A Comparative Analysis of Protein Stabilizing Potential of Honey and Simulated Honey Sugar Cocktail.

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.

Targeting chemical and thermal stability of ovalbumin by simulated honey sugar cocktail.

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.

Predictors of the yield of mobilized peripheral blood CD34+ cells in HLA-matched sibling donor.

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.

Isolation of purified autologous peripheral blood CD34+ cells with low T cell content using CliniMACS device--a local experience.

INTRODUCTION: 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. OBJECTIVE: To evaluate the purity, recovery and viability of CD34+ cells selected from harvested peripheral blood stem cells using the CliniMACS device, as well as to evaluate the T and B cell contents of these products. METHOD: Eight adult patients with malignant haematological diseases (5 non-Hodgkin's lymphomas in 2nd complete remission (CR) and 3 acute myeloid leukaemias in 1st CR) were mobilised with granulocyte colony-stimulating factor (G-CSF) with or without chemotherapeutic regimens. A total of nine leukaphereses for peripheral blood stem cell harvest using the Cobe Spectra cell separator (Cobe BCT Lakewood, CO) were performed. The harvested PBSC were then positively selected for CD34+ cells using the CliniMACS device (Milteny Biotech, Germany). RESULTS: A total of nine leukapheresis products from eight adults with a median pre-selection total CD34+ cell count of 282.2 x 10(6) (range 103.7 - 738.2 x 10(6)) were positively selected with CliniMACS. The median post-selection total CD34+ cell count was 99.5 x 10(6) (range 7.7 - 443.9 x 10(6)) with the median recovery was 66.0% (range 2 - 94%) and median purity of products of 79% (range 18 - 86%). The median total T cell count was reduced dramatically from 3.1 x 10(9) pre-selection to 7.9 x 10(6) post-selection. The selection did not affect the viability of selected cells that was tested with trypan blue exclusion method with a median pre and post selection viabilities of 98% (range 95 - 98%). CONCLUSION: We conclude that positive selection of CD34+ cells using magnetic separation technology by CliniMACS device results in low T-cell content stem cell with acceptable purity and recovery for autologous peripheral blood stem cell transplantation.