Date of Award

2010

Document Type

Thesis

Degree Name

Master of Clinical Research

College

College of Graduate Studies

First Advisor

Perry V. Halushka

Abstract

Background: Acute Graft-Versus-Host disease (acute GVH D) is an often fatal, inflammatory multi-organ disease driven by T cell alloreactivity directed toward host tissues. One of the chief obstacles to improving outcomes of acute GVHD patients is timely diagnosis. Currently, acute GVHD is diagnosed clinically, once the symptoms are fully manifested. However, it is clear that important immunologic events occur prior to the occurrence of symptoms. A biomarker that could predict disease during the asymptomatic period could improve patient outcomes by allowing for earlier intensification of immunosuppressant therapy. Exosomes are an attractive target for acute GVHD biomarker discovery. They contain a variety of cellular components and are easily obtained from bodily fluids. Furthermore, the cell types implicated in acute GVHD pathophysiology produce exosomes with strong immunomodulatory effects. Methods: A differential centrifugation protocol was developed to isolate exosomes from cryopreserved plasma. The presence of exosomes in the final pellet was confirmed by Western blotting of known exosome markers and by electron microscopy. This protocol was used to isolate exosome-enriched pellets from 5 ml plasma samples obtained on posttransplantation day 7 from 7 patients (N=7) who had undergone allogeneic hematopoietic stem cell transplantation. Three of these patients (cases) later developed severe acute GVHD (grade C or D), whereas 4 patients did not develop acute GVHD of any grade (controls). Proteomic analysis was performed on exosome pellets using liquid chromatography-tandem mass spectrometry and iTRAQ labeling, which allowed us to compare the relative quantities of identified proteins between cases and controls. Results: We made 33 protein identifications. iTRAQ analysis did not demonstrate any statistically significant differences between cases and controls. Despite this limitation, our data did show trends toward differences in the relative abundances of some proteins. Specifically, there was a trend toward increased IgG3 constant region in cases (median case:control ratio= 1.36; credible interval of case:control ratio= 0.857, 2.19), increased lambda light chain constant region in cases (median case:control ratio= 1 .35; credible interval of case:control ratio= 0.844,2.07) and decreased kappa light chain constant region in cases (median case:control ratio=O.847; credible interval of case:control ratio=.0586, 1.21). Further statistical analysis to compare the kappa:lambda ratios in cases compared controls showed that the median case:control kappa:lambda ratio was 0.63, with a 95% CI of 0.377, 1.027 (p=0.011). Conclusion: Differences in kappa:lambda ratios, early after allogeneic hematopoietic stem cell transplantation may be predictive of the development of acute GVHD. Future studies will be directed toward validation of these results.

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