Abstract
Hematopoietic cells are commonly exposed to human serum albumin (HSA) in the clinical setting and fetal bovine serum (FBS) in the research setting. Serum is utilized as a standard reagent across multiple research disciplines for tissue culture (10–20% FBS). HSA is a common reagent in the clinical setting and is routinely added to hematopoietic cells (0.5–4% HSA) in preparation for cryopreservation. Many labs also wash cryopreserved hematopoietic stem cells with HSA (1–5%) prior to autologous or allogenic transplantation to rid the cells of the DMSO used during cryopreservation. Lastly, HSA is sometimes infused directly into patients in the clinic for such indications as the short-term treatment of nephrotic syndrome. We have generated data suggesting that the peptidase CD26 (dipeptidylpeptidase IV/DPPIV) regulates HSC/HPC function, release of HPC out of the bone marrow (BM) during G-CSF induced mobilization, and engraftment of HSC/HPC into the BM during transplantation. It is within this context that we evaluated the presence of CD26/DPPIV activity in HSA. HSA of 20%, 10%, 2.5%, and 1% were prepared by diluting Flexbumin 25% (Baxter Healthcare) with 0.9% NaCl. FBS (Hyclone) concentrations were prepared likewise. Bovine Serum Albumin Fraction V (BSA) concentrations were prepared weight by volume. CD26/DPPIV activity was assayed utilizing the Gly-Pro-p-nitroanilide substrate in a 96-well format. Standard p-nitroanilide (pNA) curves were created by serially diluting pNA with 5mM Tris-HCl pH 7.5. 4mM Gly-Pro-pNA substrate was then combined with each test solution in 5mM Tris-HCl. Absorbance readings (A405) were then taken at 37° C every 5 minutes for 60 minutes. CD26 inhibition was achieved by treating with Diprotin-A (Ile-Pro-Ile). Prior to measurement of CD26 activity, HSA, BSA, or FBS were combined with Diprotin-A incubated at 37° C for 15 minutes. Final Diprotin-A concentrations during treatment were 5000μM, 500μM, 50μM, 5μM, 0.5μM, 0.05μM, 0.005μM, and 0 μM. Evaluation of CD26 activity in 1%, 2.5%, 10%, and 20% HSA solutions revealed a statistically significant and dose dependent level of CD26 activity within the serum (p<0.05). CD26 activity of 10% HSA was quantitated at 49.62±1.5 pmoles/min and activity of 10% FBS was quantitated at 53.42±1.44 pmoles/min. Evaluation of CD26 activity in 2.5%, 10%, or 20% FBS also showed a quantifiable and dose dependent CD26 activity (p< 0.05). 1%, 2.5%, 10%, and 20% BSA Fraction V had no detectable levels of CD26 activity as compared to saline alone. Furthermore, treatment with the CD26 inhibitor, Diprotin A, actively inhibits CD26 activity ex vivo in a dose dependent manner (p< 0.05). These results suggest that significant levels of CD26 activity are present in HSA (Flexbumin) that is utilized routinely for clinical purposes. Additional evaluations are indicated to test whether this is an industry wide phenomenon across multiple preparation procedures and manufactures. Additionally, these results suggest that the CD26 activity that is present is specific and can be inhibited utilizing CD26 inhibitors. No clinical protocol methodology currently exists for the treatment of HSA in the clinical or research setting. Lastly, these results suggest that the use of HSA that may contain CD26 activity during clinical freezing and thawing procedures of transplant units should be reconsidered, especially if the HSA remains with the unit during infusion. Future studies will be needed to evaluate if the results these pre-clinical experiments necessitate changes to clinical methodologies.
IMMUNOLOGIC TOLERANCE AFTER SPECIFIC IMMUNIZATION