The Langerhans cell (LC) is a histiocytic cell that represents a resident immigrant population in the epidermis and functions as a professional antigen presenting cell (APC). Langerhans cell histiocytosis (LCH) is a rare disorder that can affect both adults and children and can involve various organs. Clinically the disease ranges from a solitary lytic bone lesion, which may resolve following curettage, to a disseminated leukaemia-like disease with multiple organ involvement, which can be fatal. Despite considerable advances in our understanding of the disease, the aetiology ofLCH remains obscure. The aim of this study is to identify differentially expressed genes in LCH cell as compared to normal LC to gain novel insight into potential pathologic mechanisms involved in this disease. This has been carried out by using immunoaffinity cell purification against the LC marker CDla, a technique developed in our laboratory, together with a suppressive subtractive cDNA hybridisation (SSH) technique. Because of the limited LCH RNA material, an initial study was carried out to optimise conditions for SSH using HeLa cells and YCI cells, a derivative of HeLa in which CDla is constitutively expressed. By doing these experiments, it was found that the inclusion of an additional step to the subtraction protocol, namely size fractionation and purification of subtracted cDNA, allowed efficient recovery ofcDNA clones for the differentially expressed CDla gene. Using this optimised protocol, gene expression between normal LC and LCH cells obtained from bronchial alveolar lavage from one patient with LCH have been compared. A total of 291 differentially expressed clones were identified by differential screening and sequenced. The resulting sequences were used to search DNA sequence databases and the results used to classify sequences into three groups: (i) sequences that matched known human gene sequences, (ii) sequences that were found to originate from the pathogen Mycoplasma hyorhinis (M hyorhinis) and (iii) gene sequences of unknown origin. Using RT-PCR I have evaluated the expression of these in other LCH samples and have demonstrated one of the known human genes, argininosuccinate synthetase to be differentially expressed exclusively in 18 LCH-involved tissues from 17 LCH involved patients examined to date. Further, using RT-PCR, M hyorhinis sequences were also confirmed in six out of six additional LCH samples derived from bronchial alveolar lavage and two additional LCH samples involving gum. Taken together, these findings suggest that a discrete set of genes are differentially expressed in LCH, which may be important in the pathogenesis of this disease. Further, my data suggest the possible involvement of a novel infectious agent in the disease.