Molecular cloning of multiple forms of the ovine B7-2 (CD86) costimulatory molecule.

To facilitate analysis of the role of costimulatory molecules in the ovine immune system, we cloned and sequenced eight putative alternatively spliced transcripts of the sheep CD86 (B7-2) costimulatory molecule. Using RT-PCR and rapid amplification of cDNA ends (RACE), we cloned the sheep CD86 (B7-2) molecule that encodes eight forms, which differ in the length of the signal peptide, the presence or absence of a transmembrane region and in their cytoplasmic tails. Comparison of the deduced amino acid sequence of the largest ovine CD86 TM form (CD86-2) with the sequence of cattle, pig, human and mouse CD86 indicated that the deduced protein had a higher degree of similarity to cattle (85% of amino acid identity) than to pig (77%), human (59%), and mouse sequence (45% of identity). Our results indicate that mRNA transcripts encoding different CD86 protein forms are expressed in sheep, like in other mammals, and suggest that the expression of this gene may be regulated at the transcriptional or RNA splicing level, which could give rise to tissue-specific expression of CD86. It is possible that, in the sheep, these CD86 mRNA variants could play different regulatory roles in T cell activation.

Detection of Specific mRNAs in Culture Medium Conditioned by Human Tumour Cells: Potential for New Class of Cancer Biomarkers in Serum.

BACKGROUND: This study aimed to develop and optimise procedures to enable us to establish, in a well-controlled environment, if cancer cells routinely secrete gene transcripts potentially suitable for monitoring as biomarkers. MATERIALS AND METHODS: Aliquots of the conditioned media (CM) exposed to cancer cells (including breast, lung and nasal cancer cell lines) were removed at intervals of 24 hours over a 96-hour time period and were passed through 0.45 µm or 0.22 µm filters, to remove cellular material. Methods for subsequent RNA extraction (from CM and cells) were investigated. RT-PCR was performed for a number of mRNAs, including mdr-1, mrp-1, CK-19, HnRNP B1, GST-π, topoisomerase II, bcl-2 and ß-actin. RESULTS: Gene transcripts, amplifiable by RT-PCR, were detected in conditioned media from all human cancer cell lines studied. This RNA did not result from the presence of cells in the conditioned media and, as expected, was absent from control medium not exposed to tumour cells. CONCLUSION: The results from this study indicate that gene transcripts may be secreted from human cancer cells and are detectable in the subsequent cell-free media. The methods developed and optimised here may be suitable for analysis of mRNAs as biomarkers in serum/plasma from cancer patients.