to trace the copyright
owner(s) and in the meantime this item has been reproduced here in good faith. We
would be pleased to hear from the copyright owner(s).

Additional Information:

Thesis (Ph.D.)--University of Tasmania, 1999. Includes bibliographical references

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