| Protein and cell therapy for lecithin-cholesterol acyltransferase (LCAT) deficiency | |
Abstract/OtherAbstract
:
|
Lecithin-cholesterol acyltransferase (LCAT) is an enzyme principally secreted by the liver into the circulation where it esterifies cholesterol and plays a key role in high- density lipoprotein (HDL) metabolism. In familial and acquired (liver disease) LCAT deficiency, the failure to esterify cholesterol causes many cellular and metabolic disturbances. Here, I describe the purification of recombinant LCAT and assess two approaches to treat LCAT deficiency. Human LCAT cDNA was cloned into a selectable expression vector and used to generate a stably–transfected Chinese hamster ovary (CHO) cells secreting human LCAT tagged with 6 histidine residues. Productive clones were selected, monitoring LCAT activity by a modification of a radioactive enzymic assay for plasma, and the enzyme purified from culture medium by immobilised cobalt affinity chromatography. The pure LCAT, as judged by SDS- PAGE, was used to raise monoclonal antibodies in LCAT knockout mice for future development of a sensitive immunoassay. For therapy, I evaluated injection of pure LCAT into the peritoneal cavity of LCAT knockout mice using single and repeat dose regimes. LCAT activity was measurable in plasma post-injection and the percentage of esterified cholesterol increased, while agarose gel electrophoresis confirmed a rise in HDL levels. In a second approach, I encapsulated the recombinant CHO cells in biocompatible and semipermeable alginate-polylysine microcapsules using a syringe pump extrusion method. A study in vitro showed that, after an initial lag phase, LCAT was secreted for over 90 days with the capsules remaining intact. These microencapsulated cells were implanted into peritoneal cavities of LCAT-deficient mice. LCAT activity was detected in mice plasma one week post-implantation; the relative amount of esterified cholesterol was increased and lipoprotein profile was improved. I conclude that injection of recombinant enzyme or of encapsulated LCAT-secreting cells are feasible therapies for familial and acquired LCAT deficiency. |
Authors
:
|
Low, J.K. |
Related Documents
:
|
0001159438 - Utilization of low-grade cassava meal (gari) in the diets of egg type chicks (0-8 weeks) 0006052068 - A comprehensive dose-response study of the effects of pistachios on cardiovascular dise... |
Contributors
:
|
- |
Publication Detail
:
|
Publisher : UCL (University College London) Type : Thesis, Doctoral, NonPeerReviewed Format : application/pdf |
Date Detail
:
|
2009-01 |
Subject
:
|
- |
Coverage
:
|
- |
Relation
:
|
- |
Source
:
|
Low, J.K. (2009) Protein and cell therapy for lecithin-cholesterol acyltransferase (LCAT) deficiency. Doctoral thesis, UCL (University College London). |
Copyright Information
:
|
- |
Other Details
:
|
Languages : eng |
Export Citation
:
|
APA/MLA Format Download EndNote Download BibTex |
Previous Document: Characterization and consequences of Telomere replication gone awry
Next Document: The Voronoi diagram in structural optimisation