Search

Viruses that naturally infect cells expressing both MHC I and MHC II molecules render themselves potentially visible to both CD8+ and CD4+ T cells through the de novo expression of viral antigens. Here we use one such pathogen, the B-lymphotropic Epstein-Barr virus (EBV), to examine the kinetics of these processes in the virally-infected cell, comparing newly synthesised polypeptides versus the mature protein pool as viral antigen sources for MHC I- and MHC II-restricted presentation. EBV-transformed B cell lines were established in which the expression of two cognate EBV antigens, EBNA1 and EBNA3B, could be induced and then completely suppressed by doxycycline-regulation. These cells were used as targets for CD8+ and CD4+ T cell clones to a range of EBNA1 and EBNA3B epitopes. For both antigens, when synthesis was induced, CD8 epitope display rose quickly to near maximum within 24 h, well before steady state levels of mature protein had been reached, whereas CD4 epitope presentation was delayed by 36-48 h and rose only slowly thereafter. When antigen expression was suppressed, despite the persistence of mature protein, CD8 epitope display fell rapidly at rates similar to that seen for the MHC I/epitope half-life in peptide pulse-chase experiments. By contrast, CD4 epitope display persisted for many days and, following peptide stripping, recovered well on cells in the absence of new antigen synthesis. We infer that, in virally-infected MHC I/II-positive cells, newly-synthesised polypeptides are the dominant source of antigen feeding the MHC I pathway, whereas the MHC II pathway is fed by the mature protein pool. Hence, newly-infected cells are rapidly visible only to the CD8 response; by contrast, latent infections, in which viral gene expression has been extinguished yet viral proteins persist, will remain visible to CD4+ T cells.

Background: Previous reports indicate that the p53 yeast functional assay is a highly sensitive method of detection of p53 mutations in clinical specimens. Our earlier report (1) showed a 35.4\% p53 mutation frequency in Hong Kong colorectal carcinoma (CRC) patients, when conventional molecular screening techniques were utilized to assess the mutation rate in the hot spots in exons 5-8. Materials and methods: The yeast functional assay was used to determine if the previous mutation frequency determined by PCR-SSCP techniques was under-estimated and if so, to see if other hot spots for mutations explain this difference. Results, The p53 functional yeast assay results showed an increased mutation frequency. However, sequencing showed the mutations were confined to common hot spots for mutations in exons 6 and 7. Conclusions: The mutation frequency in CRC patients observed with the yeast assay is higher than previously reported. Forty-five percent of 20 SSCP-negative specimens were positive by the yeast assay, which this study shows is superior for detection of p53 mutations directly in clinical specimens containing varying amounts of normal tissue contamination.

Background: Previous reports indicate that the p53 yeast functional assay is a highly sensitive method of detection of p53 mutations in clinical specimens. Our earlier report (1) showed a 35.4\% p53 mutation frequency in Hong Kong colorectal carcinoma (CRC) patients, when conventional molecular screening techniques were utilized to assess the mutation rate in the hot spots in exons 5-8. Materials and methods: The yeast functional assay was used to determine if the previous mutation frequency determined by PCR-SSCP techniques was under-estimated and if so, to see if other hot spots for mutations explain this difference. Results, The p53 functional yeast assay results showed an increased mutation frequency. However, sequencing showed the mutations were confined to common hot spots for mutations in exons 6 and 7. Conclusions: The mutation frequency in CRC patients observed with the yeast assay is higher than previously reported. Forty-five percent of 20 SSCP-negative specimens were positive by the yeast assay, which this study shows is superior for detection of p53 mutations directly in clinical specimens containing varying amounts of normal tissue contamination.

Background: Previous reports indicate that the p53 yeast functional assay is a highly sensitive method of detection of p53 mutations in clinical specimens. Our earlier report (1) showed a 35.4\% p53 mutation frequency in Hong Kong colorectal carcinoma (CRC) patients, when conventional molecular screening techniques were utilized to assess the mutation rate in the hot spots in exons 5-8. Materials and methods: The yeast functional assay was used to determine if the previous mutation frequency determined by PCR-SSCP techniques was under-estimated and if so, to see if other hot spots for mutations explain this difference. Results, The p53 functional yeast assay results showed an increased mutation frequency. However, sequencing showed the mutations were confined to common hot spots for mutations in exons 6 and 7. Conclusions: The mutation frequency in CRC patients observed with the yeast assay is higher than previously reported. Forty-five percent of 20 SSCP-negative specimens were positive by the yeast assay, which this study shows is superior for detection of p53 mutations directly in clinical specimens containing varying amounts of normal tissue contamination.

A mutational spectrum for exons 5-8 of the p53 tumor suppressor gene in colorectal carcinomas in Hong Kong Chinese was established, Ninety-nine colorectal carcinomas from Dong Kong patients were analyzed for mutations in p53 gene by PCR-single-strand conformation polymorphism analysis and direct DNA sequencing, Thirty-five of the 99 tumors (35.4\%) contained mutations, Point mutations accounted for 80\% of all genetic changes and were predominantly base transitions at CpG dinucleotide sites, mutations that were also predominant in Caucasian carcinomas. The major hot spots at codons 175 and 248 of p53 in Caucasians are also hot spots in the Chinese gene, Identical mutations in codons 152 and 306 were detected in two independent tumors in the Chinese, which were reported only rarely in Caucasians, Moreover, a significantly higher frequency (20\%) of deletion and insertion mutations was observed in Dong Kong colorectal cancer patients, Distinct genetic and/or environmental factors may contribute to these findings.

A mutational spectrum for exons 5-8 of the p53 tumor suppressor gene in colorectal carcinomas in Hong Kong Chinese was established, Ninety-nine colorectal carcinomas from Dong Kong patients were analyzed for mutations in p53 gene by PCR-single-strand conformation polymorphism analysis and direct DNA sequencing, Thirty-five of the 99 tumors (35.4\%) contained mutations, Point mutations accounted for 80\% of all genetic changes and were predominantly base transitions at CpG dinucleotide sites, mutations that were also predominant in Caucasian carcinomas. The major hot spots at codons 175 and 248 of p53 in Caucasians are also hot spots in the Chinese gene, Identical mutations in codons 152 and 306 were detected in two independent tumors in the Chinese, which were reported only rarely in Caucasians, Moreover, a significantly higher frequency (20\%) of deletion and insertion mutations was observed in Dong Kong colorectal cancer patients, Distinct genetic and/or environmental factors may contribute to these findings.