Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...
Scientific Presentations Summer 2009 - Dana-Farber/Harvard ... Scientific Presentations Summer 2009 - Dana-Farber/Harvard ...
Production of ß 2 M and HLA-A2 for MHC Tetramers Formation Judith Alonzo Mentor: Karen S. Anderson MD, PhD Scientific Advisors: Jaewon Choi, MS; Jun Zhou, PhD; and Jessica Wong, BA Dana-Farber Cancer Institute Background: The major histocompatibility complex (MHC) is a genomic region that plays a crucial role in the immune system of most vertebrates. MHC tetramers are proteins that monitor T-cells in the blood and bind to T-cell receptors. They are folded by binding ß 2 -microglobulin (ß 2 M) with HLA-A2 and antigenic peptide. MHC tetramers require refolding individual sub-units of high purity, quantity, and quality. The goal of this project is to produce high yields of ß 2 M and A0201 to fold MHC tetramers with the desired peptide. Methods: Three A2 DNA constructs were tested, HLA-A2Nohis1, A2his+4, and A*0201 and one ß 2 M. Culture conditions were optimized to obtain higher yields of both, ß 2 M and HLA-A. Proteins were expressed in E. coli bacteria. Two methods were employed for ß 2 M and HLA-A production to fold MHC tetramers, sonication and B-PER Reagent. Both techniques disrupt the cell membranes and release cell contents, including proteins. Refolding reaction and biotinylation of each protein follows. Purity is monitored on 4-20% SDS-PAGE gel. Results: HLA-A*0201 was found to work better than the other DNA constructs, giving higher yields (390.80mg for 1L culture) and more pure samples; similar results have been obtained for ß 2 M but at lower yields (13.46mg for 250mL culture). Currently, sonication has proven to be more suitable for large-scale protein preparation than B-PER reagent. Conclusion: A0201 was obtained in high yields and purity, as well as ß 2 M. The sonication method is more efficient for Largescale protein production while B-PER reagent is more convenient for smaller sample sizes. No final conclusions can be made at this time since folding and biotinylation processes are ongoing.
Medical and Psychological Outcomes of Sibling Donors for Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) at a Single Institution Bethel Belai Mentor: Leslie Lehmann, MD Dana-Farber Cancer Institute Bone marrow transplant (BM) and cytokine mobilized peripheral blood stem cell (PBSC) transplantation can be curative for patients with malignant and nonmalignant hematologic diseases. Siblings are most often used as donor matches for such patients. In cases where siblings are not donor matches, unrelated donors are used as donor matches for these same patients. Much psychological assessment has been done regarding unrelated donors post-donation, but none involving sibling donors specifically. This exploratory study aims to assess the medical and psychological outcomes of sibling donors following a bone marrow harvest performed at Dana-Farber Cancer Institute from January1998 through December 2008. Donor outcome was assessed at a time, 3-9 months following donation. The first part of the study is the medical element, compiled by a list of an estimated 150 sibling donors within the past 10 years, who are between 5-19 years of age. The second part is the psychological element which examines the donors mental state pre and post-donation. Assessments were performed in person, or by phone by a member of the HSCT team who did not perform the actual harvest. Donors were asked questions regarding any medical or psychological issues related to the process of serving as a donor (guardian was present for questioning unless donor was under 18 years of age and without guardian consent). Data will be gathered based on the outcome of these assessments as documented in the donor medical record.
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Medical and Psychological Outcomes of Sibling Donors for Patients Undergoing<br />
Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) at a Single Institution<br />
Bethel Belai<br />
Mentor: Leslie Lehmann, MD<br />
<strong>Dana</strong>-<strong>Farber</strong> Cancer Institute<br />
Bone marrow transplant (BM) and cytokine mobilized peripheral blood stem cell<br />
(PBSC) transplantation can be curative for patients with malignant and nonmalignant<br />
hematologic diseases. Siblings are most often used as donor matches for such<br />
patients. In cases where siblings are not donor matches, unrelated donors are used<br />
as donor matches for these same patients. Much psychological assessment has been<br />
done regarding unrelated donors post-donation, but none involving sibling donors<br />
specifically. This exploratory study aims to assess the medical and psychological outcomes<br />
of sibling donors following a bone marrow harvest performed at <strong>Dana</strong>-<strong>Farber</strong><br />
Cancer Institute from January1998 through December 2008. Donor outcome was<br />
assessed at a time, 3-9 months following donation. The first part of the study is the<br />
medical element, compiled by a list of an estimated 150 sibling donors within the<br />
past 10 years, who are between 5-19 years of age. The second part is the psychological<br />
element which examines the donors mental state pre and post-donation. Assessments<br />
were performed in person, or by phone by a member of the HSCT team who did<br />
not perform the actual harvest. Donors were asked questions regarding any medical<br />
or psychological issues related to the process of serving as a donor (guardian was<br />
present for questioning unless donor was under 18 years of age and without guardian<br />
consent). Data will be gathered based on the outcome of these assessments as<br />
documented in the donor medical record.
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