Effect of simulated honey sugar cocktail (SHSC) on chemical and thermal stability of ovalbumin (OVA) was investigated using multiple-spectroscopic techniques. Urea-induced denaturation of OVA produced a transition, characterized by the start-, the mid- and the end-points at 3.2 M, 5.9/5.6 M and 8.5/8.0 M urea, respectively, when studied by MRE222nm and tryptophan fluorescence measurements. Presence of 10% or 20% (w/v) SHSC in the incubation mixture shifted the transition curve towards higher urea concentration in a concentration dependent manner. A comparison of far- and near-UV CD, UV-difference, ANS fluorescence and 3-D fluorescence spectral results of native OVA and 5.9 M urea-denatured OVA (U-OVA), obtained in the absence and the presence of 20% (w/v) SHSC suggested SHSC-induced stabilization of U-OVA. Furthermore, a significant shift towards higher denaturant concentration was also noticed in the GdnHCl and thermal transition curves of OVA in the presence of 20% (w/v) SHSC. Taken together, all these results suggested stabilization of OVA against chemical and thermal denaturations by SHSC.
Urea and thermal denaturations of bovine serum albumin (BSA) were studied in the absence and the presence of honey or simulated honey sugar cocktail (SHSC) using far-UV CD and ANS fluorescence spectroscopy. Presence of 20% (w/v) honey or SHSC in the incubation mixture shifted the urea transition curve towards higher urea concentrations, being higher in the presence of honey and transformed the two-step, three-state transition into a single-step, two-state transition. A comparison of the far-UV CD and the ANS fluorescence spectra of 4.6 M urea-denatured BSA (U-BSA) in the absence and the presence of 20% (w/v) honey or SHSC suggested greater stabilizing potential of honey than SHSC, as U-BSA maintained native like conformation in the presence of 20% (w/v) honey. Furthermore, thermal transition curves of BSA were also shifted towards higher temperature range in the presence of 20% (w/v) SHSC and honey, showing greater shift in the presence of honey. The far-UV CD spectra of the heat-denatured BSA also showed greater stabilization in the presence of honey. Taken together all these results suggested greater protein stabilizing potential of honey than SHSC against chemical and thermal denaturations of BSA.
Dendritic cells (DC) are professional antigen presenting cells of the immune system. Through the use of DC vaccines (DC after exposure to tumour antigens), cryopreserved in single-use aliquots, an attractive and novel immunotherapeutic strategy is available as an option for treatment. In this paper we describe an in vitro attempt to scale-up production of clinical-grade DC vaccines from leukemic cells. Blast cells of two relapsed AML patients were harvested for DC generation in serum-free culture medium containing clinical-grade cytokines GM-CSF, IL-4 and TNF-alpha. Cells from patient 1 were cultured in a bag and those from patient 2 were cultured in a flask. The numbers of seeding cells were 2.24 x 10(8) and 0.8 x 10(8), respectively. DC yields were 10 x 10(6) and 29.8 x 10(6) cells, giving a conversion rate of 4.7% and 37%, respectively. These DC vaccines were then cryopreserved in approximately one million cells per vial with 20% fresh frozen group AB plasma and 10% DMSO. At 12 months and 21 months post cryopreservation, these DC vaccines were thawed, and their sterility, viability, phenotype and functionality were studied. DC vaccines remained sterile up to 21 months of storage. Viability of the cryopreserved DC in the culture bag and flask was found to be 50% and 70% at 12 months post cryopreservation respectively; and 48% and 67% at 21 months post cryopreservation respectively. These DC vaccines exhibited mature DC surface phenotypic markers of CD83, CD86 and HLA-DR, and negative for haemopoietic markers. Mixed lymphocyte reaction (MLR) study showed functional DC vaccines. These experiments demonstrated that it is possible to produce clinical-grade DC vaccines in vitro from blast cells of leukemic patients, which could be cryopreserved up to 21 months for use if repeated vaccinations are required in the course of therapy.
Peripheral blood stem cells (PBSC) mobilised with growth factor with or without chemotherapeutic regimens, are used increasingly in both autologous and allogeneic transplantation. Previously, many PBSC harvests are used directly without ex vivo manipulation, and these PBSC have been shown to be contaminated with tumour cells, which may contribute to subsequent relapses post transplantation. Therefore, requirement for purging of malignant cells from the harvest has initiated the use of various methods to reduce tumour cell contamination of the graft by the positive selection of CD34+ progenitor cells or negative selection of tumour cells using other cell-specific antigens. We report here our local experience with the CliniMACS (magnetic-activated cell separation system) in eight adult patients with haematologic malignancies.
We examined the donor factors that may affect the yield of peripheral blood stem cell (PBSC) mobilized from healthy donors. Pre-apheresis PB-CD34(+) cell count was the only factor that correlated with PBSC yield. Leukocyte count (LC) and monocyte count (MC) correlated with PB-CD34(+) cell. Male gender and PB-CD34(+) cell count of at least 87.1/μL and 69.8/μL on day-4 and -5 of G-CSF were associated with the ability to harvest at least 5×10(6)/kg CD34(+) cells after one apheresis. We concluded that gender and PB-CD34(+) cell count are important predictors of PBSC yield. LC and MC may serve as surrogate markers for estimating the PB-CD34(+) cell count.