Primary biliary cirrhosis, macrophages and NRAMP1

• Main Author: Graham, Alexandra Mary
• Published: University of Edinburgh 2000
• Subjects: 616.3
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.651695

This thesis reports the use of immunohistochemistry to identify increased expression of the pro-apoptotic proteins, bax and bcl-x, in BECs of PBC patients which suggests these cells are inherently susceptible to this form of programmed cell death supporting the belief that the cells die by apoptosis. An increased frequency of Ki-67 expression was also observed in BECs in PBC liver sections, showing these cells are capable of proliferation, but p53, bcl-2 and Fas/CD95 expression was similar to BECs of normal liver. Expression of <i>NRAMP1</i> was detected by reverse transcription-polymerase chain reaction (RT-PCR) in PBC and normal liver tissue. A genotyping strategy, based on restriction fragment length polymorphism (RFLP) analysis, was developed to genotype PBC and control groups for the 5' microsatellite repeat polymorphism to investigate if a similar association existed in PBC patients as RA patients. Two novel alleles were identified at this site, one of which was found at significantly increased frequency in the PBC population, though it was still uncommon. The allele driving the highest levels of expression, found at increased frequency in RA, was found at a similar frequency in the PBC population and control populations studied. To investigate the effect of overexpression of <i>NRAMP1</i> MM6 and U937 cell lines were transfected with a plasmid constructed to drive high levels of <i>NRAMP1</i> expression. The presence and expression of plasmid sequence in transfectants was confirmed by PCR and RT-PCR respectively. Analysis of the transfectants' divalent cation cellular content by inductively coupled plasma mass spectrometry (ICPMS) after culture in increased concentrations of manganese provide further evidence that the NRAMP1 protein is a divalent cation transporter protein, though the specificity of its substrate(s) requires further analysis.